KR20070007817A - Self-processing plants and plant parts - Google Patents

Abstract

The present invention provides polynucleotides, preferably synthetic polynucleotides, which encode processing enzymes optimized for expression in plants. The polynucleotides encode mesophilic, thermophilic or hyperthermic processing enzymes activated under suitable activation conditions to act upon the substrate of interest. Also provided are "self-processing" transformed plants and plant parts, such as grains, having an alternative composition that expresses one or more of these enzymes and facilitates plant and grain processing. Also provided are methods of making and using these plants, for example, for preparing foods with improved taste and for producing fermentable substrates for the production of ethanol and fermented beverages.

Autologous Enzyme, Transformed Plant, Vector, Starch, Fermentable Sugar, Corn γ-Zane Promoter, Corn ADP-gpp Promoter

Description

Self-processing plants and plant parts

Related Applications

This application is a US patent application filed on Aug. 27, 2002 that claims priority to US patent application Ser. No. 60 / 315,281, filed Aug. 27, 2001, which is hereby incorporated by reference in its entirety. Partial serial application of heading 10 / 228,063.

The present invention relates to the field of plant molecular biology, and more particularly to the production of plants expressing processing enzymes that provide the desired properties to the plant or its products.

Enzymes are used to process various agricultural products such as wood, fruit and vegetables, starch, juice and the like. Typically, processing enzymes are produced and recovered on an industrial scale from various sources, such as microbial fermentation ( Bacillus α-amylase) or separation from plants (coffee β-galactosidase or papain from plant parts). Enzyme preparations are used in different processing applications by mixing enzymes and substrates under suitable conditions of humidity, temperature, time and mechanical mixing to achieve enzymatic reactions in a commercially viable manner. The method includes preparing an enzyme, preparing an enzyme preparation, mixing an enzyme with a substrate, and subjecting the mixture to conditions suitable to catalyze the enzymatic reaction. Methods of reducing or eliminating time, energy, mixing, capital expenditure, and / or enzyme manufacturing costs, or yielding improved or novel products, may be useful and beneficial. One example where such improvements are needed is in the area of corn milling.

Corn is milled today to obtain corn starch and other corn-milling by-products (eg corn gluten feed, corn gluten powder and corn oil). Starches obtained from this process are often further processed into derivatives starch and other products such as sugars or fermented to produce a variety of products including alcohols or lactic acid. Processing of corn starch often involves the use of enzymes, in particular enzymes that hydrolyze and convert starch into fermented sugars or fructose (α- and gluco-amylases, α-glucosidase, glucose isomerase, etc.). Current commercially available methods are capital intensive because the manufacture of bulk mills requires processing of corn on a scale that requires adequate cost effectiveness. In addition, the process requires an enzyme and substrate mixing machine for the separate preparation of starch-hydrolyzed or modified enzymes followed by hydrolyzed starch products.

Methods of recovering starch from corn grains are well known and include wet milling methods. Corn wet milling includes soaking corn kernels, grinding the corn kernels, and separating the corn components. The grains are immersed in countercurrent at about 120 ° F in the immersion tank and remain in the immersion tank for 24 to 48 hours. The immersion water usually contains sulfur dioxide at a concentration of about 0.2% by weight. Sulfur dioxide is used in the process to help reduce microbial growth and also to reduce disulfide bonds in endosperm proteins to promote more efficient starch-protein separation. Generally, about 0.59 gallons of immersion is used per corn bushel. Dipping water is considered waste and often contains undesirable levels of residual sulfur dioxide.

The soaked grains are then dewatered and applied to a set of friction mills. The first set of friction mills ruptures the grains to release the vessel from the rest of the grains. Commercially available friction mills suitable for wet milling operations are sold under the trade name Bauer. Centrifuge is used to separate vessels from the rest of the grain. Commercially available centrifuges are Merco centrifuges. Friction mills and centrifuges are very expensive items that require energy to operate.

In the next step of the process, the remaining grain components, including starch, shell, fiber and gluten, are applied to another set of friction mills and passed through a wash screening set to separate the fiber components from starch and gluten (endosperm protein). Starch and gluten pass through the selector, but not fiber. Starch is separated from the endosperm protein using centrifugation or centrifugation after third grinding. Centrifugation produces a dehydrated starch slurry, which is then washed with fresh water and dried to about 12% humidity. Substantially pure starch is usually further processed using enzymes.

Separation of starch from other components of the grain is carried out, which allows the removal of seed hulls, embryos and endosperm proteins to efficiently contact the starch and the processing enzymes, thereby substantially removing contaminants from other grain components. This is because it is liberated. Separation also allows for efficient recovery of other components of the grain and subsequently marketed as by-products to increase profit from milling.

After the starch is recovered from the wet milling process, the processing steps of gelatinization, liquefaction and dextrinization are usually carried out for the preparation of maltodextrins, and the succession of saccharification, isomerization and purification for the preparation of glucose, maltose and fructose Perform the steps.

Gelatinization is used for starch hydrolysis because currently available enzymes cannot rapidly hydrolyze crystalline starch. To prepare starch usable for hydrolysable enzymes, the starch is usually made into a slurry containing water (20-40% dry solids) and heated at a suitable gelling temperature. For corn starch, this temperature is about 105-110 ° C. Gelatinized starch is usually very viscous and therefore dilutes in the next step, referred to as liquefaction. Liquefaction breaks down some of the bonds between the glucose molecules of the starch and is achieved using enzymes or acids. The heat resistant endo α-amylase enzyme is used in this and subsequent dextrinization steps. The degree of hydrolysis is adjusted in the dextrinization step to yield the desired percent of the hydrolysis product of dextrose.

Further hydrolysis of the dextrin product from the liquefaction step is performed with a number of different exo-amylases and debranching enzymes, depending on the desired product. Finally, if fructose is desired, glucose is usually converted to fructose using an immobilized glucose isomerase enzyme.

The dry milling method of preparing fermentable sugars (and, for example, ethanol) from corn starch, promotes effective contact of the exogenous enzyme with the starch. These methods are less capital intensive than wet milling, but significant cost advantages are still desired because often the by-products derived from these methods are not worth as much as the by-products derived from wet milling. For example, in the case of dry milling of corn, the grains are ground into powder to promote efficient contact of the starch by enzymatic degradation. After enzymatic hydrolysis of corn powder, the residual solids have some value as feed because they contain proteins and some other ingredients. Eckhoff recently described the possibility and improvement of dry milling in a paper entitled "Fermentation and Cost of Fuel Ethanol from Corn Using Short-Pear Processing" (Appl. Biochem). Biotechnol., 94:41 (2001)]. The "short pear" method makes it possible to separate rich pears from starch using reduced soaking times.

One example where control and / or concentration of endogenous processing enzymes in plants can lead to desirable products. Typically, sweet corn varieties are distinguished from wild corn varieties due to the fact that the average level of starch biosynthesis is not possible. Gene mutations in genes encoding enzymes involved in corn biosynthesis are commonly used in sweet corn varieties to limit corn biosynthesis. Such mutations result in genes encoding starch synthase and ADP-glucose pyrophosphorylase (eg, saccharide and ultra-high sugar mutations). Fructose, glucose and sucrose, the simple sugars needed to produce the desired sugar content required by consumers of fresh edible corn, accumulate in the developing endocrine of these mutants. However, if the corn is left to mature for too long (late harvest) or stored for an excessive period before the corn is consumed, if the starch accumulation concentration is too high, the product loses sugar content and feels in a pale taste and mouth. Therefore, the harvest time for sweet corn is very narrow and the storage period is limited.

Another important disadvantage for farmers cultivating sweet corn varieties is that their usefulness is entirely limited to edible food. If a farmer wants to harvest his sweet corn ahead of time for use as an edible food during seed development, the crop will inevitably be lost. Grain yield and quality of sweet corn are poor for two fundamental reasons. The first reason is that mutations in the starch biosynthesis pathway impair starch biosynthesis mechanisms and the grains do not fully collapse, which compromises yield and quality. Second, because of the high concentrations of sugar present in the grains and their inability to sequester into the starch, the total sink strength of the seeds decreases, exacerbating the reduction in nutrient storage in the grains. The endosperm of the sweet corn varieties is contracted and collapsed, resulting in improper dehydration and increased susceptibility to disease. Poor quality of sweet corn grains further has agricultural effects such as poor seed growth, poor germination, seedling disease susceptibility and poor early seedling growth due to a combination of factors caused by inadequate starch storage. Thus, the poor quality problem of sweet corn affects consumers, farmers / cultivators, distributors and seed producers.

Therefore, for dry milling, there is a need for a method that will improve the efficiency of the process and / or increase the value of by-products. For wet milling, there is a need for a starch processing method that does not require the equipment necessary for extended dipping, grinding, milling and / or separation of the components of the grain. For example, in wet milling, it is necessary to modify or remove the immersion step, which will reduce the amount of sewage that requires disposal, saving energy and time, and increasing the milling capacity (grain will be less in the immersion tank). Will spend time). There is also a need to eliminate or improve the process of separating starch-containing endoderm from embryos.

Summary of the Invention

The present invention relates to self-processing plants and plant parts and methods of use thereof. Self-processing plants and plant parts of the invention can express and activate enzyme (s) (medium, thermophilic and / or hyperthermia). Upon activation of the enzyme (s) (medium, thermophilic or superthermophilic), the plant or plant part can self-process the substrate on which these enzymes act to obtain the desired result.

The present invention is directed to a) having a SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59 or its complement, or low stringency hybridization Under conditions hybridize with the complement of any one of SEQ ID NOs: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59, and α-amylase, pool Encoding a polypeptide having lulanase, α-glucosidase, glucose isomerase or glucoamylase activity, or b) SEQ ID NOs: 10, 13, 14, 15, 16, 18, 20, 24, 26, 27, 28, Isolated polynucleotides comprising polynucleotides encoding polypeptides comprising 29, 30, 33, 34, 35, 36, 38, 40, 42, 44, 45, 47, 49 or 51 or enzymatically active fragments thereof It is about. Preferably, the isolated polynucleotide encodes a fusion polypeptide comprising a first polypeptide and a second peptide, wherein the first polypeptide is α-amylase, pullulanase, α-glucosidase, glucose isomera Or glucoamylase activity. Most preferably, the second peptide comprises a signal sequence peptide capable of targeting the first polypeptide to vacuoles, cytoplasmic reticulum, chloroplasts, starch granules, seeds or cell walls of the plant. For example, the signal sequence can be an N-terminal signal sequence from wawy, an N-terminal signal sequence from γ-jane, a starch binding domain or a C-terminal starch binding domain. Polynucleotides that hybridize with the complement of any one of SEQ ID NOs: 2, 9 or 52 under low stringency hybridization conditions and encode a polynucleotide having α-amylase activity; Polynucleotides that hybridize with the complement of SEQ ID NO: 4 or 25 under low stringency hybridization conditions and encode a polypeptide having pullulanase activity; Polynucleotides that hybridize with the complement of SEQ ID NO: 6 and encode a polypeptide having α-glucosidase activity; Polynucleotides that hybridize with the complement of any one of SEQ ID NOs: 19, 21, 37, 39, 41 or 43 under low stringency hybridization conditions and encode a polypeptide having glucose isomerase activity; Further included are polynucleotides that hybridize with the complement of any one of SEQ ID NOs: 46, 48, 50 or 59 under low stringency hybridization conditions and encode a polypeptide having glucoamylase activity.

The present invention includes a) SEQ ID NOs: 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108 and 110 or complements thereof; The complement of any one of SEQ ID NOs: 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108 and 110 under low stringency hybridization conditions Hybridize and encode a polypeptide having xylanase, cellulase, glucanase, beta glucosidase, esterase or phytase activity or b) SEQ ID NOs: 62, 64, 66, 70, 80, 82, A polynucleotide comprising 84, 86, 88, 90, 92, 109 or 111, or an isolated polynucleotide encoding an enzymatically active fragment thereof. The isolated polynucleotide may encode a fusion polypeptide comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is xylanase, cellulase, glucanase, beta glucosidase, protease or Has phytase activity. The second peptide may comprise a signal sequence peptide capable of targeting the first polypeptide to vacuoles, cytoplasmic reticulum, chloroplasts, starch granules, seeds or cell walls of the plant. For example, the signal sequence can be an N-terminal signal sequence from waxy, an N-terminal signal sequence from γ-Zane, a starch binding domain or a C-terminal starch binding domain.

Exemplary xylanases provided and useful herein are those encoded by SEQ ID NOs: 61, 63 or 65. Exemplary proteases encoded by SEQ ID NO: 69, ie bromelain, are also provided. Exemplary cellulases include cellobiohydrolases I and II provided herein and encoded by SEQ ID NOs: 79, 81, 93, and 94. Exemplary glucanases are provided as 6GP1 described herein, encoded by SEQ ID NO: 85. Exemplary beta glucosidases include beta glucosidases 2 and D as described herein and encoded by SEQ ID NOs: 96 and 97. Exemplary esterases, ie ferulic acid esterases encoded by SEQ ID NO: 99, are also provided. And also provided is an exemplary phytase, Nov9X, encoded by SEQ ID NOs: 109-112.

And have SEQ ID NO: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59 or complements thereof, or under low stringency hybridization conditions 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52 or 59 hybridize with the complement and α-amylase, pullulanase, α-glucose Encoding a polypeptide having a sidase, glucose isomerase or glucoamylase activity or b) SEQ ID NOs: 10, 13, 14, 15, 16, 18, 20, 24, 26, 27, 28, 29, 30, 33, Expression cassettes comprising polynucleotides encoding polypeptides comprising 34, 35, 36, 38, 40, 42, 44, 45, 47, 49, or 51 or enzymatically active fragments thereof are included. The expression cassette further comprises a promoter operably linked to a polynucleotide, such as an inducible promoter, a tissue-specific promoter or preferably an endosperm-specific promoter. Preferably, the endosperm-specific promoter is a corn γ-zein promoter or corn ADP-gpp promoter or corn Q promoter or rice glutelin-1 promoter. In a preferred embodiment, the promoter comprises SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 67 or SEQ ID NO: 98. Further, in another preferred embodiment, the polynucleotides are oriented in the sense direction with respect to the promoter. The expression cassette of the present invention may further encode a signal sequence operatively linked to a polypeptide encoded by the polynucleotide. The signal sequence preferably targets the operably linked polypeptide to the vacuoles, cytoplasmic reticulum, chloroplasts, starch granules, seeds or cell walls of the plant. Signal sequences include N-terminal signal sequences from waxy, N-terminal signal sequences from γ-Zane or starch binding domains.

In addition, a) has SEQ ID NOs: 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108 and 110 or complements thereof; The complement of any one of SEQ ID NOs: 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108 and 110 under low stringency hybridization conditions Encode a polypeptide that hybridizes and has xylanase, cellulase, glucanase, beta-glucosidase, esterase or phytase activity, or b) SEQ ID NOs: 62, 64, 66, 70. 80, 82 , Expression cassettes comprising polypeptides encoding 84, 86, 88, 90, 92, 109 or 111 or polypeptides encoding enzymatically active fragments thereof. The expression cassette further comprises a promoter operably linked to a polynucleotide, such as an inducible promoter, a tissue-specific promoter or preferably an endosperm-specific promoter. The endosperm-specific promoter may be a corn γ-zein promoter or a corn ADP-gpp promoter or a corn Q promoter or a rice glutelin-1 promoter. In one embodiment, the promoter comprises SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 67 or SEQ ID NO: 98. Further, in other embodiments, the polynucleotides are directed in the sense direction relative to the promoter. The expression cassette of the present invention may further comprise a signal sequence operably linked to a polypeptide encoded by a polynucleotide. The signal sequence targets the operably linked polypeptide, preferably to plant vacuoles, cytoplasmic reticulum, chloroplasts, starch granules, seeds or cell walls. Signal sequences include N-terminal signal sequences from waxy, N-terminal signal sequences from γ-Zane or starch binding domains.

The invention further relates to a vector or cell comprising the expression cassette of the invention. The cells may be selected from the group consisting of grain cells such as Agrobacterium , monocotyledonous cells, dicotyledonous cells, Liliposida cells, Panicoideae cells, corn cells and rice cells.

In addition, the present invention encompasses plants stably transformed with the vectors of the present invention. Α-amylase having an amino acid sequence of any one of SEQ ID NOs: 1, 10, 13, 14, 15, 16, 33, 35 or 88 or encoded by a polynucleotide comprising any of SEQ ID NOs: 2, 9 or 87 It provides a plant stably transformed with a vector comprising a.

In another embodiment, a plant having an amino acid sequence of any one of SEQ ID NOs: 24 or 34, or stably transformed with a vector comprising a pullulanase encoded by a polynucleotide comprising any of SEQ ID NOs: 4 or 25 to provide. Further provided is a plant stably transformed with a vector having an amino acid sequence of any one of SEQ ID NOs: 26 or 27 or comprising an α-glucosidase encoded by a polynucleotide comprising SEQ ID NO: 6. A polynucleotide having an amino acid sequence of any one of SEQ ID NOs: 18, 20, 28, 29, 30, 38, 40, 42 or 44 or comprising any of SEQ ID NOs: 19, 21, 37, 39, 41 or 43 Further described herein are plants stably transformed with a vector comprising a glucose isomerase encoded by. In another embodiment, it is stable with a vector comprising a glucose amylase having an amino acid sequence of any of SEQ ID NOs: 45, 47 or 49 or encoded by a polynucleotide comprising any of SEQ ID NOs: 46, 48, 50 or 59 Transformed plants are described.

A further embodiment is stably with a vector comprising a xylanase having an amino acid sequence of any of SEQ ID NOs: 62, 64 or 66 or encoded by a polynucleotide comprising any of SEQ ID NOs: 61, 63 or 65 Provide a transformed plant. Also provided are plants stably transformed with the vector comprising the protease. The protease may be a bromelain having the amino acid sequence set forth in SEQ ID NO: 70 or encoded by a polynucleotide having SEQ ID NO: 69. In another embodiment, a plant is stably transformed with a vector comprising cellulase. The cellulase may be a cellobiohydrolase encoded by a polynucleotide comprising any of SEQ ID NOs: 79, 80, 81, 82, 93 or 94.

Another aspect provides a plant stably transformed with a vector comprising a glucanase, such as endoglucanase. Endoglucanase may be endoglucanase I having the amino acid sequence of SEQ ID NO: 84 or encoded by a polynucleotide comprising SEQ ID NO: 83. Also provided are plants stably transformed with the vector comprising beta glucosidase. Beta glucosidase may be glucosidase 2 or beta glucosidase D having the amino acid sequence set forth in SEQ ID NO: 90 or 92 or encoded by a polynucleotide having SEQ ID NO: 89 or 91. In another embodiment, a plant is stably transformed with a vector comprising an esterase. The esterase may be a ferulic acid esterase encoded by a polynucleotide comprising SEQ ID NO: 99.

Further provided are plant products such as seeds, fruits or grains from the stably transformed plants of the present invention.

In another aspect, the invention relates to a transformed plant in which the genome is enhanced with recombinant polynucleotides encoding one or more processing enzymes operably linked to a promoter sequence, wherein the sequence of the polynucleotides is optimized for expression in the plant have. The plant can be monocotyledonous or dicotyledonous, such as corn or rice. The plant may be a cereal plant or a commercially grown plant. Processing enzymes include α-amylase, glucoamylase, glucose isomerase, glucanase, β-amylase, α-glucosidase, isoamylase, pullulanase, neo-pululanase, iso-pululanase, amylo Pullulanase, cellulase, exo-1,4-β-cellobiohydrolase, exo-1,3-β-D-glucanase, β-glucosidase, endoglucanase, L-arabina Agent, α-arabinosidase, galactanase, galactosidase, mannase, mannosidase, xylanase, xylosidase, protease, glucanase, xylanase, esterase, phytase And lipases. Processing enzymes include α-amylase, glucoamylase, glucose isomerase, β-amylase, α-glucosidase, isoamylase, pullulanase, neo-pululanase, iso-pululanase and amylopululana Starch-processing enzyme selected from the group consisting of zero. The enzyme may be selected from α-amylase, glucoamylase, glucose isomerase, α-glucosidase and pullulanase. The processing enzyme may be superthermophilic. According to this aspect of the invention, the enzyme may be a non-starch degrading enzyme selected from the group consisting of proteases, glucanases, xylanases, esterases, phytases, cellulases, beta glucosidases and lipases. Such enzymes may be superthermophilic. In one embodiment, the enzyme accumulates in the vacuoles, cytoplasmic reticulum, chloroplasts, starch granules, seeds or cell walls of the plant. In addition, in other embodiments, the genome of the plant may be further enhanced with a second recombinant polynucleotide comprising a non-thermophilic enzyme.

In another aspect of the invention, α-amylase, glucoamylase, glucose isomerase, α-glucosidase, pullulase, xylanase, cellulase, protease, glucanase, whose genome is operably linked to a promoter sequence Provided are transformed plants enhanced with recombinant polynucleotides encoding at least one processing enzyme selected from the group consisting of beta glucosidase, esterase, phytase or lipase, wherein the sequence of polynucleotides is expressed in a plant Optimized for

In another embodiment, α-amylase, glucoamylase, glucose isomerase, α-glucosidase, pullulanase, xylanase, cellulase, protease, glucanase, phytase, To a transformed corn plant enhanced with recombinant polynucleotides encoding at least one processing enzyme selected from the group consisting of beta glucosidase, esterase or lipase, the sequence of the polynucleotides optimized for expression in corn plants It is.

Provided are transformed plants in which the genome has been enhanced with recombinant polynucleotides having SEQ ID NO: 83 operably linked to a promoter and signal sequence. In addition, a transgenic plant is described wherein the genome is enhanced with recombinant polynucleotides having SEQ ID NOs: 93 or 94 operably linked to a promoter and signal sequence. In another aspect, a transformed plant is described wherein the genome is enhanced with recombinant polynucleotides having SEQ ID NO: 95 operably linked to a promoter and signal sequence. In addition, it describes transformed plants whose genome has been enhanced with recombinant polynucleotides having SEQ ID NO. Also described are transformed plants whose genome has been augmented with recombinant polynucleotides having SEQ ID NO. It also describes a transformed plant whose genome has been enhanced with a recombinant polypeptide having SEQ ID 99.

The product of the transformed plant is further contemplated herein. For example, the product includes seeds, fruits or grains. The product may alternatively be a processing enzyme, starch or sugar.

Further described are plants obtained from the stably transformed plants of the present invention. In this aspect, the plant may be a hybrid plant or a suburban plant.

Starch compositions are a further aspect of the invention comprising one or more processing enzymes that are proteases, glucanases or esterases.

The grains are α-amylase, pullulanase, α-glucosidase, glucoamylase, glucose isomerase, xylanase, cellulase, glucanase, beta glucosidase, esterase, protease, lipase or phytase And one or more processing enzymes.

In another embodiment, treating a grain comprising one or more non-starch processing enzymes, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding one or more enzymes, under conditions that activate one or more enzymes, starch A method comprising the steps of yielding a mixture comprising granules and non-starch decomposition products, and separating starch granules from the mixture. Here, the enzyme may be a protease, glucanase, xylanase, phytase, lipase, beta glucosidase, cellulase or esterase. In addition, the enzyme is preferably superthermophilic. The grains may be copper granules and / or may be processed under low or high humidity conditions. Alternatively, the grain can be processed into sulfur dioxide. The invention may further comprise the step of separating the non-starch product from the mixture. The starch products and non-starch products obtained in this way are further described.

In another embodiment, under conditions of activating one or more enzymes, the corn is processed by enhancing the transformed corn or a portion thereof, an expression cassette encoding one or more starch-degrading or starch-isomerizing enzymes, and processing the genome expressing it in endocrine. There is provided a method of producing ultra-high sugar corn, including the step of converting the polysaccharide in the sugar to sugar, and obtaining ultra-high sugar corn. The expression cassette may further comprise a promoter operably linked to the polynucleotide encoding the enzyme. The promoter may be, for example, a constitutive promoter, seed-specific promoter or endocrine-specific promoter. The enzyme may be superthermophilic and may be α-amylase. As used herein, an expression cassette may further comprise a polynucleotide encoding a signal sequence operably linked to one or more enzymes. The signal sequence can direct the enzyme to, for example, apoplast or cytoplasmic net. The enzyme comprises any of SEQ ID NOs: 13, 14, 15, 16, 33 or 35. The enzyme may also include SEQ ID NO: 87.

In the most preferred embodiment, under conditions of activating one or more enzymes, the polysaccharide in maize is processed by processing a transformed maize or part thereof whose genome is enhanced with an expression cassette encoding α-amylase and expressing the expression cassette in endocrine. It describes a method of producing ultra-high sugar corn, including the step of converting the sugar to sugar, and obtaining the ultra high sugar corn. The enzyme may be superthermophilic and the superthermophilic α-amylase will comprise the amino acid sequence of any of SEQ ID NOs: 10, 13, 14, 15, 16, 33 or 35 or an enzymatically active fragment thereof having α-amylase activity. Can be. The enzyme comprises SEQ ID NO: 87.

Starch granules and plant parts comprising one or more processing enzymes obtained from a transformed plant whose genome has been enriched with an expression cassette encoding one or more processing enzymes are treated under conditions that activate one or more enzymes and thus starch granules Processing to form an aqueous solution comprising the hydrolyzed starch product; And collecting an aqueous solution comprising the hydrolyzed starch product is described herein. Hydrolyzed starch products may include dextrins, maltooligosaccharides, glucose and / or mixtures thereof. The enzyme may be α-amylase, α-glucosidase, glucoamylase, pullulanase, amylopululase, glucose isomerase or any combination thereof. In addition, the enzyme may be superthermophilic. In another aspect, the genome of the plant part can be further enhanced with an expression cassette encoding a non-superthermotropic starch processing enzyme. The non-superthermotropic starch processing enzyme may be selected from the group consisting of amylase, glucoamylase, α-glucosidase, pullulanase, glucose isomerase or combinations thereof. In another aspect, the processing enzyme is preferably expressed in endocrine. The plant part may be grain and may be from corn, wheat, barley, rye, oats, sugar cane or rice. One or more processing enzymes are operably linked to a promoter and signal sequence that targets the enzyme to starch granules or cytoplasmic reticulum or cell walls. The method may further comprise separating the hydrolyzed starch product and / or fermenting the hydrolyzed starch product.

In another aspect of the invention, a plant portion comprising starch granules and one or more starch processing enzymes, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding one or more α-amylases, activates one or more such enzymes. Processing under such conditions and thereby processing the starch granules to form an aqueous solution comprising the hydrolyzed starch product; And collecting an aqueous solution comprising the hydrolyzed starch product. α-amylases are superthermophilic and superthermophilic α-amylases comprise the amino acid sequence of SEQ ID NO: 1, 10, 13, 14, 15, 16, 33 or 35, or active fragments thereof having α-amylase activity. The expression cassette hybridizes with any one of SEQ ID NOs: 2, 9, 46, or 52, polynucleotide selected from its complement, or any of SEQ ID NOs: 2, 9, 46, or 52 under low stringency hybridization conditions and has α-amylase activity. Polynucleotides encoding a polypeptide having: In addition, the present invention further provides for the genome of a transformed plant further comprising a polynucleotide encoding a non-thermophilic starch-processing enzyme. Alternatively, plant parts may be treated with non-superthermotropic starch-processing enzymes.

The invention further relates to a transformed plant portion comprising one or more starch-processing enzymes present in plant cells obtained from a transformed plant whose genome has been enhanced with an expression cassette encoding one or more starch processing enzymes. will be. Preferably, the enzyme is α-amylase, glucoamylase, glucose isomerase, β-amylase, α-glucosidase, isoamylase, pullulanase, neo-pululanase, iso-pululanase and amyloful Starch-processing enzyme selected from the group consisting of lulanase. In addition, the enzyme may be superthermophilic. The plant may be any plant, for example corn or rice.

Another aspect of the invention is one wherein the genome is from a transformed plant obtained from a transformed plant with an expression cassette encoding at least one non-starch processing enzyme or at least one non-starch polysaccharide processing enzyme. A transformed plant part comprising the above non-starch processing enzyme. The enzyme may be superthermophilic. In addition, the non-starch processing enzyme may be a protease, glucanase, xylanase, esterase, phytase, beta glucosidase, cellulase or lipase. The plant part may be any plant part, but is preferably ear, seed, fruit, grain, stover, bran or baggasse.

The invention also relates to a transformed plant part. For example, by a polynucleotide having an amino acid sequence of any one of SEQ ID NOs: 1, 10, 13, 14, 15, 16, 33 or 35, or comprising any of SEQ ID NOs: 2, 9, 46 or 52 A transformed plant part comprising an encoded α-amylase, comprising an α-glucosidase having an amino acid sequence of any of SEQ ID NO: 5, 26 or 27 or encoded by a polynucleotide comprising SEQ ID NO: 6 A poly having an amino acid sequence of any of the transformed plant parts, SEQ ID NOs: 28, 29, 30, 38, 40, 42 or 44 or comprising any of SEQ ID NOs: 19, 21, 37, 39, 41 or 43 Transformed plant part comprising a glucose isomerase encoded by nucleotides, having the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49, or comprising any of SEQ ID NOs: 46, 48, 50, or 59 Ha A transformed plant portion comprising a glucoamylase encoded by a polynucleotide and a transformed plant portion comprising a pullulanase encoded by a polynucleotide comprising any of SEQ ID NOs: 4 or 25 are described. .

The present invention further relates to a transformed plant part. For example, a transformed plant having an amino acid sequence of any one of SEQ ID NOs: 62, 64, or 66 or comprising a xylanase encoded by a polynucleotide comprising any of SEQ ID NOs: 61, 63, or 65 Part. Also provided is a transformed plant portion comprising a protease. The protease may be a bromelain having the amino acid sequence set forth in SEQ ID NO: 70 or encoded by a polynucleotide having SEQ ID NO: 69. In another aspect, a transformed plant portion comprising cellulase is provided. The cellulase may be a cellobiohydrolase encoded by a polynucleotide comprising any of SEQ ID NOs: 79, 80, 81, 82, 93 or 94.

Further embodiments provide a transformed plant portion comprising a glucanase, such as endoglucanase. Endoglucanases may be endoglucanas I having the amino acid sequence set forth in SEQ ID NO: 84 or encoded by a polynucleotide comprising SEQ ID NO: 83. Also provided are transformed plant parts comprising beta glucosidase. Beta glucosidase may be stated as SEQ ID NO: 90 or 92, or beta glucosidase 2 or beta glucosidase D encoded by a polynucleotide having SEQ ID NO: 89 or 91. In another aspect, a transformed plant portion comprising an esterase is provided. The esterase may be a ferulic acid esterase encoded by a polynucleotide comprising SEQ ID NO: 99.

Another aspect is a method of converting starch in a transformed plant part, comprising activating starch processing enzymes contained in the transformed plant part. Starch, dextrins, maltooligosaccharides or sugars prepared according to the process are further described.

The invention further comprises one or more transformed plant parts comprising one or more non-starch polysaccharide processing enzymes obtained from a transformed plant whose genome is enhanced with an expression cassette encoding one or more non-starch polysaccharide processing enzymes. Treating under enzyme activating conditions, thereby degrading the non-starch polysaccharide to form an aqueous solution comprising oligosaccharides and / or sugars; And collecting an aqueous solution comprising oligosaccharides and / or sugars, wherein the method comprises using a transformed plant portion comprising one or more non-starch processing enzymes in the cells of the cell wall or plant portion. Non-starch polysaccharide processing enzymes may be superthermophilic.

A transgenic seed comprising one or more proteases or lipases, obtained from a transformed plant whose genome is enriched with an expression cassette encoding one or more enzymes, is subjected to conditions comprising activating one or more enzymes to contain amino acids and fatty acids. Obtaining an aqueous mixture; And harvesting the aqueous mixture, describing a method of using a transgenic seed comprising one or more processing enzymes. Preferably, amino acids, fatty acids or both are separated. One or more proteases or lipases may be hyperthermia.

A plant portion comprising one or more polysaccharide processing enzymes, obtained from a transformed plant whose genome is enriched with an expression cassette encoding one or more polysaccharide processing enzymes, is subjected to treatment under conditions that activate one or more enzymes to degrade the polysaccharides to oligosaccharides or Forming a fermentable sugar; And incubating the fermentable sugars under conditions that facilitate the conversion of the fermentable sugars or oligosaccharides to ethanol. Plant parts may be grains, fruits, seeds, stems, wood, vegetables or roots. Plant parts can be obtained from plants selected from the group consisting of oats, barley, wheat, berries, grapes, rye, corn, rice, potatoes, sugar beets, sugar cane, pineapples, grasses and trees. In another preferred embodiment, the polysaccharide processing enzyme is α-amylase, glucoamylase, α-glucosidase, glucose isomerase, pullulanase or combinations thereof.

Α-amylase, glucoamylase, α-glucosidase, glucose isomerase or pullulanase or combinations thereof, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding one or more enzymes Treating the plant portion comprising one or more enzymes by heating for a sufficient time under conditions activating the one or more enzymes to degrade the polysaccharides to form fermentable sugars; And incubating the fermentable sugar under conditions that promote the conversion of the fermentable sugar to ethanol. One or more enzymes may be superthermophilic or mesophilic.

In another embodiment, a plant portion comprising one or more non-starch processing enzymes, obtained from a transformed plant whose genome is enriched with an expression cassette encoding one or more enzymes, is subjected to a non- Degrading starch polysaccharides into oligosaccharides and fermentable sugars; And incubating the fermentable sugar under conditions that promote the conversion of the fermentable sugar to ethanol. The non-starch processing enzyme may be xylanase, cellulase, glucanase, beta glucosidase, protease, esterase, lipase or phytase.

Α-amylase, glucoamylase, α-glucosidase, glucose isomerase or pullulanase or combinations thereof, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding one or more enzymes Treating the plant portion comprising one or more enzymes under conditions activating the one or more enzymes to degrade the polysaccharides to form fermentable sugars; And incubating the fermentable sugar under conditions that promote the conversion of the fermentable sugar to ethanol. The enzyme may be superthermophilic.

In addition, plant parts comprising one or more starch processing enzymes, obtained from transformed plants whose genome has been enriched with expression cassettes encoding one or more enzymes, are subjected to starch granules in the plant parts by subjecting them to conditions that activate one or more enzymes. Processing it to sugar to form a high sugar content product; And processing the high sugar product into a starch food, thereby describing a method for producing a high sugar starch food without the addition of additional sweeteners. High sugar content foods can be formed from high sugar content and water. In addition, high sugar content foods may contain malts, flavors, vitamins, minerals, colorants or any combination thereof. One or more enzymes may be superthermophilic. The enzyme may be selected from α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase or any combination thereof. The plant may further be selected from the group consisting of soybean, rye, oats, barley, wheat, corn, rice and sugar cane. The starch food may be cereals, breakfast foods, convenience foods or baked foods. Processing may include baking, boiling, heating, steaming, electric discharge, or any combination thereof.

The invention further provides that starches comprising one or more starch processing enzymes, obtained from transformed plants whose genome is enhanced with an expression cassette encoding one or more enzymes, are subjected to conditions under which one or more enzymes are activated. Decomposing starch to form sugar to form high sugar starch; And adding high sugar starch to the product to produce a high sugar starch containing food, thereby sweetening the starch-containing product without the addition of sweeteners. Transformed plants can be selected from the group consisting of corn, soybean, rye, oats, barley, wheat, rice and sugar cane. One or more enzymes may be superthermophilic. One or more enzymes may be α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase or any combination thereof.

Starch foods and high sugar starch-containing products are provided herein.

The invention also provides for the treatment of fruits or vegetables comprising one or more polysaccharide processing enzymes obtained from a transformed plant whose genome has been enhanced with an expression cassette encoding one or more polysaccharide processing enzymes under conditions that activate one or more enzymes. The present invention relates to a method of sweetening a polysaccharide-containing fruit or vegetable, including processing the polysaccharide in the fruit or vegetable to form a sugar, and obtaining a high sugar fruit or vegetable. The fruit or vegetable is selected from the group consisting of potatoes, tomatoes, bananas, pumpkins, peas and beans. One or more enzymes may be superthermophilic.

The present invention further relates to a process for producing an aqueous solution comprising sugars, including treating the starch granules obtained from the plant part under conditions activating one or more enzymes to yield an aqueous solution comprising sugars.

In another embodiment, a plant portion comprising starch granules and one or more starch processing enzymes, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding one or more starch processing enzymes, is subjected to conditions under which one or more enzymes are activated. Thereby processing starch granules to form an aqueous solution comprising dextrin or sugar; And collecting an aqueous solution comprising the starch derived product, wherein the starch derived product is produced from the grain without wet or dry milling the grain prior to recovery of the starch derived product. One or more starch processing enzymes may be superthermophilic.

culturing the transformed plant containing α-amylase, glucoamylase, glucose isomerase, α-glucosidase or pullulanase and from therefrom α-amylase, glucoamylase, glucose isomerase, α-glucose There is further provided a method of separating α-amylase, glucoamylase, glucose isomerase, α-glucosidase and pullulanase, comprising the step of isolating cedarase or pullulanase. In addition, cultured transformed plants containing xylanase, cellulase, glucanase, beta glucosidase, protease, esterase, phytase or lipase and cultured xylanase, cellulase, glucanase, beta glucose A method for separating xylanase, cellulase, glucanase, beta glucosidase, protease, esterase, phytase or lipase, including isolating sidase, protease, esterase, phytase or lipase to provide.

Provided are methods for preparing maltodextrin, including mixing the transformed grains with water, heating the mixture, separating solids from the prepared dextrin syrup, and collecting maltodextrin. The transformed grains comprise one or more starch processing enzymes. The starch processing enzyme may be α-amylase, glucoamylase, α-glucosidase and glucose isomerase. Further provided are maltodextrins prepared by the process and compositions prepared by the process.

A plant part comprising starch granules and one or more starch processing enzymes, obtained from a transformed plant whose genome is enriched with an expression cassette encoding one or more processing enzymes, is treated under conditions that activate one or more enzymes, and thus starch Processing the granules to form an aqueous solution comprising dextrin or sugar; And collecting an aqueous solution comprising sugars and / or dextrins, thereby providing a method for preparing dextrins or sugars from the grains without requiring mechanical shredding of the grains prior to recovery of the starch-derived product.

The invention further provides that a plant portion comprising starch granules and one or more starch processing enzymes, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding one or more processing enzymes, under conditions that activate one or more enzymes. Processing, thereby processing the starch granules to form an aqueous solution comprising dextrin or sugar; And collecting an aqueous solution containing fermentable sugars.

In addition, provided herein are corn plants stably transformed with a vector comprising a superthermophilic α-amylase. For example, corn plants preferably transformed with a vector comprising a polynucleotide sequence encoding α-amylase that is greater than 60% identical to SEQ ID NO: 1 or SEQ ID NO: 51 is included.

1A and 1B show the activity of α-amylase expressed in corn kernels and endosperm by separating T1 kernels from pNOV6201 plants and six pNOV6200 strains.

2 shows the activity of α-amylase in separating T1 kernels from the pNOV6201 strain.

FIG. 3 shows the amount of ethanol produced upon fermentation of a mash of transgenic maize containing heat resistant 797GL3 alpha amylase applied at liquefaction times of up to 60 minutes at 85 ° C. and 95 ° C. FIG. The figure shows that the ethanol yield at 72 hours of fermentation hardly changed during the liquefaction of 15 to 60 minutes. In addition, this shows that the mash prepared by liquefaction at 95 ° C. produces more ethanol at each time point than the mash prepared by liquefaction at 85 ° C.

FIG. 4 shows the percentage of residual starch remaining after fermentation of a mash of transgenic maize containing heat resistant alpha amylase applied to liquefaction times of up to 60 minutes at 85 ° C. and 95 ° C. FIG. The figure shows that the ethanol yield at 72 hours of fermentation hardly changed during the liquefaction of 15 to 60 minutes. In addition, this shows that the mash prepared by liquefaction at 95 ° C. produces more ethanol at each time point than the mash prepared by liquefaction at 85 ° C.

FIG. 5 shows ethanol yields for mash of transgenic maize, control maize and various mixtures thereof prepared at 85 ° C. and 95 ° C. FIG. This figure illustrates that transgenic corn containing α-amylase results in a significant improvement in the production of fermentable starch due to the reduction of the starch remaining after fermentation.

FIG. 6 shows the amount of residual starch measured in dried dregs after fermentation on mash of transgenic grains, control corn and various mixtures thereof prepared at 85 ° C. and 95 ° C. FIG.

FIG. 7 shows ethanol yield as a function of fermentation time of a sample comprising 3% transgenic corn over a period of 20 to 80 hours at various pH ranges from 5.2 to 6.4. This figure illustrates that fermentation performed at low pH proceeds faster than at pH 6.0 or higher.

FIG. 8 shows the ethanol yield during fermentation of a mash comprising 0-12 wt% of varying wt% transgenic corn at various pH ranges from 5.2 to 6.4. This figure illustrates that ethanol yield was independent of the amount of transgenic grain included in the sample.

9 shows analysis of T2 seeds from different events transformed with pNOV 7005. Compared to the non-transformation control, high expression of pullulanase activity can be observed in various events.

10A and 10B show the results of HPLC analysis of hydrolysis products produced by expressing pullulanase from starch in transgenic corn powder. Incubation of the powder of pullulanase expressing corn for 30 minutes in a reaction buffer at 75 ° C. results in production of heavy chain oligosaccharides (degree of polymerization (DP) ˜10 to 30} and amylose short chain (DP to 100-200) from corn starch. do. 10A and 10B also show the effect of added calcium ions on the activity of pullulanase.

11A and 11B show data derived from HPLC analysis of starch hydrolysis products from two reaction mixtures. The first reaction, denoted 'amylase', contained a mixture [1: 1 (w / w)] of corn powder samples of α-amylase expressing transformed corn and non-transformed corn A188; The second reaction mixture 'amylase + pullulanase' contains a mixture [1: 1 (w / w)] of corn powder samples of α-amylase expressing transformed corn and pullulanase expressing transformed corn.

FIG. 12 shows the amount of sugar product in μg in 25 μl of the reaction mixture for the two reaction mixtures. The first reaction, denoted 'amylase', contained a mixture [1: 1 (w / w)] of corn powder samples of α-amylase expressing transformed corn and non-transformed corn A188; The second reaction mixture 'amylase + pullulanase' contains a mixture [1: 1 (w / w)] of corn powder samples of α-amylase expressing transformed corn and pullulanase expressing transformed corn.

13A and 13B show starch hydrolysis products from two sets of reaction mixtures at the end of incubation at 85 ° C. and 95 ° C. for 30 minutes. There are two reaction mixtures for each set; The first reaction, denoted 'amylase X flulanase', contained a powder from transgenic maize expressing both α-amylase and pullulanase; The second reaction mixture, denoted 'amylase', is observed in a mixture of a mixture of corn powder samples of α-amylase expressing transformed corn and non-transformed corn A188 at a rate where the same amount of α-amylase activity is obtained (amylase X Pullulanase).

FIG. 14 shows degradation of starch into glucose using non-transformed corn seed (control), transgenic maize containing 797GL3 α-amylase, and a combination of Mal A α-glucosidase and 797GL3 transgenic maize seed. will be.

15 shows the conversion of raw starch at room temperature or 30 ° C. In this figure, reaction mixtures 1 and 2 are a combination of water and starch at room temperature and 30 ° C., respectively. Reaction mixtures 3 and 4 are combinations of barley α-amylase and starch at room temperature and 30 ° C., respectively. Reaction mixtures 5 and 6 are combinations of thermophilic anaerobic bacterial glucoamylase and starch at room temperature and 30 ° C., respectively. Reaction mixtures 7 and 8 are a combination of barley α-amylase and thermophilic anaerobic bacterial glucoamylase and starch at room temperature and 30 ° C., respectively. Reaction mixtures 9 and 10 are a combination of barley alpha-amylase control and starch at room temperature and 30 ° C., respectively. The degree of polymerization (DP) of the product of thermophilic anaerobic bacterial glucoamylase is shown.

FIG. 16 depicts the preparation of fructose from amylase transgenic corn powder using alpha amylase, alpha glucosidase and glucose isomerase as described in Example 19. FIG. Amylase corn powder was mixed with enzyme solution added water or buffer. All reactions contained 60 mg of amylase powder and 600 μl of total liquid and were incubated at 90 ° C. for 2 hours.

FIG. 17 shows the peak area of the reaction product with 100% amylase powder from self-processing grains as a function of incubation time 0-1200 minutes at 90 ° C.

FIG. 18 shows the peak area of the reaction product with self-processed grains and 10% transformed amylase powder from 90% control corn powder as a function of incubation time 0-1200 minutes at 90 ° C.

FIG. 19 provides the results of HPLC analysis of transformed amylase powder incubated at 70 ° C., 80 ° C., 90 ° C. or 100 ° C. for up to 90 minutes to assess the effect of temperature on starch hydrolysis.

FIG. 20 shows the ELSD peak areas for samples containing 60 mg of transformed amylase powder mixed with enzyme solution to which water or buffer was added under various reaction conditions. One set of reactions was buffered with 50 mM MOPS, 10 mM MgSO ₄ and 1 mM CoCl ₂ at pH 7.0; In the second set of reactants the metal-containing buffer was replaced with water. All reactions were incubated at 90 ° C. for 2 hours.

According to the present invention, a "self-processing" plant or plant part comprises therein an isolated polynucleotide encoding a processing enzyme that can process, for example, modify starch, polysaccharides, lipids, proteins, etc. in the plant. Wherein the processing enzyme may be mesophilic, thermophilic or superthermophilic and may be activated by polishing, water addition, heating or other advantageous conditions provided for the action of the enzyme. Isolated polynucleotides encoding processing enzymes are incorporated into a plant or plant part for expression in the plant or plant part. Depending on the expression and activation of the processing enzyme, the plant or plant part of the present invention processes the substrate under the action of the processing enzyme. Thus, the plant or plant part of the present invention is generally capable of self-processing the substrate of an enzyme upon activation of the processing enzyme contained therein, with or without the reduced external source required to process these substrates. As such, transformed plants, transformed plant cells, and transformed plant parts have the "built-in" processing capacity to process the desired substrate via the enzymes contained therein according to the present invention. Preferably, the processing enzyme-encoding polynucleotide is “genetically stable”, ie the polynucleotide remains stable within the transformed plant or plant part of the invention and is stably inherited to the progeny through successive generations. do.

According to the present invention, methods using these plants and plant parts may obviate the need for milling or otherwise physically crushing the intact plant parts before harvesting the starch-derived product. For example, the present invention provides an improved method of processing corn or other grains to harvest starch-derived products. The present invention also provides a method for enabling the collection of starch granules containing concentrations of starch degrading enzymes in or on granules suitable for hydrolysis of specific bonds in starch without the need of adding exogenously produced starch hydrolase. To provide. The present invention also provides improved products from self-processing plants or plant parts obtained by the process of the invention.

In addition, "self-processing" transformed plant parts, such as grains and transformed plants, are a major problem with existing techniques (ie, processing enzymes typically do not require separation of enzymes from the culture supernatant). Prepared by fermentation of expensive, costly microorganisms, the isolated enzyme needs to be formulated for a particular application, and processes and mechanisms for the addition, mixing and reaction of the enzyme with its substrate have been developed. Should be avoided). Transformed plants of the present invention or parts thereof are also a source of processing enzymes themselves and substrates and products of enzymes such as sugars, amino acids, fatty acids and starch and non-starch polysaccharides. The plants of the present invention can also be used to produce progeny plants such as hybrids and suburbs.

Processing enzymes and polynucleotides encoding them

A polynucleotide encoding a processing enzyme (meteophilic, thermophilic or superthermophilic) is introduced into a plant or plant part. The processing enzyme is selected based on the substrate of interest on which it acts, as found in the plant or transformed plant and / or the desired end product. For example, the processing enzyme may be a starch-processing enzyme such as starch-degrading or starch-isomerase or a non-starch processing enzyme. Suitable processing enzymes include, for example, α-amylase, endo or exo-1,4, or 1,6-α-D, glucoamylase, glucose isomerase, β-amylase, α-glucosidase and other exo- Starch degradation or isomerization enzymes including amylases; And glycosyl transferases, exo, such as starch debranching enzymes such as isoamylase, pullulanase, neo-pululanase, iso-pululanase, amylopululase, cyclodextrin glycosyltransferase, and the like. Cellulase such as -1,4-β-cellobiohydrolase, exo-1,3-β-D-glucanase, hemicellulase, β-glucosidase and the like; Endoglucanases such as endo-1,3-β-glucanase and endo-1,4-β-glucanase; L-arabinases such as endo-1,5-α-L-arabinase, α-arabinosidase and the like; Galactanases such as endo-1,4-β-D-galactanase, endo-1,3-β-D-galactanase, β-galactosidase, α-galactosidase and the like; Mannaases such as endo-1,4-β-D-mannase, β-mannosidase, α-mannosidase, and the like; Xylanases such as endo-1,4-β-xylanase, β-D-xylosidase, 1,3-β-D-xylanase and the like; And pectinase; And non-starch processing enzymes including, but not limited to, proteases, glucanases, xylanases, thioredoxin / thioredoxin reductases, esterases, phytase and lipases.

In one embodiment, the processing enzyme is a starch-degrading enzyme selected from the group consisting of α-amylase, pullulanase, α-glucosidase, glucoamylase, amylopululase, glucose isomerase or combinations thereof. In accordance with this embodiment, the starch-degrading enzyme may cause degradation of the self-processing plant or plant part upon activation of the enzyme contained in the plant or plant part and will be further described herein. Starch-degrading enzyme (s) are selected according to the desired end product. For example, glucose-isomerase can be selected to convert glucose (hexose) to fructose. Alternatively, the enzyme may be selected based on the desired starch-derived end product, for example, having a variety of chain lengths or a variety of branching types desired based on a function of the degree of processing. For example, α-amylase, glucoamylase or amylopululase can be used under short incubation times to produce dextrin products and longer incubation times to produce short chain products or sugars. Pullulanase can be used to specifically hydrolyze the branching points in the starch to yield high-amylose starches, or to prepare starches with stretches of α 1,4 bonds with scattered α 1,6 bonds. Neopululanase can be used. Glucosidase can be used to make limited dextrins, or a combination of different enzymes can be used to make other starch derivatives.

In other embodiments, the processing enzyme is a non-starch processing enzyme selected from protease, glucanase, xylanase, phytase, lipase, cellulase, beta glucosidase and esterase. These non-starch degrading enzymes allow the incorporation of the self-processing plant or plant part of the invention into the target area of the plant and, upon activation, causes the plant to shred while the starch granules remain intact therein. do. For example, in a preferred embodiment, the non-starch degrading enzyme targets the endosperm substrate of the plant cell and, upon activation, disrupts the endosperm substrate while leaving the starch granules intact and more intact from the material obtained. Makes it easy to recover.

Combination of processing enzymes is further contemplated by the present invention. For example, starch-processed and non-starch processed enzymes can be combined and used. Combination of processing enzymes can be obtained utilizing the use of multiple gene constructs encoding each enzyme. Alternatively, individual transformed plants stably transformed with enzymes can be crossed by known methods to obtain plants containing both enzymes. Another method involves the use of transformed plants and exogenous enzyme (s).

The processing enzyme may be isolated or derived from any source and the corresponding polynucleotides may be identified by one skilled in the art. For example, processing enzymes such as α-amylase may be pyrococcus (e.g. Pyrococcus furiosus ), thermomus, thermococcus {e.g. Thermococcus hydrothermalis )}, sulfolobus {e.g. Sulfolobus solfataricus }, turmotoga {e.g. Thermotoga maritima and Turmotoga neapolitana ( Thermotoga neapolitana)}, thermophilic anaerobic bacteria (e. g., teomo anae fishing bakteo X konjen sheath (Thermoanaerobacter tengcongensis)}, AAS peogil Russ {e.g., Aspergillus peogil Russ swiro Usami (Aspergillus shirousami) and Aspergillus peogil Russ nigeo (Aspergillus niger )}, lycopus {for example Rhizopus oryzae }, thermophiles, desulfurococcus {for example, desulfurococc amylolyticus } , Metanobacterium Methanobacterium thermoautotrophicum ), Methanococcus jannaschii ) , Methanopyrus kandleri , Thermosynechococcus elongatus ), Thermoplasma ashdofilum acidophilum ), Thermoplasma volcanium , Aeropyrum pernix ) and plants such as corn, barley and rice.

The processing enzyme of the present invention can be activated after being introduced and expressed in the genome of a plant. Conditions for the activation of enzymes are determined for each individual enzyme and may include various conditions such as temperature, pH, hydration, the presence of metals, activating compounds, inactivating compounds and the like. For example, temperature-dependent enzymes may include mesophilic, thermophilic and hyperthermic enzymes. Mesophilic enzymes usually have maximum activity at temperatures between 20 ° C. and 65 ° C. and are inactivated at temperatures above 70 ° C. The mesophilic enzyme has significant activity at 30 ° C. to 37 ° C., and the activity at 30 ° C. is preferably at least 10% of the maximum activity, more preferably at least 20% of the maximum activity.

Thermophilic enzymes have maximum activity at temperatures between 50 ° C and 80 ° C. The mesophilic enzyme will preferably have less than 20% of the maximum activity at 30 ° C., more preferably less than 10% of the maximum activity.

"Superthermic" enzymes are active at higher temperatures. Superthermolytic enzymes have a maximum activity at temperatures above 80 ° C. and maintain activity at temperatures above 80 ° C., more preferably maintain activity at temperatures above 90 ° C., and most preferably maintain activity at temperatures above 95 ° C. . Superthermic enzymes also have reduced activity at low temperatures. The hyperthermic enzyme may have activity at 30 ° C., which is less than 10% of the maximum activity and preferably less than 5% of the maximum activity.

Polynucleotides encoding processing enzymes are preferably modified to include codons optimized for expression in selected individuals, such as plants (see, eg, Wada et al., Nucl. Acids Res., 18: 2367 ( 1990), Murray et al., Nucl.Acids Res., 17: 477 (1989), US Pat. Nos. 5,096,825, 5,625,136, 5,670,356 and 5,874,304. Codon optimization sequences are synthetic sequences, that is, they do not occur in nature, preferably the same polypeptide (or substantially the same activity as the full length polypeptide) encoded by a non-codon optimized parent polynucleotide encoding a processing enzyme. Enzymatically active fragments of full length polypeptides). The polypeptide is preferably biochemically distinguished or improved, for example, via regression mutagenesis of DNA encoding a particular processing enzyme from the parent polypeptide, thereby improving performance in its processing method. Preferred polynucleotides are optimized for expression in the target host plant and encode processing enzymes. Methods for preparing these enzymes include mutations such as regression mutagenesis and screening. Mutagenesis and nucleotide sequence alteration methods are well known in the art. See, eg, Kunkel, Proc. Natl. Acad. Sci. USA, 82: 488, (1985); Kunkel et al., Methods in Enzymol., 154: 367 (1987); US Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing, New York) and references and references cited herein by Arnold et al., Chem. Eng. Sci., 51: 5091 (1996). Methods for optimizing the expression of nucleic acid fragments in a target plant or organism are well known in the art. Briefly, codon usage tables representing the optimal codons used by the target individual are obtained and the optimal codons are selected to replace them with target polynucleotides, followed by chemical synthesis of the optimized sequence. Preferred codons for corn are described in US Pat. No. 5,625,136.

Further contemplated are the complementary nucleic acids of the polynucleotides of the present invention. An example of low stringency conditions for hybridization of complementary nucleic acids with 100 or more complementary residues on a filter in Southern or Northern blots is 50% formamide, for example 50% at 37 ° C. Formamide, 1M NaCl, hybridization in 1% SDS, and washing in 0.1 × SSC at 60 ° C. to 65 ° C. Examples of low stringency conditions include hybridization with a buffer of 30-35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ° C, and 1X-2X SSC (20X SSC = 3.0M at 50-55 ° C). Washing in NaCl / 0.3M trisodium citrate). Exemplary medium stringency hybridization conditions include 40 to 45% formamide at 27 ° C., 1.0 M NaCl, hybridization at 1% SDS, and washing at 0.5 × 1 × SSC at 55 ° C. to 60 ° C.

In addition, polynucleotides encoding "enzymatically active" fragments of processing enzymes are further contemplated. As used herein, “enzymatically active” means a polypeptide fragment of a processing enzyme that has substantially the same biological activity as the processing enzyme in modifying the substrate as the processing enzyme normally functions under suitable conditions.

In a preferred embodiment, the polynucleotides of the invention are corn-optimized polynucleotides encoding α-amylase, as provided in SEQ ID NOs: 2, 9, 46 and 52. In another preferred embodiment, the polynucleotides are corn-optimized polynucleotides encoding pullulanases, as provided in SEQ ID NOs: 4 and 25. In another preferred embodiment, the polynucleotide is a corn-optimized polynucleotide encoding an α-glucosidase as provided in SEQ ID NO: 6. Another preferred polynucleotide is a corn-optimized polynucleotide encoding glucose isomerase having SEQ ID NOs: 19, 21, 37, 39, 41 or 43. In another embodiment, corn-optimized polynucleotides encoding glucoamylases as set forth in SEQ ID NOs: 46, 48 or 50 are preferred. In addition, a corn-optimized polynucleotide for the glucanase / mannase fusion polypeptide is provided as SEQ ID NO: 57. The present invention further hybridizes under moderate or preferably low stringency hybridization conditions, optionally with α-amylase, pullulanase, α-glucosidase, glucose isomerase, glucoamylase, glucanase or The complement of such polynucleotides that encodes mannase activity is provided.

Polynucleotides can be used interchangeably with "nucleic acid" or "polynucleic acid" and are single- or double-helix deoxyribonucleotides consisting of monomers (nucleotides) containing sugars, phosphoric acids and bases (purines or pyrimidines) Or ribonucleotides and polymers thereof. Unless specifically limited, the term includes nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, certain nucleic acid sequences also implicitly include conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary and implicitly shown sequences. In particular, degenerate codon substitutions can be achieved by preparing a sequence in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

“Variants” or substantially similar sequences are further included herein. For nucleotide sequences, variants include those sequences that encode the same amino acid sequence of a native protein because of the degeneracy of the genetic code. Naturally occurring allelic variants such as these can be identified by well known molecular biology techniques (eg, polymerase chain reaction (PCR), hybridization techniques, and linkage assembly techniques). Variant nucleotide sequences may also be used, for example, synthetically derived nucleotide sequences encoding natural proteins, prepared using site-directed mutagenesis, and synthetically derived nucleotides encoding polypeptides having amino acid substitutions. It includes. In general, the nucleotide sequence variants of the present invention are at least 40%, 50%, 60%, preferably 70%, more preferably 80%, even more preferably 90%, of these classes of native nucleotide sequences, Most preferably will have 99% and single unit identity. For example, 71%, 72%, 73%, etc. to 90% or more. Variants may also include full-length genes corresponding to the identified gene fragments.

Regulatory Sequences: Promoter / Signal Sequences / Selection Markers

Polynucleotide sequences encoding the processing enzymes of the invention, for example, polycoding the localizing signal or signal sequence (N- or C-terminus of the polypeptide) that targets the superthermophilic enzyme to a specific site within the plant. It can be operatively linked to nucleotides. Examples of such targets include, but are not limited to, vacuoles, cytoplasmic reticulum, chloroplasts, starch, starch granules or cell walls or specific tissues such as seeds. With the use of tissue-specific or inducible promoters in plants, the expression of polynucleotides encoding a processing enzyme with a signal sequence can yield high levels of localized processing enzymes in the plant. Many signal sequences are known to affect the expression of polynucleotides or targeting to or from specific sites. Suitable signal sequences and targeting promoters are known in the art and include, but are not limited to, those provided herein.

For example, where expression in specific tissues or organs is desired, tissue specific promoters can be used. In contrast, where gene expression in response to stimulation is desired, an inducible promoter is the choice of regulatory element. If sustained expression across plant cells is desired, a constitutive promoter is used. Additional regulatory sequences upstream and / or downstream of the core promoter sequence can be included in the expression construct of the transformation vector to induce the expression of various levels of heterologous nucleotide sequences in the transformed plant.

Many plant promoters have been described with various expression properties. Examples of some of the constitutive promoters described include rice actin 1 (Wang et al., Mol. Cell. Biol., 12: 3399 (1992); US Pat. No. 5,641,876), CaMV 35S (see, eg, Odell et al., Nature, 313: 810 (1985)), CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh ( Walker et al., 1987), sucrose synthase (Yang & Russell, 1990) and ubiquitin promoters.

Vectors for use in tissue-specific targeting of genes in transformed plants will typically include tissue specific promoters and may also include other tissue specific regulatory elements such as enhancer sequences. Promoters that direct specific or enhanced expression in specific plant tissues will be known to those of skill in the art in light of the present disclosure. These include, for example, the rbcS promoter specific for green tissue, the ocs, nos and mas promoters with high activity in root or injured leaf tissue, truncated (-90 to +8) to direct enhanced expression in the roots. Promoters derived from the 35S promoter, the α-tubulin gene that directs expression in roots and the zein storage protein gene that directs expression in endosperm.

Tissue specific expression can be functionally performed by introducing the genes that are essentially expressed (all tissues) together with antisense genes expressed only within these tissues, if the gene product is not desired. For example, genes encoding lipases can be introduced and expressed in all tissues using a 35S promoter from the cauliflower mosaic virus. For example, expression of the antisense transcript of the lipase gene in corn kernels using the zein promoter will prevent the accumulation of lipase protein in the seed. Thus, the protein encoded by the introduced gene will be present in all tissues except grain.

In addition, several tissue-specific regulatory genes and / or promoters in plants have been reported. Some reported tissue-specific genes include genes or fatty acids encoding seed storage proteins (eg, nappine, cruciferin, beta-conglycinin and pazeolin) zein or oil body proteins (eg, oleosin). Genes that perform biosynthesis (including acyl carrier proteins, stearoyl-ACP desaturase and fatty acid desaturase (fad 2-1)) and other genes expressed during embryonic development (eg, Bce4, e.g. See, eg, European Patent No. EP 255378 and Kridl et al., Seed Science Research, 1: 209 (1991). Examples of tissue-specific promoters described include lectins (Vodkin, Prog. Clin. Biol. Res., 138; 87 (1983); Lindstrom et al., Der. Genet., 11: 160 (1990) ], Corn alcohol dehydrogenase I (Vogel et al., 1989; Dennis et al., Nucleic Acids Res., 12: 3983 (1984)), corn light harvesting complex (Simpson , 1986; Bansal et al., Proc. Natl. Acad. Sci. USA, 89: 3654 (1992)), corn heat shock protein (Odell et al., 1985; Rochester et al., 1986) Pea subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid monopine synthase (Langridge et al., 1989), Ti Plasmid nopalin synthase (Langridge et al., 1989), petunia challenge corn isomerase (vanTuene et al., EMBO J /. 7: 1257 (1988)), soybean glycine enriched protein 1 (documents) (Keller et al., Genes Dev., 3: 1639 (1989)), truncated CaMV 35s (Odell et al., Nature, 313: 810 (1985)), potato patatin (Wenzler et al., Plant Mol. Biol., 13: 347 (1989)), root cells (Yamamoto et al., Nucleic Acids Res., 18: 7449 (1990)), corn zein (Reina et al., Nucleic Acids Res., 18: 6425 (1990); Kriz et al., Mol. Gen. Genet., 207 : 90 (1987); Wandelt et al., Nucleic Acids Res., 17: 2354 (1989); Langridge et al., Cell, 34: 1015 (1983); Reina et al., Nucleic Acids Res., 18: 7449 (1990)), globulin-1 (Belanger et al., Genetics, 129: 863 (1991)), α-tubulin, cap ( Sullivan et al., Mol. Gen. Genet., 215: 431 (1989)), PEPCase (Hudspeth & Grula, 1989), R gene complex-related promoters (see: Chandler et al., Plant Cell, 1: 1175 (1989)), and the Calcon synthase promoter (Franken et al., EMBO J., 10: 2605 (1991)). Particularly useful promoters for seed-specific expression are the pea vicillin promoter (Czako et al., Mol. Gen. Genet., 235: 33 (1992)). (Also cited herein by reference) See US Pat. No. 5,625,136). Another promoter useful for expression in mature leaves is a promoter that is switched on at the onset of aging, such as SAG from Arabidopsis (Gan et al., Science, 270: 1986 (1995). ].

A class of fruit-specific promoters that are expressed at flowering time or during flowering, at least until they begin to mature throughout fruit development, is discussed in US Pat. No. 4,943,674, which is incorporated herein by reference. CDNA clones selectively expressed in cotton fibers were isolated (John et al., Proc. Natl. Acad. Sci. USA, 89: 5769 (1992)) from tomatoes exhibiting differential expression during fruit growth. cDNA clones were isolated and characterized (Mansson et al., Gen. Genet., 200: 356 (1985), Slater et al., Plant Mol. Biol., 5: 137 (1985)). The promoter for galacturonase gene is activated during fruit maturation The polygalacturonase gene is disclosed in US Pat. No. 4,535,060, US Pat. No. 4,769,061, US Pat. No. 4,801,590, which is incorporated herein by reference. And US Pat. No. 5,107,065.

Other examples of tissue-specific promoters include leaf stems (eg, patatin gene promoters) and fibrous cells (developmentally-regulated) in leaf cells after injury to leaves (eg, by insect bites). Examples of fibroblast proteins that have been synthesized are E6, which includes a promoter directing expression (John et al., Proc. Natl. Acad. Sci. USA, 89: 5769 (1992)). Is the most active in fiber, although low concentrations of transcripts are found in leaves, embryos and flowers.

The tissue-specificity of some "tissue-specific" promoters may not be absolute and may be tested by one skilled in the art using diphtheria toxin sequences. It is also possible to achieve tissue-specific expression with “leaky” expression by a combination of different tissue-specific promoters (Beals et al., Plant Cell, 9: 1527 (1997). ]). One skilled in the art can isolate other tissue-specific promoters (see US Pat. No. 5,589,379).

In one embodiment, the direction of the product from a polysaccharide hydrolysis gene such as α-amylase can be targeted to specific organs such as apoplasts rather than the cytoplasm. This is exemplified by the use of the N-terminal signal sequence (SEQ ID NO: 17), a corn γ-agent that confers apoplast-specific targeting of the protein. Directing proteins or enzymes to specific compartments will allow localization of the enzymes from contact with the substrate. In this way, the enzyme's enzymatic action will not occur until the enzyme comes into contact with its substrate. An enzyme may be contacted with its substrate by a process of milling (physical disruption of cell integrity) or heating of the cell or plant tissue to destroy the physical unity of the plant cell or organ containing the enzyme. For example, mesophilic starch-hydrolase may be targeted to apoplasts or cytoplasmic reticulum and thus not in contact with starch granules in starch. Milling of the grains will destroy the integrity of the grains and the starch hydrolase will then come into contact with the starch granules. In this way, the potential negative effects of co-localization of enzymes and their substrates can be avoided.

In another embodiment, the tissue-specific promoter is a corn γ-zein promoter (illustrated as SEQ ID NO: 12) or a maize ADP-gpp promoter (illustrated in SEQ ID NO: 11, including 5 'non-toxin and intron sequences). Or endosperm-specific promoters such as the Q protein promoter (illustrated by SEQ ID NO: 98) or the rice flutein 1 promoter (illustrated by SEQ ID NO: 67). Thus, the present invention provides isolated polynucleotides comprising a promoter comprising SEQ ID NO: 11, 12, 67 or 98, polynucleotides that hybridize with their complements under low stringency hybridization conditions or, for example, at least 10%, Preferably at least 50% includes fragments thereof having the activity of a promoter having SEQ ID NO: 11, 12, 67 or 98.

In another embodiment of the invention, the polynucleotides encode a superthermophilic processing enzyme operably linked to the chloroplast (starch) transit peptide (CTP) and, for example, a starch binding domain from a waxy fusion. Exemplary polynucleotides of this embodiment encode SEQ ID NO: 10 (α-amylase linked to the starch binding domain from waxy). Another exemplary polynucleotide is a hyperthermic processing enzyme (alpha-amylase, α-glucosidase, corn isomerase, respectively) operatively linked to a signal sequence that targets the enzyme to cytoplasmic reticulum and secretes into apoplasts. A super thermophilic enzyme (operated to SEKDEL) linked to a signal sequence that retains the enzyme in the cytoplasmic network, exemplified by a polynucleotide encoding SEQ ID NO: 13, 27 or 30, including the N-terminal sequence from Jane Super thermophilic enzymes, wherein the enzymes are α-amylase, malA α-glucosidase, T. maritima glucose isomerase, T. neapolitana glucose isomerase Polynucleic encoding SEQ ID NO: 14, 26, 28, 29, 33, 34, 35 or 36, comprising an N-terminal sequence from an operably linked corn γ-Zane Coding for SEQ ID NO: 15, which includes an N-terminal starch targeting sequence from waxy operably linked to an α-amylase, linked to an N-terminal sequence that targets the enzyme to a starch N-terminal starch targeting from waxy operably linked to an α-amylase / waxy fusion polypeptide comprising a starch binding domain that targets the enzyme to starch granules, Encoding a polynucleotide encoding SEQ ID NO: 16, comprising a sequence), a super thermophilic processing enzyme (exemplified as a polynucleotide encoding SEQ ID NOs: 38 and 39) linked to an ER retention signal. In addition, the superthermophilic processing enzyme may be linked to a live starch binding site having an amino acid sequence (SEQ ID NO: 53), wherein the polynucleotide encoding the processing enzyme is a corn-optimized nucleic acid sequence (sequence that encodes the binding site). Number 54).

Several inducible promoters have been reported. Many promoters are described in Gatz, in Current Opinion in Biotechnology, 7: 168 (1996) and Gatz, C., Annu. Rev. Plant Physiol. Plant Mol. Biol., 48: 89 (1997). Examples include tetracycline inhibitor systems, Lac inhibitor systems, copper-induced systems, salicylate-induced systems (eg, PR1a systems), glucocorticoid-induced (see Aoyama T. et al. , NH Plant Journal, 11: 605 (1997)) and ecdysone-induced systems. Other inducible promoters include ABA- and swell-induced promoters, promoters of auxin-binding protein genes (Schwob et al., Plant J., 4: 423 (1993)), UDP glucose flavonoid glycosyls -Transferase gene promoter (Ralston et al., Genetics, 119: 185 (1988)), MPI proteinase inhibitor promoter (see Cordero et al., Plant J., 6) : 141 (1994)] and glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., Plant Mol. Biol., 29; 1293 (1995); Quigley et al., J. Mol. Evol., 29: 412 (1989); Martinez et al., J. Mol. Biol., 208: 551 (1989)), and also benzene sulfonamide-derived (US Pat. No. 5,364,780) and Alcohol-derived (WO 97/06269 and WO 97/06268) systems and glutathione S-transferase promoters.

Other studies have focused on genes that are inductively regulated in response to environmental stress or stimulation such as increased salinity, drought, pathogens and wounds (Graham et al., J. Biol. Chem., 260). : 6555 (1985); Graham et al., J. Biol. Chem., 260: 6561 (1985), Smith et al., Planta, 168: 94 (1986)]. Accumulation of metallocarboxypeptidase-inhibitor proteins has been reported in the leaves of wounded potato plants (Graham et al., Biochem. Biophys. Res. Comm., 101: 1164 (1981)). Other plant genes have been reported to be induced by methyl jasmonate, inducers, heat-shock, anaerobic stress or herbicides.

The regulated expression of chimeric trans agonistic viral replication proteins can be further regulated by other gene mechanisms such as, for example, Cre-mediated gene activation (Odell et al., Mol. Gen. Genet., 113: 369 (1990)). Thus, a Cre-mediated cleavage of a DNA fragment comprising a 3 'regulatory sequence bound by a lox position between a promoter and a replicating protein coding sequence that blocks expression of chimeric replication from the promoter can be eliminated by trans-functionality. Results in expression of the replicating gene. In such cases, the chimeric Cre gene, chimeric trans-acting replication gene or both can be placed under the control of tissue- and development-specific or inducible promoters. An alternative gene mechanism is the use of tRNA suppressor genes. For example, coordinated expression of a tRAN inhibitor gene can conditionally control the expression of a trans-acting replication protein coding sequence comprising a suitable stop codon (see Ulmasov et al., Plant Mol. Biol., 35: 417 (1997)]. Again, a chimeric tRNA inhibitor gene or chimeric trans-acting replication gene, or both, can be placed under control of a tissue- and development-specific or inducible promoter.

Preferably, for multicellular individuals, the promoter may be specific for a particular tissue, organ or stage of development. Examples of such promoters include, but are not limited to, Zea mays , ADP-gpp and Zea mays γ-zein promoters and Zea mays globulin promoters.

Gene expression in transformed plants may be desirable only at certain times during plant development. Developmental timing is often associated with tissue specific gene expression. For example, expression of the Jane storage protein is initiated in endosperm about 15 days after pollination.

Additionally, vectors can be constructed and used for intracellular targeting of specific gene products into cells of transformed plants and for directing the protein to the extracellular environment. This will generally be accomplished by binding the DNA sequence encoding the transfer or signal peptide sequence to the coding sequence of a particular gene. The resulting transition or signal peptides will pass the protein to specific intracellular or extracellular destinations, respectively, and then be removed after translation. Metastasis or signal peptides act by promoting protein transport through intracellular membranes such as vacuoles, vesicles, plastids and mitochondrial membranes, while signal peptides direct proteins through extracellular membranes.

Signal sequences, such as the corn γ-zein N-terminal signal sequence for targeting to cytoplasmic reticulum and secretion of apoplasts, can be operably linked to polynucleotides encoding superthermophilic processing enzymes according to the present invention. See Torrent et al., 1997]. For example, SEQ ID NOs: 13, 27 and 30 provide for a polynucleotide encoding a super thermophilic enzyme operably linked to an N-terminal sequence from a corn γ-zein protein. Another signal sequence is the amino acid sequence SEKDEL for retention of polypeptides in the cytoplasmic reticulum (Munro and Pelham, 1987). For example, a polynucleotide encoding SEQ ID NO: 14, 25, 28, 29, 33, 34, 35 or 36 may be an N-terminal sequence from corn γ-Zane operably linked to a processing enzyme operably linked to SEKDEL. It includes. Polypeptides can be targeted to starches by fusing to waxy starch targeting peptides (Klosgen et al., 1986) or starch granules. For example, polynucleotides encoding superthermophilic processing enzymes can be operatively linked to chloroplast (starch) transferase (CTP) and starch binding domains (eg, from the waxy gene). SEQ ID NO: 10 illustrates an α-amylase linked to a starch binding domain from waxy. SEQ ID NO: 15 illustrates an N-terminal sequence starch targeting sequence from waxy operably linked to α-amylase. In addition, polynucleotides encoding processing enzymes can be fused to target starch granules using waxy starch binding domains. For example, SEQ ID NO: 16 illustrates a fusion polypeptide comprising an N-terminal starch targeting sequence from waxy operably linked to an α-amylase / waxy fusion polypeptide comprising a waxy starch binding domain.

In addition to the processing signals, the polypeptides of the present invention may further comprise other regulatory sequences known in the art. "Regulatory sequence" and "suitable regulatory sequence" are each located upstream of the coding sequence (5 'non-coding sequence), inside or downstream of the coding sequence (3' non-coding sequence), and transcription, RNA processing or stabilization, or It refers to a nucleotide sequence that affects the translation of the relevant coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. These include sequences that can be natural and synthetic sequences and combinations of synthetic and natural sequences.

Selection markers well known in the art can also be used in the present invention to select transformed plants and plant tissues. It may be desirable to use selectable or differential marker genes or in addition to expressable genes of interest. A "marker gene" is a gene that gives a distinct phenotype to cells expressing a marker gene, so that these transformed cells are distinguished from cells without markers. Such genes may be used to determine whether the marker confers traits that can be selected through chemical methods, i.e. the use of selective agents (e.g., herbicides, antibiotics, etc.), or simply observed or tested, i.e. Depending on whether or not the trait can be identified by the R-left trait), a selection or differential marker can be encoded. Of course, many examples of suitable marker genes are known in the art and can be used in the practice of the present invention.

Included within the term selection or differentiation marker gene are also genes encoding "selection markers" whose secretion can be detected by identification or selection methods for transforming cells. Examples include markers encoding secretable antigens that can be identified by antibody interaction or even secretable enzymes that can be detected by their catalytic activity. Secretable proteins include, for example, small diffuse proteins detectable by ELISA; Small active enzymes detectable in extracellular solution (eg, α-amylase, β-lactamase, phosphinothricin acetyltransferase); And proteins that are inserted or captured in the cell wall (eg, proteins comprising leader sequences as found in expression units of extensin or tobacco PR-S).

In view of selectable secretory markers, it is considered particularly advantageous to use genes that are sequestered to the cell wall and that encode proteins comprising native epitopes. Such secreted antigen markers will theoretically use epitope sequences that will provide a low background in plant tissue, efficient expression and targeting through the plasma membrane, and will use promoter-leader sequences to make proteins that bind to the cell wall and moreover are accessible to antibodies. Normally secreted wall proteins modified to contain unique epitopes will meet all of these requirements.

One example of a protein suitable for modification in this way is extensin or hydroxyproline rich glycoprotein (HPRG). For example, maize HPRG (Steifel et al., The Plant Cell, 2: 785 (1990)) molecules are well characterized in terms of molecular biology, expression and protein structure. However, any one of a variety of extensin and / or glycine-rich wall proteins (Keller et al., EMBO Journal, 8: 1309 (1989)) was modified by addition of antigenic sites to produce wall markers. Could be manufactured.

a. Screening marker

Selectable markers that can be used in connection with the present invention include neo or ntpII genes that encode kanamycin resistance and can be screened for use with kanamycin, G418 and the like (Potrykus et al., Mol. Gen. Genet., 199: 183 (1985)); Bar gene that confers resistance to herbicide phosphinothricin; Genes encoding modified EPSP synthase proteins to confer glyphosate resistance (Hinchee et al., Biotech., 6: 915 (1988)); Nitrile genes such as bxn from Klebsiella ozaenae that confer resistance to bromoxynil (Stalker et al., Science, 242: 419 (1988)); Mutant acetolactate synthase gene (ALS) that confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (European Patent Application No. 154,204, 1985); Methotrexate-resistant DHFR gene (Thillet et al., J. Biol. Chem., 263: 12500 (1988)); The dalapon dehalogenase gene that confers resistance to the herbicide dalapon; Phosphomannose isomerase (PMI) gene; Mutated anthranilate synthase genes that confer resistance to 5-methyl tryptophan; Hph gene conferring resistance to antibiotic hygromycin; Or the mannose-6-phosphate isomerase gene (also referred to herein as the phosphomannose isomerase gene) which provides the ability to metabolize mannose (US Pat. Nos. 5,767,378 and 5,994,629) Included, but not limited to. One skilled in the art can select a selection marker gene suitable for use in the present invention. When using the mutant EPSP synthase gene, additional benefits can be realized through the incorporation of a suitable chloroplast transfer peptide, CTP (European Patent Application No. 0,218,571, 1987).

Exemplary embodiments of selectable marker genes that can be used within the system to select transformants include genes encoding the enzyme phosphinothricin acetyltransferase (eg, from Streptomyces hygroscopicus ). bar gene or pat gene from Streptomyces viridochromogenes }. The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide vialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase resulting in rapid accumulation of ammonia and cell death (Murakami et al., Mol. Gen. Genet., 205: 42 (1986); Twell et al., Plant Physiol. , 91: 1270 (1989). The success of using this selection system with monocotyledonous plants was particularly surprising because of the major difficulties reported in the transformation of grains (Potrykus, Trends Biotech., 7: 269 (1989)).

Where it is desired to use a vialaphos resistance gene in the practice of the present invention, a gene particularly useful for this purpose is the bar or pat gene obtainable from Streptomyces spp. (Eg ATCC 21,705). Cloning of the bar gene has the use of the bar gene in terms of plants other than monocotyledonous plants (De Block et al., EMBO Journal, 6: 2513 (1987); De Block et al., Plant Phhsiol., 91: 694 (1989)] (Murakami et al., Mol. Gen. Genet., 205: 42 (1986); Thompson et al., EMBO Journal, 6: 2519 (1987)) .

b. Differential marker

Differentiation markers that can be used include β-glucuronidase or uidA gene (GUS), which encodes enzymes for which various chromogenic substrates are known; R-left genes encoding products that regulate the production of anthocyanin pigment (red) in plant tissues (Delaporta et al., In Chromosome Structure and Function, pp. 263-282 (1988)) ; [Beta] -lactamase gene, in which various chromogenic substrates (eg PADAC, chromogenic cephalosporin) encode known enzymes (Sutcliffe, PNAS USA, 75: 3737 (1978)); XylE gene encoding catechol deoxygenase capable of converting chromogenic catechol (Zukowsky et al., PNAS USA, 80: 1101 (1983)); α-amylase gene (Ikuta et al., Biotech., 8: 241 (1990)); Tyrosinase gene (Katz et al., J. Gen. Microbiol., 129: 2703), which encodes an enzyme that oxidizes tyrosine to DOPA and dopaquinone and then condenses to form the easily detectable compound melanin 1983)]); Β-galactosidase gene encoding an enzyme that is a chromogenic substrate; Luciferase (lux) gene that enables bioluminescence detection (Ow et al., Science, 234: 856 (1986)); Or aquarin genes that can be used for calcium-sensitive bioluminescence detection (Praser et al., Biochem. Biophys. Res. Comm., 126: 1259 (1985)) or green fluorescent protein genes (see: Niedz et al., Plant Cell Reports, 14: 403 (1995)].

Genes from the maize R gene complex are thought to be particularly useful as differential markers. The R gene complex in maize encodes a protein that functions to regulate the production of anthocyanin pigments in most seed and plant tissues. Genes from the R gene complex are useful for maize transformation because the expression of these genes in transformed cells does not harm the cells. Thus, the R gene introduced into these cells will cause the expression of the red pigment and, if stablely incorporated, can be visually recorded as a red zone. If the maize strain retains the dominant allele for the gene encoding the enzymatic intermediate in the anthocyanin biosynthetic pathway (Ce, A1, A2, Bz1 and Bz2), but has a recessive allele at the R locus, Transformation of any cell will result in red pigment formation. Exemplary notes include K55 derivatives of Wisconsin 22, r-g, b, P1 containing the rg-Stadler allele and TR112. Alternatively, if the C1 and R alleles are introduced together, any genotype of corn can be used. A further differentiation marker contemplated for use in the present invention is firefly luciferase encoded by the lux gene. The presence of the lux gene in the transformed cells can be detected using, for example, X-ray films, scintillation counting, fluorescence spectrophotometry, low light video cameras, photon counting Makera or multiwell luminescence. It is also contemplated that the system can be developed for bioluminescence such as tissue culture plates or even for population differentiation for the whole plant.

Polynucleotides used to transform plants include, but are not limited to, DNA from plant genes and non-plant genes (eg, genes from bacteria, yeast, animals, or viruses). The introduced DNA may include chimeric genes, including modified genes, portions of genes or genes from the same or different maize genotypes. The term “chimeric gene” or “chimeric DNA” refers to a species from a species that is located or bound in a manner that does not bind DNA under natural conditions or that does not normally occur in the natural genome of a plant whose DNA sequence or fragment is untransformed. It is defined as a gene or DNA sequence or fragment comprising two or more DNA sequences or fragments of.

There is further provided an expression cassette comprising a polynucleotide encoding an ultra thermophilic processing enzyme, and preferably a codon-optimized polynucleotide. A regulatory sequence such as a polynucleotide (first polynucleotide) in an expression cassette, a promoter, enhancer, intron, termination sequence, or any combination thereof, and optionally, an enzyme encoded by the first polynucleotide to a particular cell or subcellular location It is preferred to operably link to a second polynucleotide encoding a signal sequence (N- or C-terminus). Thus, a promoter and one or more signal sequences can be provided for high levels of enzyme expression at specific locations within a plant, plant tissue or plant cell. The promoter may be a constitutive promoter, an inducible (conditional) promoter or a tissue-specific promoter, for example a corn γ-zein promoter (illustrated in SEQ ID NO: 12) or a corn ADP-gpp promoter (5 ′ overdose and introns). Endocrine-specific promoters, such as those exemplified by SEQ ID NO: 11, comprising the sequence. The invention also relates to isolated polynucleotides comprising promoters comprising SEQ ID NOs: 11 or 12, polynucleotides hybridized to their complements under low stringency hybridization conditions, or examples of promoters having SEQ ID NOs: 11 or 12 Fragments thereof having at least 10% and preferably at least 50% activity. Also provided are vectors comprising the expression cassette or polynucleotide of the present invention and transformed cells comprising the polynucleotide, expression cassette or vector of the present invention. The vector of the invention may comprise a polynucleotide sequence encoding one or more of the superthermophilic processing enzymes of the invention (the sequence may be in the sense or antisense direction), and the transformed cell may comprise one or more vectors of the invention. It may include. Preferred vectors are those useful for introducing nucleic acids into plant cells.

Transformation

An expression cassette or expression construct comprising an expression cassette can be inserted into a cell. Expression cassettes or vector constructs can be transported in the episomal state or inserted into the genome of a cell. The transformed cells can then be cultured with the transformed plants. Thus, the present invention provides a product of a transformed plant. Such products include, but are not limited to, products of seeds, fruits, progeny, and progeny of transformed plants.

Various techniques for the introduction of the constructs into the cell host are available and are known to those skilled in the art. Transformation of bacteria and many eukaryotic cells can be performed using polyethylene glycol, calcium chloride, viral infections, phage infections, electroporation and other methods known in the art. Techniques for transforming plant cells or tissues include a. A. tumefaciens or A. Transformation to DNA using A. rhizogenes , electroporation, DNA injection, microblast bombardment, particle acceleration, and the like (see, for example, EP 295959 and EP 138341). .

In one aspect, a binary vector of Ti and Ri plasmids of Agrobacterium sp. Ti-derived vectors are used to transform a wide variety of higher plants, including monocotyledonous plants and dicotyledonous plants (e.g., soybean, cotton, rapeseed, tobacco and rice) (Pacciotti et al. Bio / Technology. 3: 241 (1985): Byrne et al. Plant Cell Tissue and Organ Culture, 8: 3 (1987); Sukhapinda et al. Plant Mol. Biol., 8: 209 (1987); Lorz et al. Mol. Gen. Genet., 199: 178 (1985); Potrykus Mol. Gen. Genet., 199: 183 (1985); Park et al., J. Plant Biol., 38: 365 (1985): Hiei et al., Plant J., 6: 271 (1994)]. The use of T-DNA, to transform plant cells, has been the subject of extensive research and has been fully described (European Patent EP 120516; Hoekema, In: The Binary Plant Vector System.Offset-drukkerij Kanters BV; Alblasserdam (1985), Chapter V; Knauf, et al., Genetic Analysis of Host Range Expression by Agrobacterium In: Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. ed., Springer-Verlag, New York, 1983, p. 245; and An. Et al., EMBO J., 4: 277 (1985)).

Those skilled in the art will appreciate the direct acceptance of foreign DNA constructs (see European Patent No. EP 295959), electroporation techniques (Fromm et al., Nature (London), 319: 791 (1986)) or Other transformation methods, such as high speed ballistic impact using metal particles coated with nucleic acid constructs (Kline et al., Nature (London) 327: 70 (1987) and US Pat. No. 4,945,050), can be used. have. Once transformed cells can be regenerated by those skilled in the art. Of particular relevance are the hereditary genes for rape seed (De Block et al., Plant Physiol. 91: 694-701 (1989)), sunflower (Everett et al., Bio / Technology. 5: 1201 (1987)), soybeans (Mccabe et al., Bio / Technology, 6: 923 (1988); Hinchee et al., Bio / Technology, 6: 915 (1988); Chee et al., Plant Physiol., 91: 1212 (1989); Christou et al., Proc. Natl. Acad. Sci USA, 86: 7500 (1989), European Patent No. EP 301749]), rice (Hei et al. , Plant J., 6: 271 (1994), and corn (Gordon Kamm et al., Plant Cell, 2: 603 (1990); Fromm et al., Biotechnology, 8: 833, (1990) It is a recently described method of transforming a commercially important crop such as)]).

Expression vectors comprising genomic or synthetic fragments can be introduced into protoplasts or intact tissues or isolated cells. Preferably the expression vector is introduced into intact tissue. General plant tissue culture methods are described, for example, in Maki et al. "Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology & Biotechnology, Glich et al. (Eds.), Pp. 67-88 CRC Press (1993); And Phillips et al. "Cell-Tissue Culture and In-Vitro Manipulation" in Corn & Corn Improvement, 3rd Edition 10, Sprague et al. (Eds.) Pp. 345-387, American Society of Agronomy Inc. (1988).

In one embodiment, expression vectors can be introduced into maize or other plant tissue using direct gene transfer methods such as microprojectile-mediated delivery, DNA injection, electroporation, and the like. The expression vector is introduced into plant tissue using microprojectile media delivery using biolistics equipment. See, eg, Tomes et al. "Direct DNA transfer into intact plant cells via microprojectile bombardment" in Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer Verlag, Berlin (1995). Nevertheless, the present invention contemplates the transformation of plants using super thermophilic processing enzymes according to known transformation methods. In addition, Weissinger et al., Annual Rev. Genet., 22: 421 (1988); Sanford et al., Particulate Science and Technology, 5:27 (1987) (onion); Christou et al., Plant Physiol., 87: 671 (1988) (soy); McCabe et al., Bio / Technology, 6: 923 (1988) (soybean); Datta et al., Bio / Technology, 8: 736 (1990) (rice); Klein et al., Proc. Natl. Acad. Sci. USA, 85: 4305 (1988) (corn); Klein et al., Bio / Technology, 6: 559 (1988) (corn); Klein et al., Plant Physiol., 91: 440 (1988) (corn); Fromm et al., Bio / Technology, 8: 833 (1990) (corn); And Gordon-Kamm et al., Plant Cell, 2, 603 (1990) (corn); Svab et al., Proc. Natl. Acad. Sci. USA, 87: 8526 (1990) (potato chloroplast); Koziel et al., Biotechnology, 11: 194 (1993) (corn); Shimamoto et al., Nature, 338: 274 (1989) (rice); Christou et al., Biotechnology, 9: 957 (1991) (rice); European Patent Application EP 0 332 581 (Duck and other Poapuaceae); Vasil et al., Biotechnology, 11: 1553 (1993) (wheat); Weeks et al., Plant Physiol., 102: 1077 (1993) (wheat); Methods in Molecular Biology, 82. Arabidopsis Protocols Ed. Martinez-Zapater and Salinas 1998 Humana Press (Baby Pole).

Plant transformation can be performed using a single DNA molecule or multiple DNA molecules (ie, co-transformation), both of which are suitable for use with expression cassettes and constructs of the invention. Many transformation vectors can be used for plant transformation and the expression cassettes of the invention can be used with any such vector. The selection of the vector will depend on the desired transformation technique and the target species for transformation.

Ultimately, the most preferred DNA fragments for introduction into the monocot genome encode the desired trait (e.g., hydrolysis of proteins, lipids or polysaccharides), novel promoters or enhancers, etc., or perhaps even homology or Can be a homologous gene or gene family introduced under the control of a tissue-specific (eg, root-, age / leaf vine-, stratum-, stem-, chock-, grain- or leaf-specific) promoter or control element have. In fact, it is contemplated that certain uses of the present invention may be gene targeting of constitutive or inductive methods.

Examples of suitable transformation vectors

Many transformation vectors that can be used for plant transformation are known to those of ordinary skill in the art of plant transformation, and the genes involved in the present invention can be used with any such vectors known in the art. The selection of the vector will depend on the desired transformation technique and the target species for transformation.

a. Vectors Suitable for Agrobacterium Transformation

Many vectors are available for transformation with Agrobacterium lithium passance. These typically have one or more T-DNA sequences and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). Below, the construction of two conventional vectors suitable for Agrobacterium transformation are described.

pCIB200 and pCIB2001

Binary vectors pCIB200 and pCIB2001 are used for the construction of vectors of recombinant vectors for use with Agrobacterium and are constructed in the following manner. pTJS75 was digested with NarI (Schmidhauser & Helinski, J. Bacteriol., 164: 446 (1985)) to cleave the tetracycline-resistant gene, followed by insertion of an AccI fragment from pUC4K carrying NPTII ( See Messing & Vierra, Gene, 19: 259 (1982): Bevan et al., Nature, 304: 184 (1983): McBride et al., Plant Molecular Biology, 14: 266 (1990)) to prepare pTJS75kan. do. The XhoI connector was attached to the EcoRV fragment of pCIB7, including the left and right T-DNA borders, the plant selected nos / nptII chimeric gene and the pUC multi-linker (Rothstein et al., Gene, 53: 153 (1987)). Ligation and cloning XhoI-digestion fragments into SalI-digestion pTJS75kan to prepare pCIB200 (see also EP 0 332 104, Example 19). pCIB200 contains the following unique multilinker restriction sites: EcoRI, SstI, Kpnl, BgIII, Xbal, and Sall. Unique restriction sites in the multi-linker of pCIB2001 are EcoRI, SstI, KpnI, BgIII, XbaI, Sall, Mlul, Bell, AvrII, Apal, HpaI and StuI. In addition to these unique restriction sites, pCIB2001 also includes plant and bacterial kanamycin selection, left and right T-DNA borders for E. cobacterium-mediated transformation, E. coli. RK2-derived trfA function for maneuver between E. coli and other hosts, and also OriT and OriV functions from RK2 . pCIB2001 multilinkers are suitable for cloning plant expression cassettes containing their own regulatory signals.

pCIB10 and its hygromycin selection derivatives:

The binary vector pCIB10 comprises genes encoding kanamycin resistance for selection in plants and T-DNA left and right border sequences, comprising two. It includes sequences from a broad host-range plasmid pRK252 that allows replication in both E. coli and Agrobacterium. Its construct is described by the author Rothstein et al. (Gen, 53: 153 (1987)). Various derivatives of pCIB10 are constructed that include genes for the hygromycin B phosphotransferase described in the author, Gritz et al. (Gen, 25: 179 (1983)). These derivatives only allow for the selection of transformed plant cells on hygromycin (pCIB743) or hygromycin and kanamycin (pCIB715, pCIB717).

b. Vectors Suitable for Non-Agrobacterium Transformation

Transformation without using Agrobacterium lithium passivation does not require the T-DNA sequence of the selected transformation vector, resulting in a vector lacking these sequences in addition to the vector as described above containing the T-DNA sequence. Can be used. Transformation techniques that do not depend on Agrobacterium include particle bombardment, protoplast uptake (eg PEG and electroporation) and transformation via microinjection. The choice of vector depends largely on the desired selection of the species to be transformed. Non-limiting examples of construction of conventional vectors suitable for non-Agrobacterium transformation are further described.

pCIB3064

pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques with selection by the herbicide Vaster (or phosphinothricin). Plasmid pBIC246 is E. coli. CaMV 35S promoter and CaMV 35S transcription terminators of operative fusion to the E. coli GUS gene and are described in PCT Publication No. WO 93/07278. The 35S promoter of these vectors comprises two ATG sequences 5 'of the starting site. These sites are mutated using standard PCR techniques in order to remove ATG and yield restriction sites SspI and PvuII. The new restriction site is 96 and 37 bp from the unique SalI site and 101 and 42 bp from the actual starting site. The derivative of pCIB246 obtained is named pCIB3025. The GUS gene is then digested with SalI and SacI to cleave from pCIB3025, leave the ends smooth and reconnect to prepare plasmid pCIB3060. Plasmid pJIT82, available from the John Innes Center, Norwich, cuts 400 bp long SmaI fragments containing the bar gene from Streptomyces viridochromogens and inserts into the HpaI site of pCIB3060. See Thompson et al., EMBO J, 6: 2519 (1987)]. This includes pCIB3064, which includes the bar gene under the control of the CaMV 35S promoter and terminator for selection of herbicides, the ampicillin resistance gene (for selection in E. coli) and multiple connectors in the unique sites SphI, PstI, HindIII and BamHI. Was generated. The vectors are suitable for cloning plant expression cassettes containing their own regulatory signals.

pSOG19 and pSOG35:

Plasmid pSOG35 is a screening marker that confers resistance to methotrexate. Transformation vector using E. coli gene dihydrofolate reductase (DHFR). PCR was used to amplify the 35S promoter (-800bp), intron 6 (-550bp) from the maize Adh1 gene and 18bp of the GUS non-poisonous leader sequence from pSOG10. this. 250-bp fragments encoding E. coli dihydrofolate reductase type II genes were also amplified by PCR and these two PCR fragments were SacI- from pB1221 (Clontech) containing the pUC19 vector backbone and nopaline synthase terminator. Assemble with PstI fragment. Assembly of these fragments results in pSOG19 comprising a 35S promoter in a fusion with the Intron 6 sequence, the GUS leader, the DHFR gene, and the nopaline synthase terminator. Replacement of the GUS leader sequence of pSOG19 with the leader sequence from maize whitening spot virus (MCMV) produces the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have HindIII, SphI, PstI and EcoRI sites that allow cloning of foreign material.

c. Vectors Suitable for Chloroplast Transformation

For expression of the nucleotide sequences of the present invention in plant plastids, the plastiform transformation vector pPH143 (WO 97/32011, Example 36) is used. The nucleotide sequence is inserted into pPH143 to replace the PROTOX coding sequence. The vector is then used for chromatin transformation and screening for transformants for spectinomycin resistance. Alternatively, the nucleotide sequence is inserted into pPH143 to replace the aadH gene. In this case, the transformants are selected for resistance to PROTOX inhibitors.

Plant host applied to the transformation method

Regardless of whether they are organogenic or embryogenic, any plant tissue capable of subsequent clonal proliferation can be transformed with the constructs of the invention. The term organogenesis refers to the process by which shoots or roots develop sequentially from the meristem center, whereas the term embryogenicity refers together in a planned manner (out of order), regardless of whether the shoots and roots are somatic or germline derived. It means the process of development. The particular tissue selected will be available for the particular species to be transformed and will vary depending on the most suitable clone propagation system. Exemplary tissue targets include leaf beds, roots, stems, shoots, leaves, pollen, seeds, embryos, cotyledon, hypocotyls, female embryoid bodies, union tissues, existing meristems (eg, apical meristem, side eyes, and roots) Meristem) and induced meristems (eg, cotyledonic and embryonic meristems), tumor tissues and various forms of cells and cultures such as single cells, protoplasts, embryos and callus tissues, including but not limited to Non-differentiated and undifferentiated tissues or plants are included. Plant tissue may be a plant or organ, tissue or cell culture.

The plant of the present invention may take various forms. The plant can be a chimera of transformed and non-transformed cells; The plant may be a clonal transformant (eg, all cells transformed to contain an expression cassette); Plants may include implants of transformed and non-transformed tissues (eg, transgenic roots roots implanted in ciliated species in untransformed scion). Transformed plants can be propagated by various methods such as clonal propagation or typical breeding techniques. For example, a first generation (or T1) transformed plant can be self-proliferated to provide a homogenous second generation (or T2) transformed plant, and the T2 plants can be further propagated through classical breeding techniques. Can be. Preferred selection markers (eg nptII) can be associated with expression vectors to support sarcoma.

The present invention relates to corn (zea mays), Brassica ssp. B. napus, b. B. rapa, b. B. junciea}, especially those brassica species useful as a source of seed oil, Medicago sativa, Oryza sativa, Rye (Secale cereale), Sorghum bicolor (Sorghum vulgare), millet { For example, the pearl millet (Pennisetum glaucum), the proso millet (Panicum miliaceum), the foxtail millet (Setaria italica), the finger millet (Eleusine coracana)}, the sunflower (Helianthus annuus), the safflower (Carthamus tinctorius), Wheat (Triticum aestivum), Soybean (Glycine max), Tobacco (Nicotiana tabacum), Potatoes (Solanum tuberosum), Peanuts (Arachis hypogaea), Cotton (Gossypium barbadense, Gossypium hirsutum), Sweet potato (Ipomoea batatus), Cassava (Manihot esculenta , Coffee (Cofea spp.), Coconut (Cocos nucifera), Pineapple (Ananas comosus), Citrus tree (Citrus spp.), Cocoa (Theobroma cacao), Tea (Camellia sinensis), Banana (Musa spp.), Avocado (Persea) americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europa ea, Carica papaya, Cashew (Anacardium occidentale), Macadamia (Macadamia integrifolia), Almond (Prunus amygdalus), Sugar beet (Beta vulgaris), Saccharum spp., Oats, Barley, Vegetables, Ornamental, Wood Plants (e.g. conifers and deciduous trees), squash, pumpkins, hemp, zucchini, apples, pears, quince, melons, plums, cherries, peaches, nectarines, apricots, strawberries, grapes, raspberries, blackberries, soybeans Can be used for transformation of any plant species, including, but not limited to, sorghum, sugar cane, rape, clover, carrots and Arabidopsis thaliana .

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lyca beans (Phaseolus limensis), peas (Lathyrus spp.), Cauliflower, broccoli, turnips, radishes, spinach, Asparagus, onions, garlic, peppers, celery, and members of the genus Melon, such as cucumber (C. sativus), C. cantalupensis, and C. melo. Ornamental plants include Rhododendron spp., Hydrangea (Macrophylla hydrangea), Hibiscus (Hibiscys rosasanensis), Rose (Rosa spp.), Tulip (Tulipa spp.), Narcissus spp., Petunia hybrida, Carnations (Dianthus caryophyllus), Poinsettia (Euphorbia pulcherrima) and chrysanthemum. Conifers that may be used in the practice of the present invention include, for example, Pinus taeda, Pinus elliotii, Pinus ponderosa, Pinus contorta, and Monterey pine (Pinus). pines such as radiata, Douglas fir (Pseudotsuga menziesii); Tsuga canadensis; Sitka spruce (Picea glauca); American cedar (Sequoia sempervirens); Veterans such as silver fir (Abies amabilis) and Abies balsamea; And cedars such as the American Red Cedar (Thuja plicata) and Alaska Yellow Cedar (Chamaecyparis nootkatensis). Legumes include beans and peas. Soybeans include guar, soybeans, fenugreek, soybeans, silver beans, madgi, green beans, lima beans, paba beans, lentils, and chickpeas. Legumes include arachis (e.g. peanuts), bisia (e.g. golden leeks, hairy bitches, red beans, mung beans and chickpeas), lupine (e.g. lupines, trifolium) Ruth (e.g., soybeans and lima beans), P. sume (e.g., alfalfa), Melilotus (e.g. clover), Medicago (e.g. alfalfa), Lotus (e.g. trefoil) ), But are not limited to lenses (eg lentils and cyanobacteria). Preferred grasses and turfgrass for use in the method of the present invention include alfalfa, stalk grass, laver hair, perennial ryegrass, snow ear grass and red ear wheat.

Preferably, the plant of the present invention is a crop, for example, corn, alfalfa, sunflower, brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, barley, rice, tomato, potato, squash , Melon, legumes and more. Other preferred plants include the Liliaceae and Elderaceae.

Once the desired DNA sequence is transformed into a particular plant species, it can be propagated within that species using conventional breeding techniques or transferred to various other identical species (including commercial varieties in particular).

The following is a description of representative techniques and representative chromatin transformation techniques for transforming both dicotyledonous and monocotyledonous plants.

a. Transformation of Dicotyledonous Plants

Transformation techniques for dicotyledonous plants are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium techniques include the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated reception, particle impact-mediated delivery or microinjection. Examples of these techniques are described in Paszkowski et al., EMBO J, 3: 2717 (1984), Potrykus et al., Mol. Gen. Genet., 199: 169 (1985), Reich et al., Biotechnology, 4: 1001 (1986), and Klein et al., Nature, 327: 70 (1987). In each case, the transformed cells are regenerated into whole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique for the transformation of dicotyledonous plants because of its high transformation efficiency and its wide utility with many different species. Agrobacterium transformation is usually a suitable Agro that can be followed by the complement of the vir gene carried by a co-stable Ti plasmid or a chromosomal host Agrobacterium strain (eg strain CIB542 for pCIB200 and pCIB2001). Delivery of a binary vector (eg, pCIB200 or pCIB2001) that carries foreign DNA of interest to the Vectrium strain (Uknes et al., Plant Cell, 5: 159 (1993)). Delivery of the recombinant binary vector to Agrobacterium results in E. coli carrying the recombinant binary vector. E. coli, which has a plasmid such as pRK2013 and which is capable of transferring a recombinant binary vector to a target Agrobacterium. Achieved by the maternal breeding process using E. coli strains. Alternatively, recombinant binary vectors can be delivered to Agrobacterium by DNA transformation (Hofgen & Willmitzer, Nucl., Acids Res., 16: 9877 (1988)).

Transformation of target plant species by recombinant Agrobacterium generally involves co-culture of explants and Agrobacterium from plants, and the following protocols are well known in the art. Transformed tissue is regenerated in selection medium containing antibiotic or herbicide resistance markers present between the binary plasmid T-DNA boundaries.

The vector can be introduced into plant cells by known methods. Preferred cells for transformation include Agrobacterium, monocotyledonous cells and dicotyledonous cells (including celiac cells and millet pediatric cells). Preferred monocotyledonous cells are cereal cells such as corn (corn), barley and wheat, and starch accumulating dicotyledonous cells such as potatoes.

Another method of transforming plant cells with genes includes propagating inert or biologically active particles in plant tissues and cells. This technique is disclosed in US Pat. Nos. 4,945,050, 5,036,006 and 5,100,792. In general, the method includes propelling the inert or biologically active particles into the cell under conditions effective to penetrate and incorporate into the outer surface of the cell. When inert particles are used, the vector can be introduced into cells by coating the particles with a vector containing the gene of interest. Alternatively, the target cells can be surrounded by a vector to allow the vector to be carried into the cell along the trajectory of the particle. Biologically active particles (eg, anhydrous enzyme cells, anhydrous bacteria or bacteriophages, each comprising DNA to be introduced) may also be propagated into plant cell tissue.

b. Transformation of monocotyledonous plants

Transformation of most monocot species is also now common. Preferred techniques include direct delivery of genes into protoplasts using polyethylene glycol (PEG) or electroporation techniques and particle bombardment into callus tissue. Transformation can be performed using a single DNA species or multiple DNA species (ie co-transformation), both of which are suitable for use with the present invention. Co-transformation has the advantage of being able to avoid complete vector construction and transformed plants with unassociated loci for genes of interest and selection markers, and to remove the selection markers in later generations (if this is deemed desirable). Can have. However, a disadvantage of the use of co-transformation is that the frequency of integration of isolated DNA species into the genome is less than 100% (Schocher et al., Biotechnology, 4: 1093, 1986).

EP 0 292 435, EP 0 392 225 and WO 93/07278 describe the preparation of callus and protoplasts from the superior suburbs of corn, transformation of protoplasts using PEG or electroporation, and Techniques for the regeneration of corn plants from transgenic protoplasts are described. Author Gordon-Kamm et al. (Plant Cell, 2: 603 (1990)) and author Fromm et al. (Biotechnology, 8: 833 (1990)) describe A188-derived corn RPduf using particle bombardment. Transformation technique was published. In addition, WO 93/07278 and author Koziel et al. (Biotechnology, 11: 194 (1993)) describe a technique for transforming maize's stormwater system by particle bombardment. The technique uses 1.5-2.5 mm long immature corn embryos and impacted PDS-1000He bioballistic equipment excised from corn ears 14 to 15 days after pollination.

Rice transformation can also be accomplished by direct gene transfer techniques using protoplasts or particle bombardment. Protoplast-mediated transformation has Jockey pony car is (Japonica) is described with respect to the types and Indica (Indica) type (as described in Reference: Zhang et al, Plant Cell Rep , 7: 379 (1988); Shimamoto et al,.. Nature, 338: 274 (1989); Datta et al., Biotechnology, 8: 736 (1990)). In addition, both forms are commonly transformable using particle bombardment (Christou et al., Biotechnology, 9: 957 (1991)). In addition, WO 93/21335 describes a technique for transforming rice through electroporation. Patent application EP 0 332 581 describes techniques for the generation, transformation and regeneration of poapuraceae protoplasts. These techniques enable the transformation of Dactylis and wheat. In addition, wheat transformation uses particle bombardment of cells of type C organ regenerative callus into cells (Vasil et al., Biotechnology, 10: 667 (1992)), and also immature embryos and immature embryo-derived Using particle bombardment of fusion cells, see Vasil et al. (Biotechnology, 11: 1553 (1993)) and Weeks et al. (Plant Physiol., 102: 1077 (1993)), but preferred wheat conversion techniques include the transformation of wheat by particle bombardment of immature embryos. And high sucrose or high maltose steps prior to gene transfer Any number of embryos (0.75-1 mm long) prior to impact were subjected to 3% sucrose (Murashiga & Skoog, Physiologia Plantarum, 15: 473 (1962)]) and plate culture on MS medium with 2,4-D 3 mg / l for induction of somatic embryos, which can proceed in the dark .. On the day of impact, embryos are removed from the incubation medium and osmotic Place on medium (ie, induction medium with sucrose or maltose added at the desired concentration, typically 15%) Embryos are protoplasted for 2-3 hours and then impacted.20 per target plate Dog embryos are common but not critical A suitable gene-bearing plasmid (eg pCIB3064 or pSG35) is deposited on micrometer size gold particles using standard methods Explosion of about 1000 psi using a standard 80 mesh screen on each embryo plate Shoot using pressured DuPont Biolostics ^R helium equipment After impact, the embryo is placed back in the dark for about 24 hours (still on osmotic medium) to recover, after 24 hours, the embryo is removed from the osmotic medium and again Place on induction medium and allow to stay for one month prior to regeneration After approximately one month, embryo explants with developing embryogenic callus are further selected as suitable selection agents (10 mg / 1 vastar for pCIB3064 and methotre for pSOG35). Transfer medium containing MS 2 mg / l) (MS + 1 mg / liter NAA, 5 mg / liter GA) After approximately one month, the developed shoots are medium-strength MS, 2% sucrose and copper. Transferred to a larger sterile containers known as "GA7s" containing a selection agent for the concentration.

Transformation of monocotyledonous plants with Agrobacterium is also described. See WO 94/00977 and US Pat. No. 5,591,616, which are incorporated herein by reference.

c. Transformation of the Chromosome

Nicotiana tabacum cv. Seeds of "Xanthi nc" are germinated 7 per plate in 1 "cyclic alignment on T agar medium and essentially with DNA from plasmids pPH143 and pPH145 as described. Shock is applied 12 to 14 days after sowing with coated 1 μm tungsten particles (M10, Biorad, Hercules, Calif.) (Svab and Maliga, PNAS, 90: 913 (1993)). Seedlings were removed from leaves and RMOP medium (Svab, Hajdukiewicz and Maliga, PNAS, 87: 8526 (1990) containing 500 μg / ml of spectinomycin dihydrochloride (Sigma, St. Louis, MO) Incubate in T medium for 2 days with the embryonic axis facing upwards under bright light (350-500 μmol photons / m ² / s) on a plate of 3). The order of resistance shown below was obtained from the same selection medium. Passage cloning to form a callus, separation and passage cloning of the second order Complete isolation of the copy (homologous chromosome) of the transformed plastid genome of an independent passage clone is assessed by standard techniques of Southern blotting. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (1989)). BamHI / EcoRI-lysed total cellular DNA (Mettler, IJ Plant Mol Biol Reporter). , 5: 346 (1987)]) were separated on a 1% tris-borate (TBE) agarose gel and transferred to a nylon membrane (Amersham) 0.7 kb BamHI / from pC8 containing the site of the rps7 / 12 chromatin targeting sequence. Probing with ³² P-labeled randomly primed DNA sequences corresponding to HindIII DNA fragments. Homologous chromosomal order is sterilely adhered on spectinomycin-containing MS / IBA medium (McBride et al., PNAS, 91: 7301 (1994)) and transferred to a greenhouse.

Preparation and Characterization of Stably Transformed Plants

The transformed plant cells are then placed in a selection medium suitable for the selection of the transformed cells, which are then grown into callus tissue. The shoots are grown from the callus and seedlings are generated from the shoots as they are cultured in lubricating medium. In general, various constructs will bind to markers for selection in plant cells. Typically, the marker may be resistant to biocides (particularly antibiotics such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicides and the like). The specific marker used will allow the selection of transformed cells as compared to cells in which the introduced DNA has been deleted. Components of the DNA constructs comprising the transcription / expression cassettes of the invention can be prepared from native (endogenous) or foreign (exogenous) sequences to the host. "Exotic" means that the sequence is not found in the wild type host into which the construct was introduced. Heterologous constructs will include one or more regions from which the gene from which the transcription-initiation-region originated is not native.

Southern blot analysis can be performed using methods known to those of skill in the art to confirm the presence of transgenes in transformed cells and plants. Insertion of polynucleic acid fragments into the genome can be detected and quantified by Southern blot, as they can be readily distinguished from the construct containing the fragments by the use of suitable restriction enzymes. The expression product of the transgene is dependent on the nature of the product and can be detected by any of a variety of methods, including Western blots and enzyme assays. One particularly useful method of quantifying protein expression and detecting replication in different plant tissues is to use report genes such as GUS. Once the transformed plants are obtained, they can be cultured to produce plant tissues and parts having the desired phenotype. Plant tissue or plant parts may be harvested and / or seed collected. Seeds can be provided as a source for culturing additional plants having a portion or tissue having the desired properties.

Thus, the present invention is directed to transformed plants or plant parts (eg, ear, seed, fruit, grain, stover, bran or vargas) comprising one or more polypeptides, expression cassettes or vectors of the invention, such plants It provides a method of producing and a method of using the plant or part thereof. Transformed plants or plant parts optionally express processing enzymes localized in specific cell or blast compartments or developing grains of specific tissues. For example, the present invention provides a transformed plant portion comprising one or more starch processing enzymes present in plant cells, obtained from transformed plants whose genome is enhanced with an expression cassette encoding one or more starch processing enzymes. do. The processing enzyme does not act on the target substrate unless activated by heating, polishing or other methods of bringing the enzyme into contact with the substrate under conditions in which the enzyme is active.

Exemplary Methods of the Invention

The self-processing plants and plant parts of the present invention can be used in various methods using processing enzymes (medium, thermophilic or superthermophilic) that are expressed and activated in such self-processing plants and plant parts. According to the invention, the transformed plant part obtained from the transformed plant whose genome has been enhanced with one or more processing enzymes is placed under conditions under which the processing enzyme is expressed and activated. Upon activation, the processing enzyme is activated and functions to act on the substrate substrate to which it normally functions to achieve the desired result. For example, starch-processing enzymes can act on starch to degrade, hydrolyze, isomerize or otherwise modify to yield the desired results. Non-starch processing enzymes can be used to disrupt plant cell membranes to facilitate extraction of starch, lipids, amino acids or other products from plants. Additionally, non-superthermophilic and superthermotropic enzymes can be used with the self-processing plants or plant parts of the present invention. For example, mesophilic non-starch degrading enzymes can be activated to destroy plant cell membranes for starch extraction, and subsequently, superthermotropic starch-degrading enzymes can then be activated in self-processing plants to degrade starch. have.

Enzymes that express in grains can be activated by placing plants or plant parts containing them under conditions that enhance their activity. For example, one or more of the following techniques may be used. The plant part may be contacted with water, which provides a substrate for the hydrolase, which will activate the enzyme. The plant part can be contacted with its substrate by contacting water to be moved from the compartment where the enzyme has precipitated during the development of the plant part. Enzyme migration is possible because no compartmentalization occurs during maturation, grain drying and rehydration. Enzymes can be contacted with their substrates by contacting circular or copper alleles with water to be removed from the compartments that have settled during the development of the plant part. Enzymes can also be activated by the addition of activating compounds. For example, calcium-dependent enzymes can be activated by the addition of calcium. Other activating compounds can be determined by one skilled in the art. The enzyme can be activated by removal of the inactivating agent. For example, peptide inhibitors of amylase enzymes are known and amylases can be activated by addition of protease after co-expression with amylase inhibitors. An enzyme can be activated by changing the pH to the pH at which the enzyme is most active. Enzymes can also be activated by raising the temperature. Enzymes generally increase in activity to an optimum temperature for that enzyme. The mesophilic enzyme will increase in activity from the level of the ambient temperature of activity to the temperature at which it loses activity, which is typically below 70 ° C. Similarly, thermophilic and superthermophilic enzymes can also be activated as the temperature increases. Thermophilic enzymes can be activated by heating to a temperature below the maximum temperature of the activity or stability. The maximum temperature of stability and activity for thermophilic enzymes will generally be between 70 ° C and 85 ° C. Superthermophilic enzymes will have relatively greater activity than mesophilic or thermophilic enzymes due to large potential changes from 25 ° C to 85 ° C to 95 ° C or even 100 ° C. The temperature increase can be achieved by any method (eg, heating, such as baking, boiling, heating, steaming, electric discharge or any combination thereof). In addition, in plants expressing mesophilic or thermophilic enzyme (s), the enzyme can be polished and activated, thereby allowing the enzyme to contact the substrate.

Optimum conditions (eg, temperature, hydration, pH, etc.) may be determined by one skilled in the art and may depend on the individual enzyme to be used and the application of the desired enzyme.

The invention further provides for the use of exogenous enzymes that can assist in certain methods. For example, the use of self-processing plants or plant parts of the present invention can be used in conjunction with an exogenously provided enzyme to facilitate the reaction. For example, transgenic α-amylase maize is used in combination with other starch-processing enzymes such as pullulanase, α-glucosidase, glucose isomerase, mannase hemicellulase and the like to hydrolyze starch or produce ethanol. can do. In fact, it has been found that the combination of transgenic α-amylase corn and the enzyme unexpectedly provides an excellent degree of starch conversion compared to the use of transgenic α-amylase corn alone.

Examples of suitable methods discussed herein are provided.

a. Starch extract from plants

The present invention provides a method for promoting the extraction of starch from plants. In particular, one or more polynucleotides encoding the processing enzymes that disrupt the endosperm's physical inhibitory substrates (cell walls, non-starch polysaccharides and protein substrates) are introduced into the plant, preferably bringing the enzyme close to the starch granules in the plant. In this aspect of the invention, the transformed plant is at least one protease, glucanase, xylanase, thioredoxin / thioredoxin reductase, cellulase, phytase, lipase, beta glucosidase, esterase And the like, but do not express any starch-degrading activity, thereby maintaining the integrity of the starch granules. The expression of these enzymes in plant parts such as grains thus improves the processing properties of the grains. The processing enzyme may be mesophilic, thermophilic or hyperthermia. In one example, the grains from the transformed plants of the present invention are heat dried to suitably inactivate non-thermophilic processing enzymes and improve seed integrity. The grains (or copper grains) are immersed at high or low water content or conditions at low or high temperatures (time is most important) with or without sulfur dioxide (Primary Cereal Processing, Gordon and Willm, eds, which is incorporated herein by reference). , pp. 319-337 (1994). Optionally, under certain humidity conditions, as the increased temperature is reached, the starch granules present in the endosperm remain intact and are more easily recoverable from the material obtained, Non-starch polysaccharides are disrupted by activating enzymes that degrade (eg, proteases, xylanase, phytase or glucanase). In addition, the proteins and non-starch polysaccharides in the effluent may be at least partially degraded and highly concentrated to improve animal feed, food or as a medium component for microbial fermentation. Effluents are considered to be corn-simmers with improved compositions.

Accordingly, the present invention provides a method for preparing starch granules. The method comprises the steps of treating a grain comprising one or more non-starch processing enzymes (e.g., cohalib) under conditions that activate one or more enzymes, starch granules and non-starch degraded granules (e.g., degradation). Calculating a mixture comprising the endosperm reflux substrate product). Non-starch processing enzymes may be mesophilic, thermophilic or hyperthermic. After activation of the enzyme, starch granules are separated from the mixture. The grains are obtained from transformed plants in which the genome comprises an expression cassette encoding one or more processing enzymes (enhanced with an expression cassette). For example, the processing enzyme may be a protease, glucanase, xylanase, phytase, thioredoxin / thioredoxin reductase, esterase, cellulase, lipase or beta glucosidase. The processing enzyme may be superthermophilic. The grains can be treated in the presence or absence of sulfur dioxide, under low or high humidity conditions. Depending on the expression level of the processing enzyme in the grains from the transformed plant, the transgenic grains may be mixed with the product grains before or during processing. Also provided are products obtained from this process, such as improved immersion water comprising starch, non-starch products and one or more additional ingredients.

b. Starch-processing method

Transformed plants or plant parts of the invention can be used to degrade starch granules into dextrins, other modified starches or hexose (e.g., α-amylase, pullulanase, α-glucosidase, glucoamylase, amyloful). Lulanase) may comprise starch-degrading enzymes as disclosed herein, which convert glucose to fructose (eg, glucose isomerase). Preferably, α-amylase, α-glucosidase, glucoamylase, pullulanase, neofululanase, amylopululase, glucose isomerase, and the like, wherein the starch-degrading enzyme is used to transform the grains; Choose from combinations thereof. Further, preferably, the enzyme is operably linked to a signal sequence that plagigates the promoter and enzyme to starch granules, starch, apoplasts or cytoplasmic reticulum. Most preferably, the enzyme is expressed in endoderm, and in particular maize endocrine, and localized in one or more cell compartments or starch granules of its own. Preferred plant parts are grains. Preferred plant parts are from corn, wheat, barley, rye, oats, sugar cane or rice.

According to the starch-degradation method of the present invention, the transformed grain accumulates starch-degrading enzymes in the starch granules, soaked at conventional temperatures of 50 ° C. to 60 ° C. and wet-milled as is known in the art. Preferably, the starch-degrading enzyme is superthermophilic. Because of the subcellular targeting of the enzyme to starch granules, or the association of the enzyme with starch granules, the enzyme and starch granules are contacted during wet-milling at normal temperature, thereby co-purifying the processing enzyme with the starch granules. Starch granules / enzyme mixtures are obtained. Recovery of the starch granule / enzyme mixture is followed by activation of the enzyme under conditions favorable for the activation of the enzyme. For example, processing may be carried out at various humidity and / or temperature conditions to promote partial hydrolysis of starch to hexose (to produce induced starch or dextrin) or complete hydrolysis. Syrups containing high dextrose or fructose equivalents are obtained in this way. The method effectively saves time, energy and enzyme costs, increases the efficiency of converting starch to the corresponding hexose, and the production efficiency of products such as high sugar immersion water and high dextrose equivalent syrups.

In another embodiment, plants or products of plants, such as fruits or grains, or powders prepared from grains expressing the enzymes are treated to activate the enzymes and convert the polysaccharides expressed and contained in the plants to sugars. Preferably, as disclosed herein, the enzyme is fused to a signal sequence that targets the enzyme to starch granules, starch, apoplasts or cytoplasm. The sugars produced can then be separated or recovered from the plant or product of the plant. In another embodiment, processing enzymes capable of converting polysaccharides to sugars are placed under the control of an inducible promoter according to methods known in the art and disclosed herein. The processing enzyme may be mesophilic, thermophilic or hyperthermia. The plant is incubated to the desired stage and induces a promoter that causes the expression of enzymes and the conversion of the polysaccharides into sugars in the plant or plant product. Preferably, the enzyme is operably linked to a signal sequence that targets the enzyme to starch granules, starch, apoplasts, or cytoplasmic nets. In another embodiment, a transformed plant is produced that expresses a processing enzyme capable of converting starch into sugar. The enzyme is fused to a signal sequence that targets the enzyme to starch granules in the plant. The starch is then isolated from the transformed plant containing the enzyme expressed by the transformed plant. The starch can then be converted to sugars by activating the enzyme contained in the isolated starch. The enzyme may be mesophilic, thermophilic or hyperthermic. Provided herein are examples of superthermophilic enzymes capable of converting starch to sugar. The method can be used with any plant capable of producing polysaccharides and expressing enzymes capable of converting the polysaccharides into hydrolyzed starch products such as sugars or dextrins, maltooligosaccharides, glucose and / or mixtures thereof.

The present invention provides a method for producing dextrins and modified starches from plants, or products from plants transformed with processing enzymes that hydrolyze specific covalent bonds of polysaccharides to form polysaccharide derivatives. In one embodiment, a plant or a product of a plant, such as fruit or a grain, or a powder prepared from a grain expressing the enzyme, is subjected to conditions sufficient to activate the enzyme and convert the polysaccharides contained in the plant to a reduced molecular weight polysaccharide. Put it. Preferably, the enzyme is fused to a signal sequence that targets the enzyme to starch granules, starch, apoplasts or cytoplasmic reticulum as disclosed herein. The prepared dextrin or derived starch can then be separated or recovered from the plant or product of the plant. In another embodiment, processing enzymes capable of converting polysaccharides to dextrins or modified starches are placed under the control of an inducible promoter according to methods known in the art and disclosed herein. The plant is cultivated to the desired stage and induces a promoter that causes expression of the enzyme in the plant or plant product and conversion of the polysaccharide to dextrin or modified starch. Preferably the enzyme is α-amylase, pullulanase, iso or neo pullulanase and is operably linked to a signal sequence that targets the enzyme to starch granules, starches, apoplasts or cytoplasmic reticulum. In one embodiment, the enzyme is targeted to apoplast or cytoplasmic reticulum. In another embodiment, the enzyme produces a transformed plant capable of converting starch to dextrin or modified starch. The enzyme is fused to a signal sequence that targets the enzyme to starch granules in the plant. The starch containing the enzyme expressed by the transformed plant is then separated from the transformed plant. The elements contained in the isolated starch can then be activated under conditions sufficient to convert the starch to dextrin or modified starch. For example, provided herein are examples of superthermolytic enzymes capable of converting starch into starch hydrolyzed starch products. The method can be used with any plant capable of producing an polysaccharide and expressing an enzyme capable of converting the polysaccharide to a sugar.

In another embodiment, starch-decomposes the binding of starch granules to dextran, modified starch or hexose (e.g., α-amylase, pullulanase, α-glucosidase, glucoamylase, amylopululase) Grains from the transformed plants of the present invention, which accumulate degrading enzymes, are immersed under conditions favorable for activation of starch degrading enzymes for various times. The resulting mixture may contain high concentrations of starch-derived product. The use of the grains further enhances the accessibility of the starch to enzymes by either 1) eliminating the need for grain milling or otherwise processing the grains first to obtain starch granules, and 2) placing the enzyme directly within the endocrine tissue of the grains. 3) eliminates the need for the production of starch-hydrolase by microorganisms. Thus, the entire process of wet milling before hex recovery is removed by heating only the grains, preferably corn grains, in the presence of water to enable the enzyme to act on the starch.

The method can be used for ethanol, high fructose syrup, for the production of hexose (glucose) containing fermentation medium or for any other use of starch that does not require purification of the grain components.

The present invention further comprises the steps of treating a plant part comprising starch granules and one or more starch processing enzymes under conditions activating one or more enzymes, thereby degrading the starch granules to form an aqueous solution comprising sugars. It provides a method for producing maltooligosaccharide and / or sugar. Plant parts are obtained from genomes whose genomes have been enhanced with expression cassettes encoding one or more processing enzymes. An aqueous solution comprising dextrin, maltooligosaccharide, and / or sugar is collected. In one embodiment, the processing enzyme is α-amylase, α-glucosidase, pullulase, glucoamylase, amylopululase, glucose isomerase, or any combination thereof. Preferably, the enzyme is superthermophilic. In another embodiment, the invention further comprises the step of separating the dextrin, maltooligosaccharide and / or sugar.

c. Improved Corn Varieties

The present invention also has a normal level of starch accumulation, accumulates sufficient levels of starch dehydrogenase (s) in the endocrine or starch accumulation organs, such as boiling or heating its plants or plant parts in the case of superthermolytic enzymes. Upon activation of the enzyme, the enzyme provides a method for producing improved corn varieties (and other crop varieties) which activate and promote the rapid conversion of starch into simple sugars. These simple sugars, mainly glucose, will provide sweetness to the treated corn. The corn plants obtained are improved varieties for dual use as grain preparation hybrids and sweet corn. Accordingly, the present invention is a transformed plant or its derivatives, wherein the genome is enriched with an expression cassette comprising a promoter operably linked to a first polynucleotide encoding one or more starch enzymes, the expression cassette being expressed in endocrine. A plant portion is processed under conditions that activate one or more enzymes to convert polysaccharides in corn to sugars, yielding sweet corn, thereby providing a method for producing ultra-sweet corn. The promoter is a constitutive promoter. It may be an endocrine-specific promoter linked to a polynucleotide encoding a processing enzyme such as a seed-specific promoter or α-amylase (eg, comprising SEQ ID NO: 13, 14 or 16). Preferably, the enzyme is superthermophilic. In one embodiment, the expression cassette further comprises a second polynucleotide encoding a signal sequence operably linked to an enzyme encoded by the first polynucleotide. Exemplary signal sequences of this embodiment of the invention direct enzymes to apoplasts, cytoplasmic reticulum, starch granules or starch bodies. Corn plants are grown to form grains with a grain, and the promoter is then induced to allow the enzyme to be expressed and the polysaccharides contained in the plant to be converted to sugars.

d. Self-fermenting plant

In another embodiment of the present invention, plants such as corn, rice, wheat or sugar cane can be treated with large amounts of processing enzymes (eg xylanase, cellulase, hemicellulase, glucanase, pectinase, lipase) on the cell wall. , Esterase, beta glucosidase, phytase, protease and the like (non-starch polysaccharide degrading enzyme)}. After harvesting of the grain component (or sugar in sugar cane), stover, bran or vargas, it is used as a source of targeted enzymes for expression and accumulation in the cell wall, and as a source of biomass. A stove (or other residual tissue) is used as feedstock in the process to recover fermentable sugars. The method of obtaining fermentable sugars consists in the activation of non-starch polysaccharide degrading enzymes. For example, activation may comprise heating the plant tissue in the presence of water for a time suitable for hydrolysis of the non-starch polysaccharides to the sugars obtained. Thus, self-processing stoves produce the enzymes necessary for the conversion of polysaccharides to monosaccharides, and since they are components of the feedstock, essentially no unit cost increases. In addition, temperature-dependent enzymes have no adverse effects on plant growth and development and cell wall targeting, even targeting to polysaccharide microfibrils by cellulose / xylose binding domains fused to proteins, enzymes of substrates. Improve accessibility

Accordingly, the present invention also provides a method of using a transformed plant portion comprising one or more non-starch polysaccharide processing enzymes of cells of the cell wall or plant portion. The method comprises the steps of transforming a plant part comprising one or more non-starch polysaccharide processing enzymes obtained from a transformed plant whose genome is enhanced with an expression cassette encoding one or more non-starch polysaccharide processing enzymes. Treating under active conditions, thereby degrading starch granules to form an aqueous solution comprising sugar; And collecting an aqueous solution containing sugar. The invention also includes a transformed plant or plant part comprising one or more non-starch polysaccharide processing enzymes present in the cells or cell walls of the plant or plant part. The plant part may have one or more non-starch processing enzymes (eg, xylanase, cellulase, glucanase, pectinase, lipase, esterase, beta glucosidase, phytase, protease or its genome). From a transformed plant enhanced with an expression cassette encoding any combination).

e. Aqueous phase with high protein and sugar content

In other embodiments, the protease and lipase are engineered to accumulate in seeds, such as soybean seeds. After activation of the protease or lipase (eg by heating), these enzymes in the seed hydrolyze lipids and storage proteins present in the soy during processing. Thus soluble products comprising amino acids can be obtained which can be used as feed, food or fermentation medium. Polysaccharides are commonly found in insoluble fractions of processed grains. However, by combining polysaccharide degrading enzyme expression and accumulation in seeds, proteins and polysaccharides can be hydrolyzed and found in the aqueous phase. For example, starch polysaccharides from zein and soybeans from corn and stock proteins can be solubilized in this way. The components of the aqueous and hydrophobic phases can be easily separated by extraction with organic solvents or supercritical carbon dioxide. Thus, there is provided a process for the preparation of an aqueous extract of grains containing high concentrations of proteins, amino acids, sugars or monosaccharides.

f. Self-Processing Fermentation

The present invention provides a process for preparing ethanol, fermented beverages or other fermentation-derived product (s). The method comprises obtaining a plant, or a plant derivative, such as a product or plant part of a plant, or grain powder, wherein a processing enzyme is expressed that converts the polysaccharide into a sugar. The plants, or products thereof, are treated to produce sugars by conversion of polysaccharides as described above. The components of sugar and other plants are then fermented according to methods known in the art to form ethanol or fermented beverages or fermentation-derived products. See, for example, US Pat. No. 4,929,452. Briefly, sugars prepared by the conversion of polysaccharides are incubated with yeast under conditions that promote the conversion of sugars to ethanol. Suitable yeasts include high alcohol-resistant and high-sugar resistant strains of yeast (eg yeast, S. aureus). S. cerevisiae ATCC 20867}. The strain was deposited on September 17, 1987 by the American Type Culture Collection, Rockville, Midland, and was assigned ATCC 20867. The fermentation product or fermentation beverage is then distilled off to separate the ethanol or distillation beverage or the fermentation product is recovered. The plant used in the method may be any plant that contains polysaccharides and can express the enzymes of the present invention. Many such plants are disclosed herein. Preferably, the plant is commercially grown. More preferably, the plants are typically used to make ethanol or fermented beverages or fermentation products, such as wheat, barley, corn, oats, potatoes, grapes or rice.

The method includes treating a plant portion comprising one or more polysaccharide processing enzymes under conditions that activate one or more enzymes, thereby degrading the polysaccharides of the plant portion to form fermentable sugars. Polysaccharide processing enzymes may be mesophilic, thermophilic or superthermophilic. Plant parts are obtained from transformed plants whose genome is enhanced with an expression cassette encoding at least one polysaccharide processing enzyme. Plant parts for this aspect of the invention include, but are not limited to, grains, fruits, seeds, stems, wood, vegetables or roots. Plants include, but are not limited to oats, barley, wheat, berries, grapes, rye, corn, rice, potatoes, sugar beets, sugar cane, pineapples, grasses and trees. The plant part can be combined with commodity grains or other commercially available substrates, and the source of the substrate for processing can be a source other than a self-processing plant. The fermentable sugars are then incubated under conditions that promote the conversion of fermentable sugars to ethanol, for example using yeast and / or other microorganisms. In one embodiment, the plant part is from corn transformed with α-amylase, which has been found to reduce fermentation time and cost.

For example, when the transgenic maize prepared according to the present invention expressing thermophilic α-amylase was used for fermentation, the amount of residual starch was found to decrease. This indicates that more starch is solubilized during fermentation. The reduced amount of residual starch results in distillation grains having higher protein weight content and higher value. In addition, the fermentation of the transformed corn of the present invention allows the liquefaction process to be carried out at low pH, so that at high temperatures (eg, at least 85 ℃, preferably at least 90 ℃, more preferably at least 95 ℃) This results in saving the chemical cost needed to adjust the pH and results in shorter liquefaction time and more complete starch solubilization and reduction of liquefaction time, all leading to an efficient fermentation reaction with high yield of ethanol.

In addition, it has been found that even contacting small parts of the transformed plants produced according to the invention with conventional plant parts can reduce fermentation time and the costs associated therewith. As such, the present invention involves applying a transformed plant portion from a plant comprising a polysaccharide processing enzyme that converts the polysaccharide to a sugar, as compared to the use of a plant portion that does not include the polysaccharide processing enzyme. It is about time reduction.

g. Raw starch processing enzymes and polynucleotides encoding them

Polynucleotides encoding mesophilic processing enzyme (s) are introduced into a plant or plant part. In one embodiment, the polynucleotides of the present invention are corn-optimized polynucleotides as provided in SEQ ID NOs: 48, 50 and 59, encoding glucoamylases as provided in SEQ ID NOs: 47 or 49. In another embodiment, the polynucleotides of the present invention are corn-optimized polynucleotides as provided in SEQ ID NO: 52, encoding the alpha-amylase as provided in SEQ ID NO: 51. In addition, the fusion products of the processing enzymes are further considered. In one embodiment, the polynucleotide of the present invention is a corn-optimized polynucleotide as provided in SEQ ID NO: 46, encoding an alpha-amylase and a glucoamylase fusion as provided in SEQ ID NO: 45. Further in the present invention, the combination of the processing enzyme is considered. For example, combinations of starch-processing enzymes and non-starch processing enzymes are discussed herein. Combinations of such processing enzymes can be obtained using the use of multiple gene constructs encoding each of the genes of interest. Alternatively, individual transformed plants stably transformed with the enzyme can be crossed by known methods to obtain plants comprising both enzymes. Another method involves the use of exogenous enzyme (s) with the transformed plant.

Sources of starch-processed and non-starch processed enzymes can be isolated or derived from any source and the corresponding polynucleotides can be identified by one skilled in the art. α-amylases may be derived from plants such as aspergillus (eg, Aspergillus Schlossami and Aspergillus niger), lycopus (eg lycopus duck) and corn, barley and rice . Glucoamylases include aspergillus (e.g. Aspergillus Schlossami and Aspergillus niger), lycopus (e.g. lycopus duck), and thermophilic bacteria (e.g., Thermomobacter termosa) Carolithic).

In another embodiment of the invention, the polynucleotide is a mesophilic starch-processing enzyme operably linked to a corn-optimized polynucleotide as provided in SEQ ID NO: 54, encoding a biostarch binding domain as provided in SEQ ID NO: 53. Encrypt it.

In another embodiment, the tissue-specific promoter is a corn γ-zein promoter (illustrated as SEQ ID NO: 12) or a maize ADP-gpp promoter (illustrated in SEQ ID NO: 11, including 5 'non-toxin and intron sequences) or Q Endosperm-specific promoters such as protein promoters (illustrated by SEQ ID NO: 98) or rice glutelin promoter (illustrated by SEQ ID NO: 67). Thus, the present invention provides isolated polynucleotides comprising a promoter comprising SEQ ID NO: 11, 12, 67 or 98, a polynucleotide that hybridizes with its complement under low stringency hybridization conditions, or SEQ ID NOs: 11, 13, 67 Or a fragment thereof having the activity of a promoter having a sequence of 98 (eg, at least 10% and preferably at least 50%).

In one embodiment, products from starch-hydrolysing genes such as α-amylase, glucoamylase or α-amylase / glucoamylase fusions can be targeted to specific organs or locations, such as cytoplasmic reticulum or apoplasts, rather than cytoplasm. . This is operative for the use of the n-terminal signal sequence (SEQ ID NO: 17), a corn γ-agent conferring apoplast-specific targeting of the protein, and a processing enzyme operably linked to the sequence SEKDEK for retention in the cytoplasmic network. Is illustrated by the use of a γ-zein N-terminal signal sequence (SEQ ID NO: 17) linked to Guidance to specific compartments of a protein or enzyme will allow the enzyme to be localized in a manner that will not contact the substrate. In this method, the enzymatic action of the enzyme will not occur until the enzyme is in contact with its substrate. The enzyme can be contacted with its substrate by methods of milling (physical disruption of cell integrity) and hydration. For example, mesophilic starch-hydrolase may be targeted to apoplasts or cytoplasmic reticulum, and thus will be in contact with starch granules in apoplasts. Milling of the grains will break the integrity of the grains, and then the starch hydrolase will come into contact with the starch granules. In this method, it is possible to bypass the potential negative effects of co-localization of enzymes and their substrates.

h. Sweetener-free food

It also provides a method for preparing high sugar starch foods without the addition of additional sweeteners. Examples of starch products include, but are not limited to, breakfast foods, convenience foods, baked goods, pasta, and cereal foods (eg, breakfast grains). The method treats plant parts comprising one or more processing enzymes under conditions that activate starch processing enzymes, thereby processing the starch granules in the plant parts into sugars, for example plant parts that do not contain superthermolytic enzymes. Processing the starch granules from to form a product that is highly sugary as compared to the product produced. Preferably, the starch processing enzyme is superthermophilic and activated by heating (eg, baking, boiling, heating, steaming, electric discharge or any combination thereof). The plant part is an expression cassette in which the genome encodes one or more superthermotropic starch processing enzymes (e.g., α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase or any combination thereof). It is obtained from transformed plants (e.g., transgenic soybean, rye, oats, barley, wheat, corn, rice or sugar cane) which have been fortified with. The high sugar content product is then processed into starch foods. The present invention also provides starch foods (eg, grain foods, breakfast foods, convenience foods or baked foods) prepared by the process. The starch food may be formed from high sugar content products and water and may contain malts, flavors, vitamins, minerals, colorants or any combination thereof.

The enzyme can be activated to convert the polysaccharides contained in the plant material into sugars prior to inclusion of the plant material into the grain product or during processing of the grain food. Thus, the polysaccharides contained in the plant material can be converted to sugars by activating the material (eg, in the case of a superthermophilic enzyme by heating) prior to inclusion in the starch product. The sugar-containing plant material prepared by the conversion of polysaccharides is then added to the product to produce a high sugar content product. Alternatively, polysaccharides can be converted to sugars by enzymes during processing of the starch product. Examples of processing methods used to make grain products are well known in the art and are described in US Pat. No. 6,183,788; 6,159,530; 6,159,530; 6,149,965; 6,149,965; Heating, baking, boiling and the like as described in US Pat. Nos. 4,988,521 and 5,368,870.

Briefly, various anhydrous ingredients and water may be mixed together and cooked to prepare a dough, to gelatinize the starch ingredients and add cooking flavor. The cooked material is then mechanically kneaded to form cooking dough (eg, grain dough). The anhydrous component may include various additives such as sugars, starches, salts, vitamins, minerals, colorants, flavors and the like. In addition to water, various liquid components such as corn or malt syrup can be added. Starch materials may include coarse grains or powders from grain grains, wheat, rice, corn, oats, barley, rye or other grain grains and mixtures thereof from the transformed plants of the invention. The dough can then be processed into the desired shape by methods such as extrusion or stamping, and further cooked using means such as a James cooker, oven or electric discharge device.

In addition, a method is provided for sweetening starch-containing products without the addition of sweeteners. The method treats starch comprising one or more starch processing enzymes under conditions activating one or more enzymes, thereby degrading the starch to form sugars, and thus, for example, starch that does not contain superthermolytic enzymes. Forming treated (high sugar) starch as compared to the product produced by treatment. Starches of the invention are obtained from transformed plants in which the genome is enhanced with an expression cassette encoding one or more processing enzymes. The enzyme vhg ma α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase or any combination thereof. The enzyme may be superthermophilic and may be activated by heat. Preferred transformed plants include corn, soybean, rye, oats, barley, wheat, rice and sugar cane. The treated starch is then added to the product to produce a high sugar starch containing the product (eg, starch food). Also provided are high sugar starch containing products produced by the process.

The present invention further comprises processing a fruit or vegetable comprising one or more polysaccharide processing enzymes under conditions activating the one or more enzymes, thereby processing the polysaccharides in the fruit or vegetable to form a sugar, eg, polysaccharide processing. It provides a method of sweetening a polysaccharide-containing fruit or vegetable, comprising the step of producing a high sugar fruit or vegetable compared to the fruit or vegetable from a plant that does not contain enzymes. Fruits or vegetables of the present invention are obtained from transformed plants whose genome has been enhanced with an expression cassette encoding at least one polysaccharide processing enzyme. Fruits and vegetables include potatoes, tomatoes, bananas, squash, peas and beans. Enzymes include α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. The enzyme may be superthermophilic.

i. Sweetening of Polysaccharide-Containing Plants or Plant Products

The method involves obtaining a plant expressing a polysaccharide processing enzyme that converts the polysaccharide to a sugar as described. Thus, the enzyme is expressed in plants and plants such as fruits or vegetables in the product. In one embodiment, the enzyme is placed under the control of an inducible promoter to allow expression of the enzyme to be induced by external stimulation. Such inducible promoters and constructs are well known in the art and described herein. Expression of enzymes in plants or their products causes conversion of the polysaccharides contained in the plants or their products to sugars and sweetens the plants or their products. In other embodiments, the polysaccharide processing enzyme is expressed essentially. Thus, plants or their products may be activated under conditions sufficient for the activation of the enzymes to convert the polysaccharides to sugars through the action of the enzymes to sweeten the plants or their products. As a result, self-processing of polysaccharides in fruits or vegetables to form sugars yields fruits or vegetables that are highly sugary (for example, as compared to fruits or vegetables from plants that do not contain polysaccharide processing enzymes). Fruits or vegetables of the present invention are obtained from transformed plants whose genome has been enhanced with an expression cassette encoding at least one polysaccharide processing enzyme. Fruits and veggies include potatoes, tomatoes, bananas, pumpkins, peas and beans. Enzymes include α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. Polysaccharide processing enzymes can be superthermophilic.

j. Isolation of Starch from Transformed Grains Containing Enzymes That Disrupt Endocrine Substrate

The present invention provides a method for separating subages from transformed grains in which an enzyme that destroys an endosperm substrate is expressed. The method includes obtaining a plant that expresses an enzyme that destroys an endosperm substrate, for example by variant of cell walls, non-starch polysaccharides and / or proteins. Examples of such enzymes include, but are not limited to, proteases, glucanase, thioredoxin, thioredoxin reductase, phytase, lipase, cellulase, beta glucosidase, xylanase and esterase. Since the enzyme does not contain any enzyme that exhibits starch-degrading activity, the integrity of the starch granules is maintained. The enzyme can be fused to a signal sequence that targets the enzyme to starch granules. In one embodiment, the grains are heat dried to activate the enzymes and the endogenous enzymes contained within the grains are inactivated. The heat treatment triggers the activation of the enzyme, destroying the endosperm substrate and then easily separating from the starch granules. In another embodiment, the grains are immersed at low or high temperatures, with or without sulfur dioxide, at high or low humidity. The grains are then heat treated to destroy the endosperm matrix and to facilitate separation of the starch granules. In another embodiment, suitable temperature and humidity conditions are established to allow the protease to enter starch granules to degrade the proteins contained in the granules. This treatment will produce starch granules with high yield and low contaminating protein.

k. Use of syrups for the production of syrups with high equivalent weight and ethanol or fermented beverage

The method comprises obtaining a plant expressing a polysaccharide processing enzyme that converts the polysaccharide to a sugar as described above. The plant or product thereof is immersed in an aqueous stream under conditions in which the expressed enzyme converts the polysaccharides contained in the plant or product thereof into dextrins, maltooligosaccharides and / or sugars. The aqueous stream containing dextrin, maltooligosaccharides and / or sugars prepared through the conversion of polysaccharides is then separated to produce syrups with high sugar equivalents. The method may or may not include the additional step of wet-milling the plant or product thereof to obtain starch granules. Examples of enzymes that can be used in the method include, but are not limited to, α-amylase, glucoamylase, pullulanase, and α-glucosidase. The enzyme may be superthermophilic. Sugars prepared according to the method include, but are not limited to, hexose, glucose and fructose. Examples of plants that can be used in the method include, but are not limited to, corn, wheat or barley. Examples of plant products that can be used include, but are not limited to, fruits, grains, and vegetables. In one embodiment, the polysaccharide processing enzyme is placed under the control of an inducible promoter. Thus, before or during the dipping process. The promoter is induced to induce the expression of an enzyme, which then converts the polysaccharide to a sugar. Examples of inducible promoters and constructs containing them are well known in the art and provided herein. Thus, if the polysaccharide processing is superthermophilic, soaking is performed at high temperatures to activate the hyperthermic enzymes and to inactivate the endogenous enzymes found in plants or their products. In other embodiments, superthermophilic enzymes capable of converting polysaccharides to sugars are expressed essentially. The enzyme may or may not be targeted to a compartment in the plant using the signal sequence. The plant or product thereof is immersed under high temperature conditions causing conversion of the polysaccharides contained in the plant to sugars.

Also provided are methods of making ethanol or fermented beverages from syrups having a high equivalent weight. The method includes incubating the syrup and yeast together under conditions that allow the conversion of sugars contained in the syrup to ethanol or fermented beverages. Examples of such fermented beverages include, but are not limited to, beer and wine. Fermentation conditions are well known in the art and are described in US Pat. No. 4,929,452 and herein. Preferably the yeast is S. It is a high alcohol-resistant and high sugar resistant strain of yeast such as Seribizia ATCC 20867. Fermentation products of fermentation beverages can be distilled to separate ethanol or distilled beverages.

l. Accumulation of Superthermolytic Enzymes in Plant Cell Walls

The present invention provides a method for accumulating superthermophilic enzymes in the cell wall of a plant. The method includes expressing a superthermophilic enzyme fused with a cell wall targeting signal in a plant to accumulate the targeted enzyme in the cell wall. Preferably, the enzyme can convert polysaccharides to monosaccharides. Examples of targeting sequences include, but are not limited to, cellulose or xylose binding domains. Examples of superthermophilic enzymes include those listed as SEQ ID NOs: 1, 3, 5, 10, 13, 14, 15 or 16. The cell wall containing the plant material may be added as a source of enzyme for conversion of a polysaccharide from a source of the desired enzyme or other source of polysaccharide derived from another source in the process for recovering sugars from the feedstock. In addition, the cell wall can be provided as a source from which the enzyme can be purified. Methods for purifying enzymes are well known in the art and include, but are not limited to, gel filtration, ion-exchange chromatography, chromatographic focusing, isoelectric focusing, affinity chromatography, FPLC, HPLC, salt precipitation, dialysis, and the like. Does not. Accordingly, the present invention also provides purified enzymes isolated from the cell walls of plants.

m. Preparation and Separation Methods of Processed Enzymes

According to the present invention, a recombinantly prepared processing enzyme of the present invention transforms a plant tissue or plant cell comprising the processing enzyme of the present invention that can be activated in a plant selected for the transformed plant tissue or cell, Transformed plant tissues or cells may be cultured into transformed plants and prepared by separating the processing enzymes from the transformed plant or parts thereof. Recombinantly prepared enzymes include α-amylase, glucoamylase, glucose isomerase, α-glucosidase, pullulanase, xylanase, protease, glucanase, beta glucosidase, esterase, lipase or pitase You can be the best. The enzyme has SEQ ID NOs: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, 59, 61, 63, 65, 79, 81, 83, 85 , 87, 89, 91, 93, 94, 95, 96, 97 or 99 can be encoded by a polynucleotide selected from.

The invention will be further described by the following examples, which are not intended to limit the scope of the invention in any way.

Example 1

Construction of Corn-Optimized Genes for Superthermic Starch-Processing / Isomerase

Enzymes α-amylase, pullulanase, α-glucosidase and glucose isomerase, which are involved in starch degradation or glucose isomerization, were chosen from their desired active properties. These include, for example, minimal activity at ambient temperature, high temperature activity / stability and activity at low pH. Corresponding genes were then designed using corn preferred codons as described in US Pat. No. 5,625,136 and synthesized by the manufacturer (Integrated DNA Technologies, Inc., Coralville, Iowa).

797GL3 α-amylase having the amino acid sequence of SEQ ID NO: 1 was selected for its supertherogenic activity. The nucleic acid sequence of the enzyme was deduced and corn-optimized and represented by SEQ ID NO: 2. Similarly, 6gp3 pullulanase was selected having the amino acid sequence set forth in SEQ ID NO: 3. Nucleic acid sequences for 6gp3 pullulanase were inferred, corn optimized and represented by SEQ ID NO: 4.

The amino acid sequence for malA α-glucosidase from sulfolobus solfataricus is described in J. Bact. 177: 482-485 (1995); J. Bact. 180: 1287-1295 (1998). Based on the published amino acid sequence of the protein (SEQ ID NO: 5), a corn-optimized synthetic gene (SEQ ID NO: 6) encoding malA α-glucosidase was designed.

Several glucose isomerase enzymes were selected. Turmoto predicted the amino acid sequence for glucose isomerase derived from Marittima (SEQ ID NO: 18) based on the published DNA sequence with accession number NC_000853 and designed a corn-optimized synthetic gene (SEQ ID NO: 1). Number 19). Similarly, the amino acid sequence (SEQ ID NO: 20) for glucose isomerases derived from Niapolitana is described in Appl. Envir. Microbiol. 61 (5): 1867-1875 (1995)], on the basis of published DNA sequences from Accession No. L38994. Turmoto designed a corn-optimized synthetic gene encoding niapolitana or glucose isomerase (SEQ ID NO: 21).

Example 2

this. Expression of 797GL3 α-amylase fusions in E. coli and expression of starch encapsulation region fusions

Constructs encoding super-thermophilic 797GL3 α-amylase fused to starch encapsulation regions (SER) from corn granule-bound starch synthase (waxy). It was introduced into E. coli and expressed. Corn granules-binding starch synthase cDNA (SEQ ID NO: 7) encoding the amino acid sequence (SEQ ID NO: 8) (Klosgen RB, et al., 1986) was added to the starch binding domain or starch encapsulation region (SER). Cloned as a source of. Full length cDNA from RNA prepared from corn seeds using primers SV57 (5 'AGCGAATTCATGGCGGCTCTGGCCACGT 3') (SEQ ID NO: 22) and SV58 (5 'AGCTAAGCTTCAGGGCGCGGCCACGTTCT 3') (SEQ ID NO: 23) designed as GenBank Accession No. X03935 Amplification by RT-PCR. The complete cDNA was cloned into pBluescrip as an EcoRI / HindIII fragment and the plasmid was named pNOV4022.

The C-terminal portion of the waxy cDNA containing the starch-binding domain (encoded by bp 919-1818) was amplified from pNOV4022 and the skeleton was completed at the 3 'end of the full-length maize-optimized 797GL3 gene (SEQ ID NO: 2). Fused. A fused gene product, 797GL3 / Waxy, having nucleic acid SEQ ID NO: 9 and encoding the amino acid sequence of SEQ ID NO: 10 as a NcoI / XbaI fragment, cloned into pET28b digested with NcoI / NheI (NOVAGEN, Madison, WI) It was. In addition, only the 797GL3 gene was cloned into the pET28b vector as an NcoI / XbaI fragment.

pET28 / 797GL3 and pET28 / 797GL3 / Waxy vectors are BL21 / DE3. E. coli cells (NOVAGEN) were transformed, cultured and induced according to manufacturer's instructions. Analysis by PAGE / Kumasi staining revealed induced proteins corresponding to the predicted size of fused and unfused amylase in both extracts, respectively.

Total cell extracts were analyzed for hyperthermic amylase activity as follows: 5 mg of starch was suspended in 20 μl of water and diluted with 25 μl of ethanol. Samples to be tested for standard amylase positive control and amylase activity were added to the mixture and water was added up to 500 μl final reaction volume. The reaction was carried out at 80 ° C. for 15 to 45 minutes. The reaction was then cooled to room temperature and 500 [mu] l of o- dianisidine and glucose oxidase / peroxidase mixture (Sigma) was added. The mixture was incubated at 37 ° C. for 30 minutes. The reaction was stopped by adding 500 μl of 12N sulfuric acid. Absorbance at 540 nm was measured to quantify the amount of glucose released by amylase / sample. Assays for both fused and unfused amylase extracts showed similar levels of superthermophilic amylase activity, whereas the control extract was negative. This indicated that when 797GL3 amylase was fused to the C-terminal region of the waxy protein, it was still active (at high temperature).

Example 3

Isolation of Promoter Fragments for Endocrine-Specific Expression in Maize

The promoter from the subunit of maize species J. mays ADP-gpp (ADP-glucose pyrophosphorylase) and the 5 'non-coding region I (including the first intron) were subjected to primers designed under Genbank Accession No. M81603. Using 1515 base pair fragment (SEQ ID NO: 11) from maize genomic DNA. The ADP-gpp promoter is shown in endosperm-specific (Shaw and Hannah, 1992).

The promoter from Zea mays γ-Zane was amplified into a 673 bp fragment (SEQ ID NO: 12) from plasmid pGZ27.3 (obtained from Dr Brian Lakin). This γ-zein promoter has been shown to be endocrine-specific (Torrent et al. 1997).

Example 4

Construction of 797GL3 Superthermophilic α-amylase Transformation Vector

Expression cassettes with various targeting signals were constructed to express 797GL3 superthermic amylase in corn endosperm.

pNOV6200 (SEQ ID NO: 13) contains a corn γ-zein signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to a synthetic 797GL3 amylase as described above in Example 1 for targeting to cytoplasmic reticulum and secretion of apoplasts. (See Torrent et al. 1997). This fusion was cloned after the corn ADP-gpp promoter for specific expression in endocrine.

pNOV6201 (SEQ ID NO: 14) comprises an N-terminal signal sequence γ-agent fused to Synthetic 797GL3, with C-terminal addition of the sequence SEKDEL for targeting to cytoplasmic network (ER) and retention in cytoplasmic network (ER) Munro and Pelham, 1987). This fusion was cloned after the corn ADP-gpp promoter for specific expression in endocrine.

pNOV7013 comprises an N-terminal signal sequence, a γ-agent fused to synthetic 797GL3, with C-terminal addition of the sequence SEKDEL for targeting to and retention in cytoplasmic reticulum (ER). PNOV7013 is identical to pNOV6201 except that the corn γ-zein promoter (SEQ ID NO: 12) is used in place of the corn AD-spp promoter to express the fusion in endocrine.

pNOV4029 (SEQ ID NO: 15) includes a waxy starch targeting peptide (Klosgen et al., 1986) fused to synthetic 797GL3 amylase for targeting to starch. This fusion was cloned after the corn ADP-gpp promoter for specific expression in endocrine.

pNOV4031 (SEQ ID NO: 16) contains a waxy starch targeting peptide fused with a synthetic 797GL3 / waxy fusion protein for targeting to starch granules. This fusion was cloned after the corn ADP-gpp promoter for specific expression in endocrine.

Additional constructs were prepared by using these fusions cloned behind the corn γ-zein promoter to obtain higher levels of enzyme expression. All expression cassettes were transferred into a binary vector for transformation into maize via Agrobacterium infection. Binary vectors contain phosphomannose isomerase (PMI) which allows for the selection of transformed cells with mannose. Transformed corn plants were self-pollinated or crossbred, and analytical seeds were harvested.

Additional constructs were prepared using the targeting signal described above fused to 6gp3 pullulanase or 340g12 α-glucosidase exactly in the same manner as described above for α-amylase. This fusion was cloned behind the maize ADP-gpp promoter and / or the γ-zein promoter and transformed into maize as described above. Transformed corn plants were self-pollinated or crossbred, and analytical seeds were harvested.

Combinations of enzymes can be prepared by heterogeneously crossing plants expressing individual genes or by cloning several expression cassettes into the same binary vector capable of cotransformation.

Example 5

Construction of a Plant Transformation Vector for 6GP3 Thermophilic Fulanase

An expression cassette for expression in cytoplasmic reticulum of maize endosperm of 6GP3 thermophilic pullulanase was constructed as follows:

pNOV7005 (SEQ ID NOS: 24 and 25) comprises an N-terminal signal sequence, a corn γ-agent fused to synthetic 6GP3 pullulanase, with C-terminal addition of the sequence SEKDEL for targeting to ER and retention in ER. The amino acid peptide SEKDEL was fused to the C-terminus of the enzyme using PCR using a primer designed to amplify the synthetic gene and simultaneously add six amino acids to the C-terminus of the protein. This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

Example 6

Construction of plant transformation vector for malA

Superthermophilic α-glucosidase

Expression cassettes with various targeting signals were constructed to express sulfolobus solfataricus malA hyperthermia a-glucosidase in corn endosperm as follows.

pNOV4831 (SEQ ID NO: 26) is a corn γ-agent N-terminal signal fused to synthetic malA α-glucosidase, with C-terminal addition of the sequence SEKDEL for targeting to cytoplasmic network (ER) and retention in ER Sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) (Munro and Pelham, 1987). This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4839 (SEQ ID NO: 27) comprises an N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) which is a corn γ-agent fused to synthetic MalA α-glucosidase for targeting to cytoplasmic networks and secretion of apoplasts (Torrent et al., 1997). This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4837 contains a N-terminal signal sequence (MRVLLVALALLALAASATS), a corn γ-agent fused to synthetic malA α-glucosidase, with C-terminal addition of the sequence SEKDEL for targeting to ER and retention in ER. Include. This fusion was cloned behind the corn ADP-gpp promoter for specific expression in endocrine. The amino acid sequence for this clone is identical to the sequence of pNOV4831 (SEQ ID NO: 26).

Example 7

Construction of Plant Transformation Vectors for Superthermophilic Turmotoga Marittima and Turmotoga niapolitana Glucose Isomerase

Expression cassettes with various targeting signals were constructed to express Turmotoga maritima and Turmotoga niapolitana or hyperthermic glucose isomerase in corn endosperm as follows.

pNOV4832 (SEQ ID NO: 28) is a N-terminal signal sequence, a corn γ-agent fused to maritima glucose isomerase by Synthetic Turmoto with C-terminal addition of the sequence SEKDEL for targeting to ER and retention in the ER. MRVLLVALALLALAASATS) (SEQ ID NO: 17). This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4833 (SEQ ID NO: 29) is a N-terminal signal sequence that is a corn γ-agent fused to niapolitana or glucose isomerase by synthetic turmoto with C-terminal addition of the sequence SEKDEL for targeting to ER and retention in the ER. (MRVLLVALALLALAASATS) (SEQ ID NO: 17). This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4840 (SEQ ID NO: 30) shows the N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17), a corn γ-agent fused with synthetic turmoto niapolitana or glucose isomerase for targeting to ER and secretion of apoplasts. (See Torrent et al. 1997). This fusion was cloned behind the corn ADP-gpp promoter for specific expression in endocrine.

pNOV4838 is an N-terminal signal sequence (MRVLLVALALLALAASATS), a corn γ-agent fused to niapolitana or glucose isomerase, with C-terminal addition of the sequence SEKDEL for targeting to ER and retention in the ER Number 17). This fusion was cloned behind the corn ADP-gpp promoter for specific expression in endocrine. The amino acid sequence for this clone is identical to the sequence of pNOV4833 (SEQ ID NO: 29).

Example 8

Construction of Plant Transformation Vectors for the Expression of Superthermoglucanase EglA

pNOV4800 (SEQ ID NO: 58) comprises a barley alpha amylase AMY32b signal sequence (MGKNGNLCCFSLLLLLLAGLASGHQ) (SEQ ID NO: 31) fused with an EglA mature protein sequence for localization of apoplasts. This fusion was cloned behind a γ-zein promoter for specific expression in endocrine.

Example 9

Construction of Plant Transformation Vectors for Expression of Multiple Superthermophilic Enzymes

pNOV4841 includes a dual gene construct of the 797GL3 α-amylase fusion and the 6GP3 pullulase fusion. Both the 797GL3 fusions (SEQ ID NO: 33) and the 6GP3 fusions (SEQ ID NO: 34) include the N-terminal signal sequence and the SEKDEL sequence, which are corn γ-agents for targeting to and retention in the ER. Each fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4842 includes a dual gene construct of the 797GL3 α-amylase fusion and malA α-glucosidase fusion. Both the 797GL3 fusion polypeptide (SEQ ID NO: 35) and the malA α-glucosidase fusion polypeptide (SEQ ID NO: 36) are N-terminal signal sequences and SEKDEL sequences, which are corn γ-agents for targeting to and retention in the ER It includes. Each fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4843 includes a dual gene construct of the 797GL3 α-amylase fusion and malA α-glucosidase fusion. Both the 797GL3 fusion and the α-glucosidase fusion polypeptide (SEQ ID NO: 36) include a N-terminal signal sequence and a SEKDEL sequence, a corn γ-agent for targeting to and retention in the ER. The 797GL3 fusion was cloned behind the maize γ-zein promoter for specific expression in endocrine. The amino acid sequence of the 797GL3 fusion and malA fusion is identical to the amino acid sequence of pNOV4842 (SEQ ID NOs: 35 and 36, respectively).

pNOV4844 includes triple gene constructs of the 797GL3 α-amylase fusion, 6GP3 pullulase fusion and malA α-glucosidase fusion. 797GL3, malA and 6GP3 all comprise a corn γ-agent N-terminal signal sequence and SEKDEL sequence for targeting to ER and retention in ER. The 797GL3 and malA fusions were cloned behind two separate corn γ-zein promoters and the 6GP3 fusions were cloned behind the corn ADP-gpp promoters for specific expression in endocrine. The amino acid sequence for the 797GL3 and malA fusions is identical to the amino acid sequence of pNOV4842 (SEQ ID NOs 35 and 36, respectively). The amino acid sequence for the 6GP3 fusion is identical to the amino acid sequence of the 6GP3 fusion in pNOV4841 (SEQ ID NO: 34).

All expression cassettes set forth in this and subsequent examples were transferred to binary vector pNOV2117 for transformation into maize via Agrobacterium infection. pNOV2117 contains a phosphomannose isomerase (PMI) gene that enables the selection of transformed cells with mannose. pNOV2117 is a binary vector with both pVS1 and ColE1 replication origins. This vector is a native VirG gene from pAD1289 (Hansen, G., et al., PNAS USA 91: 7603-7607 (1994)) and spectinomycin resistance from Tn7. Contains the genes. Clones the corn ubiquitin promoter, the PMI coding region and the nopaline synthase terminator of pNOV117 into the multi-linker between the right and left boundaries (Negrotto, D., et al., Plant Cell Reports 19: 798-803 (2000)]. Transformed corn plants were self-pollinated or crossbred, and analytical seeds were harvested. Combinations of different enzymes can be prepared by heterogeneous crossing of plants expressing individual enzymes or by transforming the plants with one of a multi-gene cassette.

Example 10

Construction of Bacterial and Pichia Expression Vectors

Expression cassettes were constructed as follows to express hyperthermia a-glucosidase and glucose isomerase in Pichia or bacteria:

pNOV4829 (SEQ ID NOs: 37 and 38) contains a synthetic Turmotoga maritima glucose isomerase fusion gene with an ER retention signal in the bacterial expression vector pET29a. The glucose isomerase gene was cloned into the NcoI and SacI positions of pET29a, which resulted in the addition of an N-terminal S-tag for protein purification.

pNOV4830 (SEQ ID NOs: 39 and 40) comprises a synthetic turmotoga niapolitana or glucose isomerase fusion with an ER retention signal in the bacterial expression vector pET29a. The glucose isomerase gene was cloned into the NcoI and SacI positions of pET29a, which resulted in the addition of an N-terminal S-tag for protein purification.

pNOV4835 (SEQ ID NOs: 41 and 42) contains a synthetic Tomoto marimarima glucose isomerase gene cloned into the BamHI and EcoRI positions of the bacterial expression vector pET28C. This results in the fusion of the N-terminus of the glucose isomerase and His-tag (for protein purification).

pNOV4836 (SEQ ID NOs: 43 and 44) contains a synthetic Tomotoga niapolitana or glucose isomerase gene cloned into the BamHI and EcoRI positions of the bacterial expression vector pET28C. This results in the fusion of the N-terminus of the glucose isomerase and His-tag (for protein purification).

Example 11

Transformation of immature corn embryos was performed basically as described in Negrotto et al., Plant Cell Reports 19: 798-803. For this example, all media components were as described in Negrotto et al., Supra. However, the various media components described in this document can be replaced.

A. Transformation Plasmids and Selectable Markers

The gene to be used for transformation was cloned into a vector suitable for maize transformation. The vector used in this example included phosphomannose isomerase (PMI) for the sex of the transformants (Negrotto et al., Plant Cell Reports 19: 798-803).

B. Preparation of Agrobacterium Lithium Passenger

Agrobacterium strain LBA4404 (pSB1) comprising a plant transgenic plasmid was isolated from YEP (yeast extract (5 g / L), peptone (10 g / L), NaCl (5 g / L), 15 g / L agar, pH 6.8) solid. The medium was incubated at 28 ° C. for 2-4 days. Approximately 0.8 × 10 ⁹ Agrobacterium were suspended in LS-inf medium with 100 μM As added (Negrotto et al., Plant Cell Reports 19: 798-803). Bacteria were pre-induced in the medium for 30 to 60 minutes.

C. Inoculation

Immature embryos of A188 or other suitable genotype were cut from 8-12 day old ears and placed in liquid LS-inf + 100 μM As. Embryos were washed once with fresh infection medium. Agrobacterium solution was then added and the embryo was stirred vigorously for 30 seconds and allowed to settle with bacteria for 5 minutes. Embryos were then transferred to the LSA medium and incubated for 2 to 3 days in the dark. Subsequently, 20-25 embryos per petri plate were transferred to LSDc medium with Cytoxim (250 mg / l) and silver nitrate (1.6 mg / l) and incubated for 10 days in the dark at 28 ° C.

D. Selection of Transformed Cells and Regeneration of Transformed Plants

Immature embryos producing embryogenic callus were transferred to LSD1M0.5S medium. The cultures were selected from the medium for 6 weeks in three weeks of subculture. Surviving callus was transferred to Reg1 medium with mannose. After incubation (16 hours light / 8 hours cancer tissue) under light source, green tissue was then transferred to Reg2 medium without growth regulator and incubated for 1-2 weeks. Seedlings were transferred to a Magenta GA-7 box containing Reg3 medium (Magenta Corp, Chicago, Ill.) And cultured under light source. After two to three weeks, plants were examined by PCR for the presence of PMI genes and other genes of interest. Plants positive in the PCR assay were transferred to the greenhouse.

Example 12

Analysis of T1 Seeds from Corn Plants Expressing α-amylase Targeted with Apoplast or ER

T1 seeds from self-pollinated corn plants transformed with pNOV6200 or pNOV6201 as described in Example 4 were obtained. Starch accumulation in these grains has been shown to be normal based on visual inspection and conventional dyeing of starch using iodine solution prior to any exposure to high temperatures. Immature grains were excised and the purified endosperm was individually placed in the microcentrifuge tube and infiltrated into 200 μl of 50 mM NaPO ₄ buffer. The tube was placed in an 85 ° C. water bath for 20 minutes and then cooled on ice. 20 microliters of 1% iodine solution was added to each tube and mixed. Approximately 25% of the isolated grains were normally stained for starch. The remaining 75% failed to stain, indicating that the starch degraded to low molecular weight sugars that were not stained with iodine. It was found that T1 grains of pNOV6200 and pNOV6201 self-hydrolyzed corn starch. There was no observable decrease in starch after incubation at 37 ° C.

In addition, expression of amylase was analyzed by PAGE / Kumasi staining after separating the superthermophilic protein fraction from endocrine. An isolated protein band of suitable molecular weight (50 kD) was observed. These samples were subjected to the α-amylase assay using commercially available dyed amylose (AMYLAZYME from Amyzyme, Ireland). High levels of superthermophilic amylase activity correlated with the presence of 50kD protein.

In addition, it has been found that when the enzyme is in direct contact with starch granules, the starch in the grains of many transgenic maizes expressing superthermophilic α-amylase, targeted to starch and sufficiently activated at ambient temperature is found to be hydrolyzed. Of the 80 strains with superthermophilic α-amylase targeted to starch, four strains of starch accumulation in the grains were identified. Three of these strains were analyzed for heat-resistant α-amylase activity using colorimetric amylazyme assay (Megazyme). The amylase enzyme assay showed that when these three strains were treated under suitable humidity and heating conditions, the starch was hydrolyzed, which indicates the presence of suitable levels of α-amylase to promote auto-hydrolysis of the starches made from these strains. It was to represent.

T1 seeds from multiple independent strains of both pNOV6200 and pNOV6201 transformants were obtained. Individual grains from each strain were excised and purified endosperm was homogenized individually in 300 μl of 50 mM NaPO ₄ buffer. An aliquot of the endosperm suspension was analyzed for α-amylase activity at 85 ° C. About 80% of the strains were isolated for superthermic activity (see Figures 1A, 1B and 2).

The grains from wild type plants or plants transformed with pNOV6201 were heated at 100 ° C. for 1, 2, 3, or 6 hours and then stained for starch with iodine solution. After 3 or 6 hours, respectively, little or no starch was detected in mature grains. Thus, when incubated at high temperature, starch in mature grains from transgenic maize expressing hyperthermic amylase targeted with cytoplasmic nets was hydrolyzed.

In another embodiment, partially purified starch from mature T kernels from pNOV6201 plants soaked at 50 ° C. for 16 hours was hydrolyzed at 85 ° C. for 5 minutes. This demonstrates that after grinding the grains, the α-amylase targeted by cytoplasmic nets binds to the starch and can hydrolyze the starch upon heating. Iodine staining showed that starch remained intact in mature seeds after soaking at 50 ° C. for 16 hours.

In another experiment, mature grains isolated from plants transformed with pNOV6201 were heated at 95 ° C. for 16 hours and then dried. In seeds expressing superthermophilic α-amylase, hydrolysis of starch to sugar results in a wrinkled appearance after drying.

Example 13

Analysis of T1 Seeds from Corn Plants Expressing Targeted α-amylase

T1 seeds from self-pollinated corn plants transformed with pNOV4029 or pNOV4031 as described in Example 4 were obtained. Starch accumulation in the grains of these strains was clearly not normal. All strains with some differences in severity were separated for very low starch phenotype or no starch phenotype. Prior to exposure to high temperatures, endosperm was purified from immature grains that were only slightly stained with iodine. After 20 minutes at 85 ° C., no staining occurred. When the ear was dried, the grain was wrinkled. This particular amylase, when in direct contact with the granules, apparently had sufficient activity to hydrolyze starch at greenhouse temperatures.

Example 14

Fermentation of Grains from Corn Plants Expressing α-amylase

At various liquefaction times, 100% transgenic grains 85 ° C. to 95 ° C.

Transgenic maize (pNOV6201) containing heat-resistant α-amylase that ferments well without addition of exogenous α-amylase requires much less liquefaction time and results in more complete solubilization of starch. Laboratory scale fermentations were performed according to a protocol having the following steps (described in detail below): 1) grinding, 2) moisture analysis, 3) grinding corn, water, backset and α-amylase. Preparation of Slurry Containing, 4) Liquefaction and 5) Simultaneous Saccharification and Fermentation (SSF). In this example, the temperature and time of the liquefaction step varied as described below. Transgenic maize was liquefied with or without exogenous α-amylase and ethanol production performance was compared to control corn treated with commercially available α-amylase.

The transgenic maize used in this example was prepared according to the procedure set forth in Example 4 using a vector comprising the α-amylase gene and a PMI marker, ie pNOV6201. The transgenic corn was prepared by pollinating a commercially available hybrid (N3030BT) as a pollen from a transformant expressing a high concentration of heat-resistant α-amylase. The corn was dried to 11% moisture and stored at room temperature. The α-amylase content of the transformed corn powder was 95 units / g, where 1 unit of enzyme resulted in a 1 micromole decrease per minute from the corn powder at 85 ° C. in pH 6.0 MES buffer. The control corn used was yellow dent corn, which is known to perform well with ethanol production.

1) Polishing: Transformed corn (1180 g) was ground with a Perten 3100 hammer mill equipped with a 2.0 mm screen. Control corn was washed thoroughly to prevent contamination by transgenic corn and then ground in the same mill.

2) Moisture Analysis: Transgenic and control corn samples (20 g) were weighed by aluminum weight boat and heated at 100 ° C. for 4 hours. The sample was weighed again and the water content was calculated from the weight loss. The water content of the transformed powder was 9.26%; The water content of the control powder was 12.54%.

3) Slurry Preparation: The composition of the slurry was designed to yield a mash with 36% solids at the start of SSF. The control sample was prepared in a 100 ml plastic bottle and contained 21.50 g of control corn powder, 23 ml of deionized water, 6.0 ml of a backset (8 wt% solids), and 0.30 ml of commercial α-amylase diluted 1/50 with water. . The α-amylase dose was chosen as an example of industrial use. When assayed under the above conditions for the assay of transformed α-amylase, the control α-amylase dose was 2 U / g corn powder. pH was titrated to 6.0 by addition of ammonium hydroxide. Transformed samples were prepared in the same manner but contained 20 g of corn powder due to the low water content of the transformed powder. Slurry of the transformed powder was prepared using the same dose of α-amylase or without exogenous α-amylase as control sample.

4) Liquefaction: A bottle containing a slurry of transgenic corn powder was infiltrated in a water bath at 85 ° C. or 95 ° C. for 5, 15, 30, 45 or 60 minutes. The control slurry was incubated at 85 ° C. for 60 minutes. During the high temperature incubation, the slurry was manually vigorously mixed every 5 minutes. After the hot step, the slurry was cooled on ice.

5) Simultaneous Glycation and Fermentation: Mash prepared by liquefaction was prepared using glucoamylase (0.65 ml of a commercially available L-400 glucoamylase 0.65 ml), protease (0.60 ml of commercial protease 1,000-fold dilution), 0.2 mg lactoside & urea. (0.85 ml of a 10-fold dilution of 50% urea solution). 100 ml containing mesh were punctured with a bottle cap to allow CO ₂ to dissipate. The mash was then incubated with yeast (1.44 ml) and incubated in a water bath set to 90F. After 24 hours of fermentation, the temperature was lowered to 86F and set to 82F at 48 hours.

Yeast for inoculation is yeast (0.12 g), maltodextrin 70 g, water 230 ml, 100 ml of bag set, glucoamylase (0.88 ml of 10-fold dilution of commercial glucoamylase), protease (1.76 ml of 100-fold dilution of commercially available enzymes), urea ( 1.07 g), a mixture containing penicillin (0.67 mg) and zinc sulfate (0.13 g) was prepared and grown. Proliferation culture was started the day before it was needed and incubated with mixing at 90 ° F.

At 24, 48 & 72 hours, samples were taken from each fermentation vessel, filtered through a 0.2 μm filter and analyzed by ethanol & sugar HPLC. At 72 hours, samples were analyzed for total dissolved solids and residual starch.

HPLC analysis was performed on a binary gradient system equipped with a refractive index detector, column heater & Bio-Rad Aminex HPX-87G column. The system was equilibrated to 1 ml / min with 0.005 H ₂ SO ₄ in water. Column temperature was 50 ° C. Sample injection volume was 5 μl; Eluted in the same solvent. RI reactions were measured by injecting known standards. Both ethanol and glucose were measured at each infusion.

Residual starch was measured as follows. Samples and standards were dried at 50 ° C. in an oven and then ground to powder in a sample mill. The powder (0.2 g) was weighed into a 15 ml graduated centrifuge tube. The powder was washed three times with 10 ml aqueous ethanol (80%), then centrifuged and the supernatant removed. DMSO (2.0 ml) was added to the pellet, followed by 3.0 ml of heat resistant alpha-amylase (300 units) in MOPS buffer. After vigorous mixing, the tubes were incubated in water at 85 ° C. for 60 minutes. During incubation, the nubs were mixed four times. The sample was cooled and 4.0 ml of sodium acetate buffer (200 mM, pH 4.5) was added followed by 0.1 ml of glucoamylase (20 U). Samples were incubated at 50 ° C. for 2 hours, stirred and centrifuged at 3,500 rpm for 5 minutes. The supernatant was filtered through a 0.2 μm filter and analyzed for glucose by the HPLC method described above. An injection size of 50 μl was used for samples with low residual starch (<20% in solids).

Results: Transgenic maize fermented well without the addition of α-amylase. The yield of ethanol at 72 hours was essentially the same with or without exogenous α-amylase as shown in Table I. These data also when higher the liquid temperature is achieved a high yield of the ethanol, the enzyme of the invention as expressed in the transgenic corn Bacillus Li kwepae when Enschede (Bacillus liquefaciens ) has activity at higher temperatures than other commercially available enzymes such as α-amylase.

Liquefaction temperature ℃ Liquefaction time minutes Exogenous α-amylase Number of iterations Average ethanol% v / v Standard deviation% v / v 85 60 Yes 4 17.53 0.18 85 60 no 4 17.78 0.27 95 60 Yes 2 18.22 ND 95 60 no 2 18.25 ND

In the case of various liquefaction times, the liquefaction time required for efficient ethanol production was much less than the time required by conventional procedures. Figure 3 shows that the ethanol yield at 72 hours of fermentation changed little from 15 minutes to 60 minutes of liquefaction. In addition, liquefaction at 95 ° C. provided more ethanol at each time point than liquefaction at 85 ° C. These observations demonstrate the method improvements achieved by the use of superthermophilic enzymes.

The control corn provided a higher final ethanol yield than the transformed corn, but the control was chosen because the fermentation was very good. In contrast, transgenic maize has a genetic background chosen to promote transformation. The introduction of the α-amylase-type into good maize embryos by well known breeding techniques will eliminate this difference.

Investigation of residual starch levels of beer prepared at 72 hours (FIG. 4) results in a significant improvement in the production of starch transgenic α-amylase usable for fermentation, indicating that much less starch remains after fermentation.

Using both ethanol levels and residual starch levels, the optimum liquefaction time was 15 minutes at 95 ° C and 30 minutes at 85 ° C. In this experiment, these times were the total time the fermentation vessel was left in the water bath, thus including the time during which the temperature of the sample increased from room temperature to 85 ° C or 95 ° C. Shorter liquefaction times may be optimal for large scale industrial processes where equipment such as jet cookers heat up the mesh rapidly. Conventional industrial liquefaction processes require storage tanks that allow the mesh to be incubated at high temperatures for at least one hour. The present invention will eliminate this storage tank need and increase the productivity of the liquefaction apparatus.

One important function of α-amylase during fermentation is to reduce the viscosity of the mash. At all time points, the sample containing the transformed corn powder was noticeably less viscous than the control sample. In addition, the transformed samples did not appear to go through the gelatinous phase observed using all control samples; Gelatinization typically occurs when cooking corn slurries. Thus, the α-amylase distributed in fragments of the endosperm provides beneficial physical properties to the mash during cooking by preventing the formation of large gels that slow down diffusion and increase mesh mixing and pumping energy costs.

High doses of α-amylase in transgenic maize may also contribute to the beneficial properties of the transgenic mesh. At 85 ° C., the α-amylase activity of the transgenic maize was several times greater than the dose of exogenous α-amylase used in the control. The latter was chosen as an example of a commercial use ratio.

Example 15

Effective function of transgenic maize when mixed with control maize

The transformed corn powder was mixed with the control corn powder at various concentrations of 5% to 100% transformed corn powder. These were treated as described in Example 14. Mash containing transformed expressed α-amylase was liquefied at 85 ° C. for 30 minutes or at 95 ° C. for 15 minutes; Control meshes were prepared as described in Example 14 and liquefied at 85 ° C. for 30 or 60 minutes (once each) or at 95 ° C. for 15 minutes or 60 minutes (once each).

Data for ethanol and residual starch at 48 and 72 hours are provided in Table 2. Ethanol concentration at 48 hours is graphically represented in FIG. 5; Residual starch measurements are shown in FIG. 6. These data indicate that the transgenic expressed heat resistant α-amylase provides very good performance even in ethanol production when the transformed grain is only a small share (as low as 5%) of the total grain of the mesh. The data also show that when the transgenic grains contain at least 40% of the total grains, the residual starch is significantly less than in the control mesh.

Control sample. The value is the average of two measurements.

Example 16

Ethanol production as a function of liquefied pH when transgenic maize is used at a rate of 1.5% to 12% of total maize

Since the transformed corn is well fermented at a level of 5 to 10% of the total corn, an additional series of fermentations were performed in which the transformed corn contained 1.5 to 12% of the total corn. The pH varied from 6.4 to 5.2 and the α-amylase expressed in transgenic maize was optimized for activity at lower pH than is commonly used in industry.

This experiment was performed as described in Example 15 except for the following.

1) The transformed powder was mixed with the control powder at a percentage of total anhydrous weight ranging from 1.5% to 12.0%.

2) Control corn was N3030BT more similar to transgenic corn than the control used in Examples 14 and 15.

3) No exogenous α-amylase was added to the sample containing the transformed powder.

4) Samples were titrated to pH 5.2, 5.6, 6.0 or 6.4 before liquefaction. Five or more samples fixed in the range of 0% transgenic maize powder to 12% transgenic maize powder were prepared for each pH.

5) Liquefaction for all samples was carried out at 85 ° C. for 60 minutes.

The change in ethanol content as a function of fermentation time is shown in FIG. 7. This figure shows data obtained from a sample containing 3% transgenic maize. At lower pH, fermentation proceeds more rapidly at pH 6.0 and above; Similar action was observed in samples with different doses of transgenic grains. The pH profile of the activity of the transforming enzyme combined with high levels of expression will result in lower pH liquefaction being faster than fermentation and thus higher than is possible in conventional pH 6.0 procedures.

Ethanol yield at 72 hours is shown in FIG. As you can see, based on the yield of ethanol, the results showed some dependence on the amount of transgenic grains included in the sample. Thus, the grains contain abundant amylase to promote fermentation production of ethanol. It also proves that low pH liquefaction results in high ethanol yield.

The viscosity of the sample after liquefaction was observed, and at pH 6.0, 6% transgenic grains were observed to be sufficient for a suitable viscosity reduction. At pH 5.2 and 5.6, the viscosity was equivalent to that of the control in 12% transgenic grains, but not at the lower percentage of transgenic grains.

Example 17

Preparation of fructose from corn powder using thermophilic enzyme

Corn, which expresses superthermotropic α-amylase, 797GL3, has been shown to promote the production of fructose when mixed with α-glucosidase (MalA) and xylose isomerase (XylA).

Amylase powder was prepared by grinding seeds from pNOV6201 transformed plants expressing 797GL3 with powder in Kleco cells. Non-transformed corn kernels were ground in the same manner to prepare a control powder.

α-glucosidase, MalA (from S. solfataricus) Expressed in E. coli. The harvested bacteria were suspended in 50 mM potassium phosphate buffer at pH 7.0 containing 1 mM 4- (2-aminoethyl) benzenesulfonyl fluoride and then lysed in a high pressure cell crusher. The lysates were centrifuged at 23,000 × g for 15 minutes at 4 ° C. The supernatant solution was removed, heated at 70 ° C. for 10 minutes, cooled on ice for 10 minutes and then centrifuged at 34,000 × g for 30 minutes at 4 ° C. The supernatant solution was removed and MalA was concentrated twice in a centricon 10 instrument. The filtrate of Centricon 10 stage was preserved for use as a negative control for MalA.

Xylose (glucose) isomerase. Tea from E. coli. It was prepared by expressing the niapolitana xylA gene. The bacteria were suspended in 100 mM sodium phosphate pH 7.0 and lysed through a high pressure cell disrupter. After precipitation of cell debris, the extract was heated at 80 ° C. for 10 minutes and then centrifuged. The supernatant solution contained XylA enzyme activity. Co-vector control extracts were prepared simultaneously with the XylA extract.

Corn powder (60 mg per sample) was added to buffer and E. coli. Mixed with extract from E. coli. As shown in Table 3, the samples contained amylose corn powder (amylase) or control corn powder (control), MalA extract (+) or filtrate (-) 50 μl, and XylA extract (+) or empty vector control (-) 20 Μl was contained. All samples also contained 230 μl 50 mM MOPS, 10 mM MgSO ₄ and 1 mM CoCl ₂ ; The pH of the buffer was 7.0 at room temperature.

Samples were incubated at 85 ° C. for 18 hours. At the end of the incubation time, the sample was diluted with 0.9 ml of water at 85 ° C. and centrifuged to remove insoluble matter. The supernatant fractions were then filtered through a Cintricon 3 ultrafiltration apparatus and analyzed by HPLC using an ELSD detector.

The gradient HPLC system is equipped with an Astec polymer amino column, 5 μm particle size, 250 × 4.6 mm and an Alltech ELSD 2000 detector. The system was pre-equilibrated with a water: acetonitrile 15:85 mixture. The flow rate was 1 ml / min. Initial conditions were maintained for 5 minutes after injection, followed by 20 minutes in a 50:50 water: acetonitrile gradient followed by 10 minutes in the same solvent. The system was washed for 20 minutes with 80:20 water: acetonitrile and then re-equilibrated with starting solvent. Fructose eluted at 5.8 minutes and glucose eluted at 8.7 minutes.

HPLC results also showed the presence of large amounts of maltooligosaccharides in all samples containing α-amylase. These results demonstrate that three thermophilic enzymes can work together for the production of fructose from corn powder at high temperatures.

Example 18

Amylase Powder with Isomerase

In another example, amylase powder was mixed with purified MalA and two bacterial xylose isomerases, respectively: T. Enzyme named BD8037 obtained from XylA of Marittima and Snail. Amylase powder was prepared as described in Example 18.

S with 6His tablet tag. Solfataricus MalA. It was expressed in E. coli. Cell lysates were prepared as described in Example 18, and then purified using nickel affinity resin (Probond, Invitrogen) and clearly until homogeneous according to manufacturer's instructions for natural protein purification.

Tee with S tag and ER retention signal added. This is Marittima XylA. T. expression in E. coli and as described in Example 18. It was prepared by the same method as niapolitana or XylA.

Xylose isomerase BD8037 was obtained as lyophilized powder and resuspended in 0.4 × of original volume of water.

Amylase corn powder was mixed with enzyme solution added water or buffer. All reactions contained 60 mg amylase powder and 600 μl total liquid. One set of reactions was buffered at room temperature with 50 mM MOPS at pH 7.0 added 10 mM MgSO ₄ and 1 mM CoCl ₂ ; In the second set of reactants, the metal-containing buffer was replaced with water. The amount of isomerase enzyme varied as shown in Table 4. All reactions were incubated at 90 ° C. for 2 hours. The reaction supernatant fractions were prepared by centrifugation. The pellet was washed with additional 600 μl of H ₂ O and recentrifuged. Supernatant fractions from each reaction were combined, filtered through Centricon 10 as described in Example 17 and analyzed by HPLC using an ELSD detector. The amount of glucose and fructose observed is shown graphically in FIG. 15.

Sample Amylase powder Mal A Isomerase One 60mg + none 2 60mg + tea. Marittima, 100 μl 3 60mg + tea. Marittima, 10 μl 4 60mg + tea. Marittima, 2 μl 5 60mg + BD8037, 100 μl 7 60mg + BD8037, 2 μl C 60mg none none

If α-amylase and α-glucosidase are present in the reaction, fructose was prepared from corn powder in a dose-dependent manner, with each isomerase. These results demonstrate that grain-expressed amylase 797GL3 can work with MalA and various different thermophilic isomerases with or without metal ions added to produce fructose from corn powder at high temperatures. In the presence of added divalent metal ions, isomerase can achieve an expected fructose: glucose equilibrium of approximately 55% fructose at 90 ° C. This would be an improvement over current methods using mesogenic isomerase, which requires chromatographic separation to increase the fructose concentration.

Example 19

Expression of pullulanase in maize

Transgenic plants homologous to pNOV7013 or pNOV7005 were cross-crossed to produce transgenic corn seeds expressing both 797GL3 α-amylase and 6GP3 pullulase.

T1 or T2 seeds from self-pollinated corn plants transformed with pNOV 7005 or pNOV 4093 were obtained. pNOV4093 is a fusion of maize optimized synthetic gene for 6GP3 (SEQ ID NOs: 3, 4) with starch targeting sequence (SEQ ID NOs: 7, 8) for localization of the fusion protein into starch. The fusion protein was placed under the control of the ADPgpp promoter (SEQ ID NO: 11) for specific expression in endocrine. The pNOV7005 construct targeted the expression of pullulanase in endocytic cytoplasmic reticulum. Localization of this enzyme in the ER allows for the normal accumulation of starch in the kernel. Prior to any exposure to high temperatures, normal staining for starch with iodine solution was also observed.

As described in the case of α-amylase, expression of starul-targeted pullulanase (pNOV4093) resulted in abnormal starch accumulation in the kernel. When the corn-ears were dried, the grains were wrinkled. Clearly, the thermophilic pullulanase is sufficiently active at low temperatures and hydrolyzes starch when brought into direct contact with starch granules in the seed endosperm.

Enzyme Preparation or Extraction of Enzymes from Corn Powder: The pullulanase enzyme was extracted from the seed by grinding the transformed seed with a Kleco polisher, followed by incubation of the powder in 50 mM NaOAc buffer at pH 5.55 for 1 hour at room temperature with constant stirring. . The incubated mixture was then spun at 14000 rpm for 15 minutes. Supernatant was used as the enzyme source.

Pullulanase Assay: The assay reaction was performed in 96 well plates. The enzyme extracted from the corn powder (100 μl) was diluted 10-fold with 900 μl of 50 mM NaOAc buffer at pH 5.5 containing 40 mM CaCl ₂ . Stir the mixture vigorously, add 1 tablet of Limit-Dextrizyme (Megazyme's Azurin-crosslinked-Pullulan) to each reaction mixture and incubate at 75 ° C. for 30 minutes (or as mentioned). Treated. At the end of incubation, the reaction mixture was spun at 3500 rpm for 15 minutes. The supernatant was diluted 5 fold and transferred to a 96-well flat plate for absorbance measurement at 590 nm. Hydrolysis of the azurin-crosslinked- pullulan substrate by pullulanase produces water soluble stained fragments and their release rate (measured by increase in absorbance at 590 nm) is directly related to enzyme activity.

9 shows analysis of T2 seeds from different events transformed with pNOV7005. High expression of pullulanase activity compared to non-transforming controls can be detected in a number of events.

PH 5.5 containing 40 mM CaCl ₂ to determine the amount of dry corn powder (˜100 μg) from transformation (expressing pullulanase or amylase or both) and / or control (non-transformation). 1000 μl 50 mM NaOAc was added. The reaction mixture was vigorously stirred and then incubated for 1 hour on a stirrer. The enzymatic reaction was initiated by transferring the incubation mixture to a high temperature (75 ° C., optimal reaction temperature for pullulanase or as mentioned in the figure) for the time indicated in the figure. The reaction was terminated by placing them on ice and cooling. The reaction mixture was then centrifuged at 14000 rpm for 10 minutes. Aliquots of the supernatant (100 μl) were diluted 3-fold and filtered through a 0.2 micron filter for HPLC analysis.

The sample was analyzed by HPLC using the following conditions:

Column: Alletch Prevail Carbohydrate ES 5 Micron 250 X 4.6mm

Detector: Alltech ELSD 2000

Pump: Gilson 322

Injector: Gilson 215 Injector / Dilutor

Solvent: HPLC gradient acetonitrile (Fisher Scientific) and water (purified by Waters Millipore system)

Gradient used for oligosaccharides of low polymerization degree (DP 1 to 15)

Time% Water% Acetonitrile

0 15 85

5 15 85

25 50 50

35 50 50

36 80 20

55 80 20

56 15 85

76 15 85

Gradient used for monosaccharides of high polymerization degree (DP 20 to 100 and above)

Time% Water% Acetonitrile

0 35 65

60 85 15

70 85 15

85 35 65

100 35 65

Systems Used for Data Analysis: Gilson Unipoint Software System Edition 3.2

10A and 10B show HPLC analysis of hydrolysis products prepared by pullulanase expressed from starch in transgenic corn powder. The powder of pullulanase expressing corn is incubated for 30 minutes in 75 ° C. reaction buffer to produce heavy chain oligosaccharides (DP ˜10-30) and shorter amylose chains (DP ˜100-200) from corn starch. The figure also shows the dependence of pullulanase activity in the presence of calcium ions.

Pullulanase expressing transformed corn can be used to prepare debranched (α1-6 binding truncated) modified starch / dextrins, and thus will have high concentrations of amylose / chain dextrins. In addition, the chain length distribution of amylose / dextrin produced by pullulanase will vary depending on the type of starch used (eg, waxy, high amylose, etc.), and the properties of the modified starch / dextrin will also vary.

Hydrolysis of α1-6 bonds was also demonstrated using pullulan as substrate. Pullulanase isolated from the corn powder effectively hydrolyzed pullulan. HPLC analysis of the product prepared at the end of the incubation (as described) indicated the preparation of maltotriose as expected due to the hydrolysis of α1-6 bonds in the pullulan molecule by enzymes from corn.

Example 20

Expression of pullulanase in maize

Expression of 6gp3 pullulanase was further extracted from corn powder and further analyzed by PAGE and Coomassie staining. Seeds were ground in a Kleco grinder for 30 seconds to produce corn powder. The enzyme was extracted from about 150 mg of powder using 1 ml of 50 mM NaOAc buffer at pH 5.5. The mixture was stirred vigorously and incubated on a stirrer at RT for 1 hour and then incubated at 70 ° C. for an additional 15 minutes. The mixture was then spin down (14000 rpm for 15 minutes at RT) and the supernatant was used for SDS-PAGE analysis. A protein band of suitable molecular weight (95 kDal) was observed. These samples are subjected to pullulanase assay using commercial dye-conjugation limit dextrin (LIMIT-DEXTRIZYME, Megazyme, Ireland). High levels of hyperthermic pullulanase activity correlate with the presence of 95 kD protein.

Western blot and ELISA analysis of transgenic maize seed also demonstrates the expression of ˜95 kD protein that reacts with antibodies prepared against pullulanase (expressed in E. coli).

Example 21

Increased starch hydrolysis rate and improved yield of short-chain (fermentable) oligosaccharides by the addition of pullulanase expressing corn

Data shown in FIGS. 11A and 11B were calculated from HPLC analysis of starch hydrolysis products from two reaction mixtures as described above. The first reactant, denoted 'amylase' is a mixture of a corn powder sample of α-amylase expressing transformed corn and a non-transformed corn A188 [1: 1 (w), for example, prepared according to the method described in Example 4. / w)]; The second reaction mixture 'amylase + pullulanase' is a mixture of corn powder samples of α-amylase expressing transformed corn and pullulanase expressing transformed corn prepared according to the method described in Example 19 [1: 1 (w / w)]. The results obtained support the benefit of using pullulanase and α-amylase together during starch hydrolysis. This gain is due to increased starch hydrolysis rate (FIG. 11A) and increased yield of fermentable oligosaccharides with low DP (FIG. 11B).

Dextrins (straight or branched chain oligosaccharides) can be prepared using either α-amylase or α-amylase and pullulanase (or any other combination of starch hydrolase) expressed in maize (FIGS. 11A, 11B, 12 and 13A). Depending on the reaction conditions, the type of hydrolase and combinations thereof, and the type of starch used, the composition of the maltodextrin produced and therefore its properties will vary.

FIG. 12 shows the results of experiments performed in the same manner as described in FIG. 11. The different temperature and time schemes performed during incubation of the reactants are shown in this figure. The optimum reaction temperature for pullulanase is 75 ° C. and> 95 ° C. for α-amylase. Thus, the schemes presented follow to provide a region for carrying out catalysis by pullulanase and / or α-amylase at their respective optimum reaction temperatures. From the results shown it can be inferred that the combination of α-amylase and pullulanase performed better hydrolysis of corn starch at the end of the 60 minute incubation period.

HPLC analysis of the starch hydrolysis products from two sets of reaction mixtures at the end of the 30 minute incubation (except using ~ 150 mg corn powder for these reactions) is shown in FIGS. 13A and 13B. . The first set of reactants was incubated at 85 ° C. and the second set was incubated at 95 ° C. For each set, there are two reaction mixtures; The first reactant, represented as 'amylase X pullulanase', contains a powder from transgenic corn (generated with cross pollination) that expresses both α-amylase and pullulase, and the second reactant, represented as 'amylase' A mixture of corn powder samples of α-amylase expressing transformed maize and non-transformed maize A188 in a ratio to obtain the same degree of α-amylase activity as observed in the crossover (amylase X pullulanase). When corn powder samples were incubated at 85 ° C., the total yield of low DP oligosaccharides was higher for α-amylase and pullulanase crosses compared to corn expressing only α-amylase. The incubation temperature of 95 ° C. inactivated the pullulanase enzyme (at least in part), and thus some differences could be observed between 'amylase X pullulanase' and 'amylase'. However, data on both incubation temperatures showed that the amount of glucose produced (late 13B) at the end of the incubation period when the corn powder of the α-amylase and pullulanase cross was compared with the corn expressing only the α-amylase. ) Represents a significant improvement. Thus, the use of maize expressing both α-amylase and pullulanase may be particularly advantageous for processes in which complete hydrolysis of starch to glucose is important.

The above examples provide sufficient evidence that when used in combination with α-amylase, the pullulanase expressed in corn seeds improves the starch hydrolysis process. α1-6 binding specific pullulanase enzyme activity debranches starch more effectively than α-amylase (α-1-4 binding specific enzymes), resulting in branched oligosaccharides (eg, restriction-dextrins, panos) They are generally non-fermentable) and increase the amount of short chain oligosaccharides (easily fermentable with ethanol or the like). Second, fermentation of starch molecules by debranching-promoted pullulanase increases substrate accessibility to α-amylase, increasing the efficiency of α-amylase promoting reactions.

Example 22

Approximately 0.35 μg malA alpha glucose to determine whether 797GL3 alpha amylase and malA alpha-glucosidase can operate under similar pH and temperature conditions to yield increased amounts of glucose beyond that produced by enzyme alone. Starch purified from cedar enzyme (produced in bacteria) from 1% starch and non-transformed corn seeds (control) or 797GL3 transgenic corn seeds (co-purified with alpha amylase with starch in 797GL3 corn seeds) It was added to the containing solution. In addition, purified starch from non-transformed and 797GL3 transgenic corn seeds was added to 1% corn starch without any malA enzyme. The mixture was incubated for 1 hour at 90 ° C., pH 6.0, rotationally strengthened to remove any insoluble material, and the aqueous fraction was analyzed by HPLC for glucose concentration. As shown in FIG. 14, 797GL3 alpha-amylase and malA alpha-glucosidase act at similar pH and temperature to break down starch into glucose. The amount of glucose produced was significantly higher than that produced with enzyme alone.

Example 23

The efficacy of thermophilic anaerobic bacteria glucoamylase for raw starch hydrolysis was measured. As stated in FIG. 15, the hydrolysis conversion of raw starch was tested by identifying water, barley α-amylase (commercially available from Sigma), thermophilic anaerobic glucoamylase and combinations thereof at room temperature and 30 ° C. As shown, the combination of thermophilic anaerobic bacterium glucoamylase and barley α-amylase was able to hydrolyze raw starch to glucose. In addition, the amount of glucose produced by barley amylase and thermophilic anaerobes GA was significantly higher than that produced by enzyme alone.

Example 24

Corn-optimized genes and sequences for raw starch hydrolysis and vector for plant transformation

The enzyme was selected based on the hydrolyzability of the raw starch at temperatures ranging from approximately 20 ° C. to 50 ° C. Corresponding genes or gene fragments were then designed using the corn codons preferred for the construction of synthetic genes as set forth in Example 1.

Aspergillus shrousami α-amylase / glucoamylase fusion polypeptide (without signal sequence) was selected and described in Biosci. Biotech. Biochem., 56: 884-889 (1992); Agric. Biol. Chem. 545: 1905-14 (1990); Biosci. Biotechnol. Biochem. 56: 174-79 (1992), and has the amino acid sequence set forth in SEQ ID NO: 45.

Similarly, Biosci. Biotech. Biochem., 62: 302-308 (1998), was selected as a thermoan bacterium termosacaroticum glucoamylase having the amino acid sequence of SEQ ID NO: 47. Corn-optimized nucleic acids were designed (SEQ ID NO 48).

See Agric. Biol. Chem. (1986) 50, pg 957-964, was selected for Rizopus auria glucoamylase having an amino acid sequence (without signal sequence) (SEQ ID NO: 50). Corn-optimized nucleic acid was designed and represented by SEQ ID NO: 51.

In addition, corn α-amylase was selected and amino acid sequence (SEQ ID NO: 51) and nucleic acid sequence (SEQ ID NO: 52) were obtained from the literature. See, eg, Plant Physiol. 105: 759-760 (1994).

Aspergillus Schlossami α-amylase / glucoamylase fusion polypeptide from a corn-optimized nucleic acid, designed as shown in SEQ ID NO: 46, a turmeric from a corn-optimized nucleic acid, designed as shown in SEQ ID NO: 48 Selected Rizopus auricum glucoamylase and maize having an amino acid sequence (without signal sequence) (SEQ ID NO: 49) from a corn-optimized nucleic acid designed and represented by SEQ ID NO: 50, as shown An expression cassette for expressing α-amylase is constructed.

A plasmid comprising maize γ-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) is fused to a synthetic gene encoding the enzyme. Optionally, the sequence SEKDEL is fused to the C-terminus of the synthetic gene for targeting to and retention at the ER. This fusion is cloned behind the corn γ-zein promoter for specific expression in endocrine in plant transgenic plasmids. The fusion is delivered into maize tissue via Agrobacterium transfection.

Example 25

An expression cassette containing the selected enzyme is constructed to express the enzyme. The plasmid containing the sequence for the raw starch binding site is fused to a synthetic gene encoding the enzyme. The raw starch binding site allows the enzyme fusion to bind to non-gelatinized starch. The raw starch binding site amino acid sequence (SEQ ID NO: 53) was determined based on the literature, and the nucleic acid sequence was corn-optimized to provide the sequence of SEQ ID NO: 54. Corn-optimized nucleic acid sequences are fused to synthetic genes encoding enzymes in plasmids for expression in plants.

Example 26

Construction of Corn-optimized Genes and Vectors for Plant Transformation

Genes and gene fragments were designed using maize preference codons for the construction of synthetic genes as stated in Example 1.

Pyrococcus puriosus EGLA, the superthermophilic endoglucanase amino acid sequence (without signal sequence) was selected and this sequence was identified as in Journal of Bacteriology (1999) 181, pg 284-290. Have an amino acid sequence as set forth in No. 55. Maize-optimized nucleic acid was designed and represented by SEQ ID NO: 56.

Termus Plabus xylose isomerase was selected and has an amino acid sequence as set forth in SEQ ID NO: 57 as described in Applied Biochemistry and Biotechnology 62: 15-27 (1997).

An expression cassette was constructed to form a pyrococcus puriosus EGLA (endoglucanase) from a corn-optimized nucleic acid (SEQ ID NO: 56) and a mutus plaus from a corn-optimized nucleic acid encoding the amino acid sequence of SEQ ID NO: 57. Bus xylose isomerase was expressed. A plasmid comprising maize γ-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) is fused to a synthetic gene encoding the enzyme. Optionally, the sequence SEKDEL is fused to the C-terminus of the synthetic gene for targeting to and retention at the ER. This fusion is cloned behind the corn γ-zein promoter for specific expression in endocrine in plant transgenic plasmids. The fusion is delivered into maize tissue via Agrobacterium transfection.

Example 27

Preparation of Glucose from Corn Powder Using Thermophilic Enzyme Expressed in Corn

Expression of superthermophilic α-amylase, 797GL3 and α-glucosidase (MalA) has been shown to result in production of glucose when mixed with aqueous solutions and incubated at 90 ° C.

Transgenic maize strains expressing MalA enzyme (Note 168A10B, pNOV4831) were identified by measuring α-glucosidase activity as shown by hydrolysis of p-nitrophenyl-α-glucoside.

Amylase powder was prepared by grinding corn kernels from transformed plants expressing 797GL3 into powder in a Kleco grinder. Corn grains from transformed plants expressing MalA were ground to powder in a Kleco grinder to prepare MalA powders. Non-transformed corn kernels were ground in the same manner to prepare a control powder.

The buffer was 50 mM MES buffer at pH 6.0.

Corn Powder Hydrolysis Reaction: Samples were prepared as shown in Table 5 below. Corn powder (about 60 mg per sample) was mixed with 40 ml of 50 mM MES buffer at pH 6.0. Samples were incubated for 2.5 hours and 14 hours in a water bath set at 90 ° C. At the indicated incubation time, samples were transferred and analyzed for glucose content.

The samples were assayed for glucose with a glucose oxidase / blush peroxidase based assay. The GOPOD reagent contained 0.2 mg / ml o- dianisidine, 100 mM Tris pH 7.5, 100 U / ml glucose oxidase and 10 U / ml blush peroxidase. 20 μl of samples or diluted samples were assayed in 96 well plates using glucose standards (variable from 0 to 0.22 ml / ml). 100 μl of GOPGD reagent was mixed and added to each well and the plates incubated at 37 ° C. for 30 minutes. 100 μl of sulfuric acid (9M) was added and the absorbance at 540 nm was read. The glucose concentration of the sample was determined by reference to the standard curve. The amount of glucose observed in each sample is shown in Table 5.

These data demonstrate that the expression of superthermophilic α-amylase and α-glucosidase in maize results in corn products that will yield glucose when hydrated and heated under suitable conditions.

Example 28

Preparation of Maltodextrin

Maltodextrins were prepared using grains expressing thermophilic α-amylase. The illustrated process does not require prior separation of starch and does not require the addition of exogenous enzymes.

Corn kernels from transformed plants expressing 797GL3 were ground into powder in a Kleco grinder to produce "amylase powder". A mixture of 10% transformed / 90% non-transformed grains was ground in the same manner to prepare a “10% amylase powder”.

Amylase powder and 10% amylase powder (approximately 60 mg / sample) were mixed at a rate of 5 μl of water per mg of powder. The resulting slurry was incubated at 90 ° C. for up to 20 minutes as shown in Table 6. The reaction was terminated by addition of 0.9 ml of 50 mM EDTA at 85 ° C. and mixed by pipetting. A 0.2 ml sample of slurry was transferred and centrifuged to remove insoluble material and diluted 3 × in water. The samples were analyzed for sugar and maltodextrin by HPLC using an ELSD detector. The gradient HPLC system was equipped with a 5 micron particle size, 250 x 4.6 mm Astec polymer amino column and an Alltech ELSD 2000 detector. The system was re-equilibrated with a 15:85 mixture of water: acetonitrile. The flow rate was 1 ml / min. Initial conditions were maintained for 5 minutes after injection and 20 minutes with a 50:50 water: acetonitrile gradient followed by 10 minutes with the same solvent. The system was washed for 20 minutes with 80:20 water: acetonitrile and then re-equilibrated with starting solvent.

The peak area obtained is normalized to the volume and weight of the powder. The response factor of ELSD per μg of carbohydrates was reduced with increasing DP, thus high DP maltodextrin showed a higher total percentage than indicated by the peak area.

 The relative peak area of the reaction product using 100% amylase powder is shown in FIG. 17. The relative peak area of the reaction product using 10% amylase powder is shown in FIG. 18.

These data demonstrate that various maltodextrin mixtures can be prepared by varying the heating time. The level of α-amylase activity can be altered by mixing wild type corn and α-amylase-expressing corn to modify maltodextrin properties.

The hydrolysis reaction products described in this example were subjected to food and centrifugation, filtration, ion-exchange, gel permeation, ultrafiltration, nanofiltration, reverse osmosis, bleaching with carbon particles, spray drying and other standards known in the art. It can be concentrated and purified for other applications by the use of a variety of well-defined methods, including techniques.

Example 29

Effect of time and temperature on maltodextrin production

The composition of the autohydrolysis maltodextrin product of grains containing thermophilic α-amylase can be modified by varying the reaction time and temperature.

In another experiment, amylase powder was prepared as described in Example 28 above and mixed with water at a rate of 300 μl of water per 60 mg of powder. Samples were incubated at 70 ° C., 80 ° C., 90 ° C. or 100 ° C. for up to 90 minutes. The reaction was terminated by addition of 900 ml of 50 mM EDTA at 90 ° C., centrifuged to remove insoluble material and filtered through a 0.45 μm nylon filter. The filter was analyzed by HPLC as described in Example 28.

The analysis result is shown in FIG. The DP number designation refers to the degree of polymerization. DP2 is maltose; DP3 is maltotriose and the like. Large DP maltodextrin eluted at a single peak is near the end of the elution and is labeled "> DP12". The aggregate includes dextrin that passed through a 0.45 μm filter and protective column, but does not include any very large starch fragments trapped by the filter or protective column.

This experiment demonstrates that the maltodextrin composition of the product can be modified by varying the temperature and incubation time to obtain the desired maltooligosaccharide or maltodextrin product.

Example 30

Maltodextrin production

Compositions of maltodextrin products from transgenic corn containing thermophilic α-amylase are added with other enzymes such as α-glucosidase and xylose isomerase, and salts are added to the aqueous powder mixture prior to heat treatment. Can be modified.

Alternatively, the amylose powder prepared as described above was mixed with purified MalA and / or bacterial xylose isomerase designated BD8037. S with 6His tablet tag. Sulpotaricus MalA. It was expressed in E. coli. Cell lysates were prepared as described in Example 28 and then purified using apparent affinity with nickel affinity resin (Probond, Invitrogen) and according to manufacturer's instructions for natural protein purification. Xylose isomerase BD8037 was obtained as a lyophilized powder from Diversa and resuspended in 0.4 × of original volume of water.

Amylase corn powder was mixed with enzyme solution added water or buffer. All reactions contained 60 mg amylase powder and a total of 600 μl of solution. The first set of reactions were buffered with 50 mM MOPS, 10 mM MgSO ₄ and 1 mM CoCl ₂ at pH 7.0 at room temperature; In the second set of reactants, the metal containing buffer solution was replaced with water. All reactions were incubated at 90 ° C. for 2 hours. The reaction supernatant fractions were prepared by centrifugation. The pellet was further washed with 600 μl H ₂ O and re-centrifuged. Supernatant fractions from each reaction were combined, filtered through Centricon 10 and analyzed by HPLC using an ELSD detector as described above.

The results are shown graphically in FIG. 20. They demonstrate that grain-expressing amylase 797GL3 can be combined with other thermophilic enzymes in the presence or absence of added metal ions to produce various maltodextrin mixtures from corn powder at high temperatures. In particular, the inclusion of glucoamylase or α-glucosidase can lead to products with more glucose and other low DP products. Inclusion of enzymes with glucose isomerase activity results in products with fructose and will therefore be higher sugar than those prepared using amylases with amylase alone or with α-glucosidase. In addition, the data suggest that the proportion of DP5, DP6 and DP7 maltooligosaccharides may increase due to the inclusion of divalent cation salts such as CoCl ₂ and MgSO ₄ .

Other methods of modifying maltodextrin compositions prepared by reactions as described herein include changes in reaction pH, changes in starch type of transformed or non-transformed grains, changes in solid ratios or addition of organic solvents.

Example 31

Preparation of Dextrins or Sugars from Grains that Do Not Mechanically Crush Grains Before Recovery of Starch-Derived Products

Sugars and maltodextrins were prepared by contacting the transgenic grain, 797GL3, expressing α-amylase with water and heating to 90 ° C. overnight (> 14 hours). The liquid was then separated from the grain by filtration. The liquid product was analyzed by HPLC by the method described in Example 15. Table 6 shows the properties of the detected products.

* Quantification of DP3 includes maltotriose and may include isomers of maltotriose with α (1 → 6) bonds instead of α (1 → 4) bonds. Similarly, DP4 to DP7 quantification includes linear maltooligosaccharides of a given chain length and isomers having one or more (1 → 6) bonds instead of one or more α (1 → 4) bonds.

These data demonstrate that sugars and maltodextrins can be prepared by contacting and heating α-amylase-expressing grains with water. The product can then be separated from intact grains by filtration or centrifugation or gravity precipitation.

Example 32

Fermentation of Raw Starch in Maize Expressing Rizopus Duck Glucoamylase

Transformed corn kernels are harvested from the transformed plants prepared as described in Example 29. The grain is ground into a powder. The corn kernel expresses a protein comprising an active fragment of glucoamylase (SEQ ID NO: 49) of lyspus ducks targeted with cytoplasmic reticulum.

The corn kernels are ground to a powder as described in Example 15. A mash containing 20 g of corn powder, 23 ml of deionized water and 6.0 ml of bag set (8 wt% solids) is prepared. The pH is titrated to 6.0 by the addition of ammonium hydroxide. Add the following ingredients to the mesh: 0.2 mg of protease (0.60 ml of 1,000-fold dilution of commercial protease), lactoside & urea (0.85 ml of 10-fold dilution of 50% urea solution). 100 ml containing the mesh are punctured in the bottle cap to allow CO ₂ to escape. The mash was then incubated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C. and set to 82 ° C. at 48 hours.

Yeast for inoculation is grown as described in Example 14.

The sample is transferred as described in Example 14 and then analyzed by the method described in Example 14.

Example 33

Transformed corn kernels are harvested from the transformed plants prepared as described in Example 28. Grind the grain into powder. The corn kernel expresses a protein comprising an active fragment of glucoamylase (SEQ ID NO: 49) of lyspus ducks targeted with cytoplasmic reticulum.

The corn kernels are ground to a powder as described in Example 15. A mash containing 20 g of corn powder, 23 ml of deionized water and 6.0 ml of bag set (8 wt% solids) is prepared. The pH is titrated to 6.0 by the addition of ammonium hydroxide. Add the following ingredients to the mesh: 0.2 mg of protease (0.60 ml of 1,000-fold dilution of commercial protease), lactoside & urea (0.85 ml of 10-fold dilution of 50% urea solution). 100 ml containing the mesh are punctured in the bottle cap to allow CO ₂ to escape. The mash was then incubated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C. and set to 82 ° C. at 48 hours.

Yeast for inoculation is grown as described in Example 14.

The sample is transferred as described in Example 14 and then analyzed by the method described in Example 14.

Example 34

Example of Fermentation of Raw Starch in Whole Grains of Corn Expressing Ripuspus Duck Residue Glucoamylase Under Addition of Exogenous α-amylase

Transformed corn kernels are harvested from the transformed plants prepared as described in Example 28. The corn kernel expresses a protein comprising an active fragment of glucoamylase (SEQ ID NO: 49) of lyspus ducks targeted with cytoplasmic reticulum.

The corn kernels are contacted with 20 g of corn powder, 23 ml of deionized water, and 6.0 ml of bag set (8 wt% solids). The pH is titrated to 6.0 by the addition of ammonium hydroxide. Add the following ingredients to the mash: Barley α-amylase (2 mg), protease (0.60 ml of 1,000-fold dilution of commercial protease), lactoside & urea (10-fold dilution of 50% urea solution) from Sigma 0.85 ml) 0.2 mg. 100 ml containing the mesh are punctured in the bottle cap to allow CO ₂ to escape. The mash was then incubated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C. and set to 82 ° C. at 48 hours.

Yeast for inoculation is grown as described in Example 14.

The sample is transferred as described in Example 14 and then analyzed by the method described in Example 14.

Example 35

Fermentation of Raw Starch in Maize Expressing Rizopus Duck Glucoamylase and Zea Mays Amylase

Transformed corn kernels are harvested from the transformed plants prepared as described in Example 28. The corn kernel expresses a protein comprising an active fragment of glucoamylase (SEQ ID NO: 49) of lyspus ducks targeted with cytoplasmic reticulum. The kernel also expresses corn amylase having a live starch binding domain as described in Example 28.

The corn kernels are ground to a powder as described in Example 14. A mash containing 20 g of corn powder, 23 ml of deionized water and 6.0 ml of bag set (8 wt% solids) is prepared. The pH is titrated to 6.0 by the addition of ammonium hydroxide. Add the following ingredients to the mesh: 0.2 mg of protease (0.60 ml of 1,000-fold dilution of commercial protease), lactoside & urea (0.85 ml of 10-fold dilution of 50% urea solution). 100 ml containing the mesh are punctured in the bottle cap to allow CO ₂ to escape. The mash was then incubated with yeast (1.44 ml) and incubated in a 90 F water bath set. After 24 hours of fermentation, the temperature is lowered to 86F and set to 82F at 48 hours.

Yeast for inoculation is grown as described in Example 14.

The sample is transferred as described in Example 14 and then analyzed by the method described in Example 14.

Example 36

Example of Fermentation of Raw Starch in Maize Expressing Thermobacterium Termosacaroticum Glucoamylase

Transformed corn kernels are harvested from the transformed plants prepared as described in Example 28. The corn kernel expresses a protein comprising an active fragment of glucoamylase (SEQ ID NO: 47) of Rizopus duck, targeted with cytoplasmic reticulum.

The corn kernels are ground to a powder as described in Example 15. A mash containing 20 g of corn powder, 23 ml of deionized water and 6.0 ml of bag set (8 wt% solids) is prepared. The pH is titrated to 6.0 by the addition of ammonium hydroxide. Add the following ingredients to the mesh: 0.2 mg of protease (0.60 ml of 1,000-fold dilution of commercial protease), lactoside & urea (0.85 ml of 10-fold dilution of 50% urea solution). 100 ml containing the mesh are punctured in the bottle cap to allow CO ₂ to escape. The mash was then incubated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C. and set to 82 ° C. at 48 hours.

Yeast for inoculation is grown as described in Example 14.

The sample is transferred as described in Example 14 and then analyzed by the method described in Example 14.

Example 37

Example of Fermentation of Raw Starch in Corn Expressing Aspergillus niger glucoamylase

Transformed corn kernels are harvested from the transformed plants prepared as described in Example 28. The corn kernels are entrusted with Aspergillus niger (Fiil, NP "Glucoamylases Gland G2 from Aspergillus niger are synthesized from two different but closely related mRNAs" EMBO J. 3 (5), 1097-1102 (1984)). Protein containing an active fragment of Glucoamylase (No. P04064). The corn-optimized nucleic acid encoding the glucoamylase has SEQ ID NO: 59 and is targeted to cytoplasmic reticulum.

The corn kernels are ground to a powder as described in Example 14. A mash containing 20 g of corn powder, 23 ml of deionized water and 6.0 ml of bag set (8 wt% solids) is prepared. The pH is titrated to 6.0 by the addition of ammonium hydroxide. Add the following ingredients to the mesh: 0.2 mg of protease (0.60 ml of 1,000-fold dilution of commercial protease), lactoside & urea (0.85 ml of 10-fold dilution of 50% urea solution). 100 ml containing the mesh are punctured in the bottle cap to allow CO ₂ to escape. The mash was then incubated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C. and set to 82 ° C. at 48 hours.

Yeast for inoculation is grown as described in Example 14.

The sample is transferred as described in Example 14 and then analyzed by the method described in Example 14.

Example 38

Example of Fermentation of Raw Starch in Corn Expressing Aspergillus niger glucoamylase and Zea mays amylase

Transformed corn kernels are harvested from the transformed plants prepared as described in Example 28. The corn kernels are entrusted with Aspergillus niger (Fiil, NP "Glucoamylases Gland G2 from Aspergillus niger are synthesized from two different but closely related mRNAs" EMBO J. 3 (5), 1097-1102 (1984)). No. P04064) (SEQ ID NO: 59, corn-optimized nucleic acid) expresses a protein comprising an active fragment of glucoamylase and is targeted to cytoplasmic reticulum. The kernel also expresses corn amylase having a live starch binding domain as described in Example 28.

The corn kernels are ground to a powder as described in Example 14. A mash containing 20 g of corn powder, 23 ml of deionized water and 6.0 ml of bag set (8 wt% solids) is prepared. The pH is titrated to 6.0 by the addition of ammonium hydroxide. Add the following ingredients to the mesh: 0.2 mg of protease (0.60 ml of 1,000-fold dilution of commercial protease), lactoside & urea (0.85 ml of 10-fold dilution of 50% urea solution). 100 ml containing the mesh are punctured in the bottle cap to allow CO ₂ to escape. The mash was then incubated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C. and set to 82 ° C. at 48 hours.

Yeast for inoculation is grown as described in Example 14.

The sample is transferred as described in Example 14 and then analyzed by the method described in Example 14.

Example 39

Example of Fermentation of Raw Starch in Maize Expressing Thermobacterium Termosacaroticum Glucoamylase and Barley Amylase

Transformed corn kernels are harvested from the transformed plants prepared as described in Example 28. The corn kernel expresses a protein comprising an active fragment of glucoamylase of thermobacterium termosacarolitum (SEQ ID NO: 47) targeted to cytoplasmic reticulum. The kernel also contains a low pI barley amylase amy1 gene (Rogers, JC and Milliman, C. "Isolation and sequence analysis of a barley alpha-amylase cDNA clone" J that has been modified to target the expression of the protein to cytoplasmic reticulum. Biol. Chem. 258 (13), 8169-8174 (1983)].

The corn kernels are ground to a powder as described in Example 14. A mash containing 20 g of corn powder, 23 ml of deionized water and 6.0 ml of bag set (8 wt% solids) is prepared. The pH is titrated to 6.0 by the addition of ammonium hydroxide. Add the following ingredients to the mesh: 0.2 mg of protease (0.60 ml of 1,000-fold dilution of commercial protease), lactoside & urea (0.85 ml of 10-fold dilution of 50% urea solution). 100 ml containing the mesh are punctured in the bottle cap to allow CO ₂ to escape. The mash was then incubated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C. and set to 82 ° C. at 48 hours.

Yeast for inoculation is grown as described in Example 14.

The sample is transferred as described in Example 14 and then analyzed by the method described in Example 14.

Example 40

Example of Fermentation of Raw Starch in Whole Kernels of Corn Expressing Thermobacterus Termosacaroticum Glucoamylase and Barley Amylase

Transformed corn kernels are harvested from the transformed plants prepared as described in Example 28. The corn kernel expresses a protein comprising an active fragment of glucoamylase of thermobacterium termosacarolitum (SEQ ID NO: 47) targeted to cytoplasmic reticulum. The kernel also contains a low pI barley amylase amy1 gene (Rogers, JC and Milliman, C. "Isolation and sequence analysis of a barley alpha-amylase cDNA clone" J that has been modified to target the expression of the protein to cytoplasmic reticulum. Biol. Chem. 258 (13), 8169-8174 (1983)].

The corn kernels are contacted with 20 g of corn powder, 23 ml of deionized water, and 6.0 ml of bag set (8 wt% solids). The pH is titrated to 6.0 by the addition of ammonium hydroxide. The following ingredients are added to this mixture: Protease (0.60 ml of 1,000-fold dilution of commercial protease), 0.2 mg of lactoside & urea (0.85 ml of 10-fold dilution of 50% urea solution). 100 ml containing the mesh are punctured in the bottle cap to allow CO ₂ to escape. The mash was then incubated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C. and set to 82 ° C. at 48 hours.

Yeast for inoculation is grown as described in Example 14.

The sample is transferred as described in Example 14 and then analyzed by the method described in Example 14.

Example 41

Example of Fermentation of Raw Starch in Corn Expressing Alpha-amylase and Glucoamylase Fusions

Transformed corn kernels are harvested from the transformed plants prepared as described in Example 28. The corn kernel expresses a corn-optimized polynucleotide as provided in SEQ ID NO: 46, encoding an alpha-amylase and glucoamylase fusion as provided in SEQ ID NO: 45, targeted to cytoplasmic reticulum.

The corn kernels are ground to a powder as described in Example 14. A mash containing 20 g of corn powder, 23 ml of deionized water and 6.0 ml (8 weight percent solids) of a bag set was prepared. The pH is titrated to 6.0 by the addition of ammonium hydroxide. The following ingredients are added to this mixture: Protease (0.60 ml of 1,000-fold dilution of commercial protease), 0.2 mg of lactoside & urea (0.85 ml of 10-fold dilution of 50% urea solution). 100 ml containing the mesh are punctured in the bottle cap to allow CO ₂ to be released. The mash was then incubated with yeast (1.44 ml) and incubated in a water bath set at 90 ° C. After 24 hours of fermentation, the temperature is lowered to 86 ° C. and set to 82 ° C. at 48 hours.

Yeast for inoculation is grown as described in Example 14.

The sample is transferred as described in Example 14 and then analyzed by the method described in Example 14.

Example 42

Construction of Transformation Vectors

An expression cassette was constructed as follows to express super thermophilic beta-glucanase EglA in maize.

pNOV4800 includes barley Amy32b signal peptide (MGKNGNLCCFSLLLLLLAGLASGHQ) fused to a synthetic gene for EglA beta-glucanase for targeting to cytoplasmic reticulum and secretion of apoplasts. This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4803 contains a barley Amy32b signal peptide fused to a synthetic gene for EglA beta-glucanase for targeting to cytoplasmic reticulum and secretion of apoplasts. This fusion was cloned behind the corn ubiquitin promoter for plant-wide expression.

Expression cassettes were constructed to express superthermophilic beta-glucanase / mannase 6GP1 (SEQ ID NO: 85) in maize as follows:

pNOV4819 includes a tobacco PR1a signal peptide (MGFVLFSQLPSFLLVSTLLLFLVISHSCRA) fused to a synthetic gene for 6GP1 beta-glucanase / mannase for targeting to cytoplasmic reticulum and secretion of apoplasts. This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4820 contains a synthetic gene for 6GP1 cloned behind maize γ-zein prolomotor for cytoplasmic localization and specific expression in endocrine.

pNOV4823 comprises a tobacco PR1a signal peptide fused to a synthetic gene for 6GP1 beta-glucanase / mannase, with C-terminal addition of the sequence KDEL for targeting to and retention within cytoplasmic reticulum. This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4825 comprises a tobacco PR1a signal peptide fused to a synthetic gene for 6GP1 beta-glucanase / mannase, with C-terminal addition of the sequence KDEL for targeting to and retention within cytoplasmic reticulum. This fusion was cloned behind the corn ubiquitin promoter for plant-wide expression.

The following expression cassettes were constructed to express barley AmyI alpha-amylase (SEQ ID NO: 87) in maize:

pNOV4867 comprises the N-terminal signal sequence, a corn γ-agent fused to barley AmyI alpha-amylase, with C-terminal addition of the sequence SEKDEL for targeting to and retention within cytoplasmic reticulum. This fusion was cloned behind the corn γ-zein promoter for expression throughout the plant.

pNOV4879 comprises an N-terminal signal sequence, a corn γ-agent fused to barley AmyI alpha-amylase with a C-terminal addition of the sequence SEKDEL for targeting to and retention within cytoplasmic reticulum. This fusion was cloned behind the corn globulin promoter for expression in embryos.

pNOV4897 comprises an N-terminal signal sequence, a corn γ-agent fused to barley AmyI alpha-amylase for targeting to cytoplasmic reticulum and secretion of apoplasts. This fusion was cloned behind the corn globulin promoter for expression in embryos.

pNOV4895 contains an N-terminal signal sequence, a corn γ-agent fused to barley AmyI alpha-amylase for targeting to cytoplasmic reticulum and secretion of apoplasts. This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4901 contains genes for barley AmyI alpha-amylase cloned behind the corn globulin promoter for cytoplasmic localization and specific expression in embryos.

An expression cassette was constructed to express barley rispus glucoamylase (SEQ ID NO: 50) in maize as follows:

pNOV4872 comprises an N-terminal signal sequence, a corn γ-agent fused to a synthetic gene for rispus glucoamylase, with C-terminal addition of the sequence SEKDEL for targeting to and retention within cytoplasmic reticulum. This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4880 comprises a N-terminal signal sequence, a corn γ-agent fused to a synthetic gene for rispus glucoamylase, with C-terminal addition of the sequence SEKDEL for targeting to and retention in cytoplasmic reticulum. This fusion was cloned behind the corn globulin promoter for expression in embryos.

pNOV4889 contains an N-terminal signal sequence, a corn γ-agent, fused to a synthetic gene for lysopus glucoamylase for targeting to cytoplasmic reticulum and secretion of apoplasts. This fusion was cloned behind the corn globulin promoter for expression in embryos.

pNOV4890 contains an N-terminal signal sequence, a corn γ-agent, fused to a synthetic gene for lysopus glucoamylase for targeting to cytoplasmic reticulum and secretion of apoplasts. This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

pNOV4891 contains a synthetic gene for lysopus glucoamylase cloned behind the corn γ-zein promoter for cytoplasmic localization and specific expression in the embryo.

Example 43

Expression of Mesophilic Lycopus Glucoamylase in Corn

Various constructs for the expression of Rifupus glucoamylase in corn were constructed. Corn γ-Zane and globulin promoters were used to express glucoamylase specifically in endocrine or embryos, respectively. In addition, corn γ-zein signal sequences and synthetic ER retention signals were used to regulate the subcellular localization of the glucoamylase protein. Transformed plants with glucoamylase activity detected in all five constructs (pNOV4872, pNOV4880, pNOV4889, pNOV4890 and pNOV4891) seeds were produced. Tables 7 and 8 show the results of the individual transformed seeds (build pNOV4872) and the aggregated seeds (build pNOV4889), respectively. No adverse phenotype was observed for any transformed plant expressing Rizopus glucoamylase.

Glucoamylase Assay: Seeds were ground to a powder and the powder suspended in water. Samples were incubated at 30 ° C. for 50 minutes to allow glucoamylase to react with starch. Insoluble material was pelleted and the glucose concentration for the supernatant was measured. The amount liberated in each sample was used as an indicator of the level of glucoamylase present. The sample was mixed with GOHOD reagent (300 mM Tris / Cl pH 7.5, glucose oxidase (20 U / ml), Blush Peroxidase (20 U / ml), o-Dianisidine (0.1 mg / ml)} for 30 minutes at 37 ° C. Were incubated, 0.5 volume of 12N H 2 SO 4 was added, and OD540 was measured to determine glucose concentration.

Table 7 shows the activity of Rifupus glucoamylase in individual transgenic maize seeds (build pNOV4872).

Table 8 shows the activity of lyfupus glucoamylase in aggregated transgenic corn seeds (build pNOV4889).

All expression cassettes were inserted into the binary vector pNOV2117 for transformation into maize via Agrobacterium infection. The binary vector contained a phosphomannose isomerase (PMI) gene that allows the selection of transformed cells with mannose. Transformed corn plants were self-pollinated or crossbred and seeds were harvested for analysis.

Example 44

Expression of Superthermophilic Beta-glucanase EglA in Corn

For expression of superthermophilic beta-glucanase EglA in corn, the present inventors used the ubiquitin promoter for expression throughout the plant and the γ-zein promoter for specific expression in the endocrine of corn seeds. Barley Amy32b signal peptide was fused to EglA for localization in apoplasts. Expression of superthermophilic beta-glucanase EglA in transformed corn seeds and leaves was analyzed using enzyme assay and western blotting.

Transformed seed isolation for construct pNOV4800 or pNOV4803 was analyzed using both Western blotting and enzyme assay for beta-glucanase. Individual seeds were soaked in water for 48 hours and then endosperm was separated therefrom. Proteins were extracted by grinding the endosperm in 50 mM NaPO 4 buffer (pH 6.0). The extract was heated at 100 ° C. for 15 minutes to separate heat resistant proteins and then insoluble material was pelleted. Supernatants containing heat resistant proteins were analyzed for beta glucanase activity using the azo-barley glucan method (megazyme). Samples were preincubated at 100 ° C. for 10 minutes and assayed at 100 ° C. for 10 minutes using azo-barley glucan substrate. After incubation, 3 volumes of precipitation solution were added to each sample, the sample was centrifuged for 1 minute, and the OD590 of each supernatant was measured. In addition, 5 μg of protein was separated by SDS-PAGE and blotted into nitrocellulose for western blot using an antibody against EglA protein. Western blot assay detected specific heat resistant protein (s) in EglA positive endosperm extracts, but not negative extracts. Western blot signals related to the level of EglA activity were detected by enzymes.

EglA activity was analyzed in the leaves and seeds of plants comprising the transforming constructs pNOV4803 and pNOV4800, respectively. Assays (performed as described above) were expressed at varying levels in the leaves (Table 9) and seeds (Table 10) of heat-resistant beta-glucanase EglA transformed plants, whereas no activity was observed in nontransgenic control plants. It was not detected. Expression of EglA in maize using the constructs pNOV4800 and pNOV4803 did not result in any detectable negative phenotype.

Table 9 shows the activity of superthermophilic beta-glucanase EglA in the leaves of transgenic corn plants. Enzyme assays were performed on extracts from the leaves of pNOV4803 transformed plants to detect superthermophilic beta-glucanase activity. The assay was performed at 100 ° C. using the azo-barley glucan method (megazyme). The results indicate that the transgenic leaves have varying levels of superthermophilic beta-glucanase activity.

Table 10 shows the activity of super thermophilic beta-glucanase EglA in the seeds of transgenic maize plants. Enzyme assays were performed on extracts from individual, isolated seeds of pNOV4800 transformed plants to detect superthermophilic beta-glucanase activity. The assay was performed at 100 ° C. using the azo-barley glucan method (megazyme). The results indicate that the transgenic seeds have varying levels of super thermophilic beta-glucanase activity.

Effects of Endoglucanase EglA on the Expression of Cell Transformation and In Vitro Degradability Analysis

Five individual seeds from each of the two states, # 263 &# 266, each of which expressed or did not express Egla (pNOV4803), were grown in a greenhouse. Protein extracts prepared from small leaf samples from immature plants were used to demonstrate that the transforming endoglucanase activity is present in # 266 plants but not in # 263 plants. At the total plant maturation, about 30 days after the moisture, the entirety of the abraded plants were harvested, coarsely cut and oven dried for 72 hours. Each procedure was divided into two duplicate samples (labeled A & B, respectively) and the material was subjected to conventional procedures (except for pretreatment at 40 ° C. or 90 ° C. prior to in vitro degradability analysis). HK Goering and PJ Van Soest, Goering, H. Keith 1941 (Washington, DC): Agricultural Research Service, US Dept. of Agriculture, 1970.iv, 20 p .: ill .-- Agriculture handbook; no. 379) Feed fiber analysis instrument, reagents, procedures, and some applications). In vitro degradability analysis was performed as follows.

Samples were cut to about 1 mm wide using a wiley mill and then sub-classified into 16 weighed aliquots for analysis. The material was suspended in buffer and incubated at 40 ° C. or 90 ° C. for 2 hours and then cooled overnight. Micronutrients, tryticase & casein & sodium sulfite were added, followed by elongated rumen solution and incubated at 37 ° C. for 30 minutes. The analysis of neutral detergent fibers (NDF), acid detergent fibers (ADF), and acid detergent lignin (AD-L) was performed by standard gravimetric methods (Van Soest & Wine, Use of Detergents in the Analysis of fibrous Feeds.IV). Determination of plant cell-wall constituents.PJ Van Soest & RH Wine. (1967) .Journal of The AOAC, 50: 50-55; see also Methods for dietry fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition (1991). PJ Van Soest, JB Robertson & BA Lewis. J. Dairy Science, 74: 3583-3597].

The data show that EglA expressing transformed plants (# 266) contain more NDF than control plants (# 233), while ADF & lignin are relatively unchanged. NDF fractions of transformed plants are more readily degraded than non-transformed plants, which is consistent with the “self-degradation” of cell wall cellulose by transformatively expressing endonuclease enzymes, cellulose (NDF). -ADF-AD-L) is due to the increased degradability.

Example 45

Expression of Thermophilic Beta-Glucanase / Mannana (6GP1) in Corn

Transformed seeds for pNOV4820 and pNOV4823 were analyzed for 6GP1 beta glucanase activity using the azo-barley glucan method (megazyme). Enzyme assays performed at 50 ° C. indicated that the transgenic seeds had super thermophilic 6GP1 beta-glucanase activity, whereas no activity was detected in the non-transgenic seeds (positive signals indicated background noise associated with the assay). Indicates).

Table 11 shows the activity of thermophilic beta-glucanase / mannase 6GP1 in transgenic corn seeds. Transformed seeds for pNOV4820 (Results 1-6) and pNOV4823 (Results 7-9) were analyzed for 6GP1 beta-glucanase activity using the azo-barley glucan method (megazyme). Enzyme assays were performed at 50 ° C. and the results indicate that the transgenic seeds had super thermophilic 6GP1 beta-glucanase activity, while no activity was detected in non-transgenic seeds.

Example 46

Expression of Mesophilic Barley AmyI Amylase in Corn

Various constructs were produced for the expression of barley AmyI alpha-amylase in corn. The corn γ-Zane and globulin promoters were used to specifically express amylase in each endocrine or embryo. In addition, corn γ-zein signal sequences and synthetic ER retention signals were used to regulate the subcellular localization of amylase protein. Transformed plants with alpha-amylase activity detected in all 5 constructs (pNOV4867, pNOV4879, pNOV4897, pNOV4895 and pNOV4901) seeds were produced. Table 12 shows the activity in individual seeds for five independent, isolated results (builds pNOV4879 and pNOV4897). View All Constructs Any transformation resulted with a wrinkled seed phenotype indicating that the synthesis of AmyI amylase could affect starch formation, accumulation or degradation.

Table 12 shows the activity of barley AmyI alpha-amylase in individual corn seeds (build pNOV4879 and pNOV4897). Separate, isolated seeds for constructs pNOV4879 (seed samples 1 and 2) and pNOV4897 (seed samples 3-5) were analyzed for alpha-amylase activity as described above.

Example 47

Preparation of Xylanase Constructs

Table 13 lists nine binary vectors, each containing a unique xylanase expression cassette. The xylanase expression cassette includes a promoter, synthetic xylanase gene (coding sequence), intron (PEPC, inverted) and terminator (35S).

Two synthetic corn-optimized endo-xylanase genes were cloned into binary vector pNOV2117. These two xylanase genes were named BD7436 (SEQ ID NO: 61) and BD6002A (SEQ ID NO: 63). Additional binary vectors can be prepared comprising a third corn-optimized sequence, BD6002B (SEQ ID NO: 65).

Two promoters were used: the corn glutelin-2 promoter (27-kD gamma-jane promoter (SEQ ID NO: 12)) and the rice glutelin-1 (Osgt1) promoter (SEQ ID NO: 67). Transformed plants were prepared using the first six vectors listed in Table 1. The latter three vectors can also be prepared and used to produce transformed plants.

Vectors 11560 and 11562 encode the polypeptide represented by SEQ ID NO: 62 (BD7436). Constructs 11559 and 11561 encode polypeptides consisting of SEQ ID NO: 17 fused to the N-terminus of SEQ ID NO: 62. SEQ ID NO: 17 is the 19 amino acid signal sequence from a 27kD gamma-zein protein.

Vector 12175 encodes the polypeptide represented by SEQ ID NO: 64 (BD6002A). Construct 12174 encodes a fusion protein consisting of a gamma-zein signal sequence (SEQ ID NO: 17) fused to the N-terminus of SEQ ID NO: 64.

Vectors pWIN062 and pWIN064 encode the polypeptide represented by SEQ ID NO: 66 (BD6002B). Vector pWIN058 encodes a fusion protein consisting of the chloroplast transfer peptide (SEQ ID NO: 68) of corn waxy protein fused to the N-terminus of SEQ ID NO: 66.

Xylanase binary vector vector Promoter Signal sequence source Xylanase gene 11559 27KD Gamma-Jane 27KD Gamma-Jane BD7436 11560 27KD Gamma-Jane none BD7436 11561 OsGt1 27KD Gamma-Jane BD7436 11562 OsGt1 none BD7436 12174 27KD Gamma-Jane 27KD Gamma-Jane BD6002A 12175 27KD Gamma-Jane none BD6002A PWIN058 27KD Gamma-Jane Corn waxy protein BD6002B PWIN062 OsGt1 none BD6002B PWIN064 27KD Gamma-Jane none BD6002B

All constructs include an expression cassette for PMI, allowing positive selection of regenerated transformed tissue on mannose-containing medium.

Example 48

Xylanase Activity Assay Results

The data shown in Tables 14 and 15 shows that regeneration of stably transformed xylanase activity with a binary vector comprising the xylanase gene BD7436 (SEQ ID NO: 61 of Example 47) and BD6002A (SEQ ID NO: 63 of Example 47) Demonstrates accumulation in T1 producing seeds harvested from corn (T0) corn plants. Using the Azo-WAXY assay (Megazyme), activity was detected in extracts from both aggregated (isolated) transformed seeds and single transformed seeds.

T1 seeds were ground and water soluble proteins were extracted from the powder samples using citrate-phosphate buffer (pH 5.4). The powder suspension was stirred at room temperature for 60 minutes and insoluble material was removed by centrifugation. The xylanase activity of the supernatant fractions was determined using the Azo-WAXY assay (Mccleary, BV "Problems in the measurement of beta-xylanase, beta-glucanase and alpha-amylase in feed enzymes and animal feeds" .In proceedings of Second European Symposium on Feed Enzymes "(W. van Hartingsveldt, M. Hessing, JP van der Jugt, and WAC Somers Eds.), Noordwiijkerhout, Netherlands, 25-27 October, 1995]. Pre-incubated at 37 ° C. To 1 volume of 1 × extract supernatant, 1 volume of substrate (1% azo-mill arabinoxysilane S-AWAXP) was added and then incubated for 5 minutes at 37 ° C. Corn powder extract Xylanase activity in the depolymerized azo-mill arabinoxysilane by the endo-mechanism and produces low molecular weight stained fragments in the form of xylo-oligomers, after incubation for 5 minutes, yielding 95% EtOH 5 volumes. The reaction was terminated by the addition of alcohol. The non-depolymerized dye substrate is precipitated so that only low molecular weight xylo-oligomers remain in solution Insoluble material is removed by centrifugation The absorbance of the supernatant fractions is measured at 590 nm and xylanase per gram powder. The unit of was determined by comparing the absorbance values from the same assay using a known xylanase standard of activity The activity of this standard was determined by the BCA assay The enzymatic activity of the standard was determined using wheat arabinoxysilane as substrate The reaction was carried out by reacting 2,2'-bicincronic acid (BCA) with the reducing end to determine the release of the reducing end The substrate was milled in 100 mM sodium acetate buffer at pH 5.30 containing 0.02% sodium azide. Prepared as arabinoxysilane (Megazyme P-WAXYM) 1.4% w / w solution. BCA reagents were prepared by mixing 50 parts of reagent A and 1 part of reagent B (reagents A and B are the products of the manufacturer [Pierce], product numbers 23232 and 23422, respectively). These reagents were mixed for up to 4 hours before use. This assay was performed by mixing 200 microliters of substrate with 80 microliters of enzyme sample. After incubation for the desired time at the desired temperature, 2.80 milliliters of BCA reagent was added. The contents were mixed and left at 80 ° C. for 30 to 45 minutes. The contents were cooled and then transferred to a cuvette and the absorbance at 560 nm was measured in comparison to the known concentration of xylose. The choice of enzyme dilution, incubation time and incubation temperature may vary depending on the skill of the art.

The experimental results shown in Table 14 demonstrate the presence of recombinant xylanase activity in powders made from T1 generation corn seeds. Seeds from 12 T0 plants (derived from independent T-DNA insertion results) were analyzed. The twelve transformation results are from six different vectors as shown (see Table 13 in Example 47 for the description of the vector). Extracts of non-transformed (negative control) corn powder do not contain measurable xylanase activity (see Table 15). Xylanase activity in these 12 samples ranged from 10 to 87 units / powder gram.

The results in Table 15 demonstrate the presence of xylanase activity in corn powders derived from single grains. T1 seeds from two T0 plants comprising vectors 11561 and 11559 were analyzed. These vectors are described in Example 47. Eight seeds from each of the two plants were ground and a powder sample was extracted from each seed. This table shows the results of a single assay of each extract. No xylanase activity was found in the assay of extracts of seeds 1, 5 and 8 for both transformation results. These seeds are representative of radish isolates. Seeds 2, 3, 4, 6 and 7 for both transformation results accumulated measurable xylanase activity that could contribute to expression of the recombinant BD7436 gene. All 10 seeds tested positive for xylanase activity (> 10 units / powder gram) had a clearly wrinkled or shrunk appearance. In contrast, six seeds tested negative for xylanase activity (≦ 1 unit / powder gram) had a normal appearance. The results suggest that recombinant xylanase depolymerized the endogenous (arabino) silane substrate during seed development and / or maturation.

Example 49

Enhanced Starch Recovery from Corn Seeds Using Enzymes

Corn wet-milling includes the steps of dipping corn kernels, grinding corn kernels, and separating the kernel components. A work bench top assay (cracked corn assay) was developed to mimic the corn wet milling process.

A "cracked corn assay" was used to identify enzymes that enhance starch yield from corn seeds resulting in improved efficiency of the corn wet milling process. Enzyme delivery was by exogenous addition, transformed corn seeds or a combination of both. In addition to using enzymes to facilitate the separation of corn components, removal of SO ₂ from this process is also shown.

Crack corn black

One gram of seed was soaked overnight at 4000, 2000, 1000, 500, 400, 40 or 0 ppm SO ₂ at 50 ° C. or 37 ° C. Seeds were cut in half to remove embryos. Each half seed was cut in half again. Dipping water was preserved from each soaked seed sample and diluted to a final concentration ranging from 400 ppm to 0 ppm SO ₂ . Two milliliters of immersion water, with or without enzyme, was added to the regerminated seeds and the sample was left at 50 ° C. or 37 ° C. for 2-3 hours. Each enzyme was added at 10 units per sample. All samples were vigorously stirred approximately every 15 minutes. After 2-3 hours, the samples were filtered through a mira cloth into a 50 ml centrifuge tube. Seeds were washed with 2 ml of water and samples were collected with the first supernatant. The sample was centrifuged at 3000 rpm for 15 minutes. After centrifugation, the supernatant was poured out and the pellet was dried at 37 ° C. All pellet weights were recorded. Starch and protein measurements were also performed on the samples to determine the starch: protein ratio released during treatment (data not shown).

Analysis of T1 and T2 Seeds from Corn Plants Expressing 6GP1 Endoglucanase in Cracked Corn Assays

When analyzed by the cracked corn assay, transgenic corn (pNOV4819 and pNOV4823) containing thermophilic endoglucanase performed well. The recovery of starch from pNOV4819 strain was found to be more than two times higher in seed expressing endoglucanase when immersed in 2000 ppm SO ₂ . Addition of protease and cellobiohydrolase to endoglucanase seeds increased starch recovery approximately 7-fold of control seeds. See Table 16.

Cracked Corn Assay for Endoglucanase (pNOV4820) Expressed in Cytoplasm. Control week, A188 / HiII; pNOV4819 note, 42C6A-1-21 and 27 Corn Note process Starch pellet weight (mg) Al88 / HiII Control No enzyme 28.4 Al88 / HiII Control Bromelain / C8546 10U 109.3 42C6A-1-21 No enzyme 52.6 42C6A-1-21 Bromelain / C8546 10U 170.4 42C6A-1-27 No enzyme 60.5 42C6A-1-27 Bromelain / C8546 10U 207.5

Similar results were seen in transformed seeds containing endoglucanase targeted with ER in endosperm (pNOV4823), which again resulted in a 2-7 fold increase in starch recovery when compared to control seeds. See Table 17.

Cracked corn assay for ER expressed endoglucanase (pNOV4823). Control week, A188 / HiII; pNOV4823 note, 101D11A-1-28. week process Starch pellet weight (mg) Starch pellet weight (mg) Average weight A188 / Hill No enzyme 22.5 19.1 20.8 101D11A-1-28 No enzyme 41.2 32 36.6 A188 / Hill 10U Bromelain / C8546 78.6 73.8 76.2 101D11A-1-28 169.8 132.6 151.2

These results confirm that expression of endoglucanase enhances the separation of starch and protein components of corn seeds. In addition, this may indicate that the reduction or removal of SO2 during the soaking process typically results in starch recovery comparable to or better than that of the soaked control seeds. See Table 18. Removal of high levels of SO 2 from the wet milling process can provide a value added benefit.

Example 50

Construction of Transformation Vectors for Corn Optimized Bromelain

Expression cassettes were constructed to express maize optimized bromelain in maize endosperm with various targeting signals as follows:

pSYN11000 (SEQ ID NO: 73) comprises a synthetic Bromelain sequence fused with a Bromelain signal sequence (MAWKVQVVFLFLFLCVMWASPSAASA) (SEQ ID NO: 72) and a C-terminal addition of the sequence VFAEAIAANSTLVAE for targeting to PVS and retention in PVS. (Vitale and Raikhel Trends in Plant Science Vol 4 no. 4pg 149-155). This fusion was cloned behind the corn gamma agent promoter for specific expression in endocrine.

pSYN11587 (SEQ ID NO: 75) comprises a synthetic bromelain sequence along with a bromelain signal sequence (MAWKVQVVFLFLFLCVMWASPSAASA) and a C-terminal addition of the sequence SEKDEL for targeting to and retention in the cytoplasmic reticulum (ER) ( Munro and Pelham, 1987). This fusion was cloned behind the corn gamma agent promoter for specific expression in endocrine.

pSYN11589 (SEQ ID NO: 74) is a bromelain signal sequence (MAWKVQVVFLFLFLCVMWASPSAASA) (SEQ ID NO: 72) fused to soluble vacuole targeting sequence SSSSFADSNPIRVTDRAAST (Neuhaus and Rogers Plant Molecular Biology 38: 127-144, 1998) Synthetic bromelain for targeting to vacuoles. This fusion was cloned behind the corn gamma agent promoter for specific expression in endocrine.

pSYN12169 (SEQ ID NO: 76) includes a corn γ-zein N-terminal signal sequence (MRVLLVALALLALAASATS) (SEQ ID NO: 17) fused to synthetic bromelain for targeting to cytoplasmic networks and secretion of apoptoplasm (SEQ ID NO: 17). See Torrent et al. 1997]. This fusion was cloned behind the corn gamma agent promoter for specific expression in endocrine.

pSYN12575 (SEQ ID NO: 77) includes waxy starch targeting peptides (Klosgen et al., 1986) fused with synthetic bromelain for targeting to starch. This fusion was cloned behind the gamma agent promoter for specific expression in endocrine.

pSM270 (SEQ ID NO: 78) is used for targeting to a bromline N-terminal signal sequence and soluble vacuole fused to a soluble vacu targeting sequence sequence SSSSFADSNPIRVTDRAAST (Neuhaus and Rogers Plant Molecular Biology 38: 127-144, 1998). Synthetic bromelain. The fusions were cloned behind whistle specific promoter P19 (US Pat. No. 6392123) for specific expression in whistle.

Example 51

Expression of Bromelain in Corn

Seeds from the T1 transgenic strains transformed with the vector comprising the synthetic bromelain gene with the targeting sequence for expression at various subcellular locations of the seed were analyzed for protease activity. Seeds were ground for 30 seconds with a Kleco grinder to produce corn powder. The enzyme was extracted from 100 mg of powder using 50 mM NaOAc at pH 4.8 or 50 mM Tris buffer at pH 7.0 containing 1 mM EDTA and 5 mM DTT. The sample was vigorously stirred and then left at 4 ° C. with continued stirring for 30 minutes. Extracts from each transgenic strain were assayed using Lesoruppin labeled casein (Roche, Cat. No. 1 080 733) as outlined in the product booklet. Powders from T2 seeds were subjected to the following modifications. Methods in Enzymology Vol. 244: Pg 557-558], assayed using a bromelain specific assay as outlined in P. 557-558. Corn Seed Powder 100mg pH 7.0 50mM Na ₂ HPO ₄ / 50mM NaH ₂ P0 ₄ Extracted at 4 ° C. for 15 minutes using 1 ml, 1 mM EDTA +/− 1 μM leupeptin. The extract was centrifuged at 4 ° C. for 5 minutes at 14,000 rpm. Extraction was performed twice. Powders from T2 transgenic strains were assayed for bromelain activity using Z-Arg-Arg-NHMec (Sigma) as substrate. 100㎕ / 4 two aliquots of 100mM Na ₂ HPO, pH 7.0 per well of corn seed extract ₄ / 100mM NaH ₂ P0 ₄ It was added to a 96 well bottom plate (Corning) containing 50 μl, 2 mM EDTA, 8 mM DTT. The reaction was started by adding 50 μl of 20 μM Z-Arg-Arg-NHMec. The reaction rate was observed using SpectraFluorPlus (Tecan) adjusted with 360 nm excitation and 465 nm emission filters at 2.5 min intervals at 40 ° C.

Table 19 shows the analysis of seeds from different T1 bromelain outputs. Bromelain expression was found to be 2-7 times higher than A188 and JHAF control weeks. T1 transformed strains were replanted and T2 seeds obtained. Analysis of T2 seeds showed expression of bromelain. FIG. 21 shows a bromelain activity assay using Z-Arg-Arg-NHMec in T2 seeds for ER targeted (11587) and soluble vacuole targeted (11589) bromelain.

Analysis of T2 Seeds from Corn Plants Expressing Bromelain

Seeds from the T2 transgenic bromelain strain, 11587-2, were analyzed in a cracked corn assay for enhanced starch recovery. Previous experiments with exogenously added bromelain have been tested alone and show increased starch recovery when tested with other enzymes, especially cellulase. T2 seeds from 11587-2 showed a 1.3-fold increase in recovered starch over control seeds when soaked overnight at 37C / 2000 ppm SO2. More importantly, the addition of cellulase (C8546) increased twice as much as starch from the T2 bromelain strain when the seeds were soaked at 37 C / 2000 ppm SO2.

Transformed strains showed a similar tendency of increased starch than control seeds when the seeds were soaked in 37C / 400 ppm SO2. A 1.6-fold increase in starch recovery was seen in the transgenic seeds and a 2.1-fold increase in starch without the addition of cellulase (C8546). See Table 20.

These results are significant, indicating that this is possible at reduced temperatures and SO 2 concentrations, but also increases starch recovery during the wet milling process when using transgenic seeds expressing bromelain.

Summary of Grain Specific Expression of Bromelain in T1 Corn Week number Targeting Construct “Specifically active” bromelain ng / protein 11000-1 Vacu GZP / Probromelain / Barley PVS 252 11000-2 Vacu GZP / Probromelain / Barley PVS 277 11000-3 Vacu GZP / Probromelain / Barley PVS 284 11587-1 ER GZP / Probromelain / KDEL 174 11587-1 ER GZP / Probromelain / KDEL 153 11589-1 Soluble vacuole GZP / Allurein SS / Probromelain 547 11589-2 Soluble vacuole GZP / Allurein SS / Probromelain 223 A188 control 56 JHAF control 75

Cracked Corn Assay Results for T2 Bromelain Seeds Immersion conditions week Starch pellet weight (mg) 2000ppm SO2 2000ppm SO2 A188 A188 / C8546 (10 units) 41.3 44 2000ppm SO2 2000ppm SO2 11587-2 11587-2 / C8546 (10 units) 57.4 94.6 400ppm 400ppm A188 A188 / C8546 (10 units) 30.7 35.8 400ppm 400ppm 11587-2 11587-2 / C8546 (10 units) 50.5 86.6

Example 52

Construction of Transformation Vectors for Corn Optimized Ferulic Acid Esterase

Expression cassettes for expressing corn optimized ferulic acid esterase in corn endosperm with or without various targeting signals were constructed as follows.

Plasmid 13036 (SEQ ID NO: 101) comprises a corn optimized ferulic acid esterase (FAE) sequence (SEQ ID NO: 99). The sequence was cloned behind the corn gamma agent promoter without any target sequence for specific expression in the cytoplasm of endocrine.

Plasmid 13038 (SEQ ID NO: 103) comprises a N-terminal signal sequence (MRVLLVALALLALAASATS) (signal sequence 17), a corn γ-agent fused to synthetic FAE for targeting to cytoplasmic reticulum and secretion of apoplasts. Torrent et al. 1997]. This fusion was cloned behind the corn gamma agent promoter for specific expression in endocrine.

Plasmid 13039 (SEQ ID NO: 105) is a waxy starch targeting peptide (MLAALATSQLVATRAGLGVPDASTFRRGAAQGLRGARASAAAD TLSMRTSARAAPRHQHQQARRGARFPSLVVCASAGA) fused to synthetic FAEs for targeting to starch (see Klosgen et al., Et al., 1986 et al.). al. 1997]. This fusion was cloned behind the gamma agent promoter for specific expression in endocrine.

Plasmid 13347 (SEQ ID NO: 107) is a corn γ-agent N-terminal signal sequence (MRVLLVALALLALAASATS) fused to a synthetic FAE with C-terminal addition of the sequence SEKDEL for targeting to and retention within cytoplasmic reticulum (ER). (Signal sequence 17) (Munro and Pelham, 1987). This fusion was cloned behind the gamma agent promoter for specific expression in endocrine.

All expression cassettes were inserted into the binary vector pNOV2117 for transformation into maize via Agrobacterium infection. The binary vector contained a phosphomannose isomerase (PMI) gene that allows the selection of transformed cells with mannose. Transformed corn plants were self-pollinated or crossbred and seeds were harvested for analysis.

Combinations of these enzymes can be made by crossing plants expressing individual enzymes or by cloning several expression cassettes into the same binary vector to enable cotransformation.

Example 53

Hydrolysable Degradation of Corn Fiber by Ferulic Acid Esterase

Corn fiber is a major by-product of corn wet and dry milling. The fiber component mainly consists of coarse fibers derived from seed husks (shells) and whistle and fine fibers derived from small amounts of endocrine cell walls. Ferulic acid, hydroxycinnamic acid, which results in crosslinking of the lignin, hemicellulose and cellulose components of the cell wall, is found in high concentrations in the cell walls of grain grains. Enzymatic degradation of ferulic acid crosslinking is an important step in the hydrolysis of corn fiber and can lead to accessibility of further enzymatic degradation by other hydrolytic enzymes.

Ferulic Acid Esterase Activity Assay

Ferulic acid esterase, FAE-1 (a corn optimized synthetic gene from C. thermocelum). Expressed in E. coli. Cells were harvested and stored overnight at -80 ° C. The harvested bacteria were suspended in 50 mM Tris buffer at pH 7.5. Lysozyme was added at a final concentration of 200 ug / ml and the sample was incubated for 10 minutes at room temperature with gentle stirring. The sample was centrifuged at 4000 rpm for 15 minutes at 4 ° C. After centrifugation, the supernatant was transferred to a 50 ml conical tube and placed in a 70 ° C. water bath for 30 minutes. Samples were then centrifuged at 4000 rpm for 15 minutes and the cleared supernatant was transferred to conical tubes (Blum et al. J Bacteriology, Mar 2000, pg 1346-1351).

Recombinant FAE-1 was tested for activity using 4-methylumbeliferyl ferulate as described in Mastihubova et al (2002) Analytical Biochemistry 309 96-101. Recombinant protein FAE-1 (104-3) was diluted 10, 100 and 1000 fold and assayed. Activity assay results are shown in FIG. 22.

Preparation of Corn Seed Fiber

Corn hull coarse fibers were separated by soaking yellow maize corn # 2 grains at 2000 ppm sodium metabisulfite (Aldrich) for 48 hours at 50 ° C. The grain was mixed with equal parts of water and mixed with a Waring laboratory heavy duty mixer with reverse blades. The mixer was controlled at 50% voltage output for 2 minutes with various automatic transformers (Staco Energy). The mixed material was washed with tap water on Standard Tester # 7 (Fisher scientific) to separate the coarse fibers from the starch fraction. Coarse fibers and embryos were separated by floating fibrous diagrams from embryos with hot tap water in a 4L beaker (Fisher scientific). The fibers were then soaked in ethanol and dried at 60 ° C. overnight in a vacuum oven. Corn coarse fibers derived from corn kernel skin were milled to a 0.5 mm particle size with a laboratory mill 3100 equipped with a milling distribution line 3170 (Perten instrument).

Corn Fiber Hydrolysis Black

Coarse fibers (CF) were suspended in 30 mg / 5 ml buffer in 50 mM citrate-phosphate buffer at pH 5.2. The CF mother liquor was stirred vigorously and transferred to a 40 ml modular reservoir (Beckman, Cat. No. 372790). After well mixing the solution, 100 ul was transferred to a 96 well plate (Corning Inc., Cat. No. 9017, Polystyrene, Flat). Enzyme was added to 1-10 ul / well and the final volume was adjusted to 110 ul using buffer. The CF background control contained only 10ul of buffer. The plate was sealed with aluminum foil and kept stirring to incubate at 37 ° C. for 18 hours. Plates were centrifuged at 4000 rpm for 15 minutes. 1-10 ul of CF supernatant was transferred to a 96 well plate reloaded with 100 ul of BCA reagent (BCA-Reagent: Reagent A (Pierce, Prod. # 23223), Reagent B (Pierce, Prod. # 23224)). The final volume was adjusted to 110 ul. The plate was sealed with aluminum foil and left at 85 ° C. for 30 minutes. After incubation at 85 ° C., the plates were centrifuged at 2500 rpm for 5 minutes. Absorbance readings were read at 562 nm (Molecular Devices, Spectramax Plus). Samples were quantified with D-glucose and D-Xylose (Sigma) calibration curves. Assay results are reported in total sugars released.

Determination of Total Sugars Released by Ferulic Acid Esterase in Corn Seed Fiber Hydrolysis Assay

Recombinant FAE-1 fiber hydrolysis assay results did not show any increase in total reducing sugar (data not shown). These results were unexpected because it has been reported in the literature that only an increase in total reducing sugars is detectable when other hydrolytic enzymes are used with FAE (see Yu et al J. Agric. Food Chem. 2003, 51, 218-223). FIG. 23 shows that the addition of FAE-2 to fungal supernatants grown on corn fiber represents and increases total reducing sugars. This suggests that FAE plays an important role in corn fiber hydrolysis.

FIG. 23 shows the results of a corn fiber hydrolysis assay showing an increase in the release of total reducing sugars from corn fiber with addition of FAE-2 to fungal supernatant (FS9).

Analysis of Ferulic Acid Released from Corn Seed Fiber by FAE-1

After examining FAE activity on corn fiber, ferulic acid was described as described in a slightly modified literature (Waldron and Parr (1996) (Waldron. KW. Parr AJ 1996 Vol 7 pages 305-312 Phytochem Anal)). Released. Corn coarse fibers derived from corn kernel skin were milled to a particle size of 0.5 mm with a laboratory mill 3100 with milling distribution line 3170 (Perten instrument) and used as a substrate at a concentration of 10 mg / ml. 1 ml assay was performed in a 24-well Becton Dickenson Multiwell ^™ . Substrates were incubated in 50 mM citrate-phosphate at pH 5.4 at 50 ° C. at 110 rpm for 18 hours in the presence and absence of recombinant FAE. After the incubation period, the samples were centrifuged at 13,000 rpm for 10 minutes and then ethyl acetate extracted. All solvents and acids used were from Fisher Scientific. 0.8 ml of the supernatant was acidified with 0.5 ml of glacial acetic acid and extracted three times with the same volume of ethyl acetate. The organic fractions were mixed and dried at 40 ° C. using a speed vac (Savant). The sample was then suspended with 100 μl of methanol and used for HPLC analysis.

HPLC chromatography was performed as follows. Ferulic acid (ICN Biomedicals) was used as standard in HPLC analysis (data not shown). HPLC analysis was performed using a Hewlett Packard series 1100 HPLC system. The procedure was performed using a C ₁₈ total capped reversed phase column (XterraRp ₁₈ , 150 mm × 3.9 mm, id 5 μm particle size) operated at 1.0 ml min ⁻¹ at 40 ° C. At 32 minutes ferulic acid was eluted using a gradient of 25-70% (solvent A: H 2 O, 0.01% b TFA; solvent B: MeCN, 0.0075%).

As shown in Figure 24, FA released from corn fiber was 2-3 times higher than the control when treated with FAE-1 10 or 100ul. These results clearly show that FAE-1 can hydrolyze corn fiber.

Example 54

Functionality during Fermentation of Corn Expressed Glucoamylase and Amylase

This example demonstrates that corn-expressed enzymes will support fermentation of starch in corn slurries in the absence of added enzymes and without cooking corn slurries. Corn kernels comprising Rizopus duck glucoamylase (ROGA) (SEQ ID NO: 49) were prepared as described in Example 32. Corn kernels containing barley low-PI α-amylase (AMYI) (SEQ ID NO: 88) were prepared as described in Example 46. The following materials are used in this example:

Aspergillus niger glucoamylase (ANGA) was purchased from Sigma.

Rizopus spp. Glucoamylase (RxGA) was purchased as anhydrous crystalline powder from the manufacturer Wako and prepared at 10 mg / ml in 10 mM sodium acetate, 5 mM CaCl ₂ at pH 5.2.

MAMYI Microbial Preparation AMYI was prepared at approximately 0.25 mg / ml in 10 mM sodium acetate, 5 mM CaCl ₂ at pH 5.2.

Yeast was Saccharomyces cerevisiae.

YE was a sterile 5% solution of yeast extract in water.

Yeast starter contained 50 g maltodextrin, 1.5 g enzyme extract, 0.2 mg ZnSO ₄ in a total volume of 300 ml water. The medium was prepared and then autoclaved and sterilized. After cooling to room temperature, 1 ml of tetracycline (10 mg / ml in ethanol), 100 μl of AMG300 glucoamylase and 155 mg active anhydrous yeast were added. The mixture was then stirred at 30 ° C. for 22 hours. As described in Current Protocols in Molecular Biology, yeast cultures stirred overnight were diluted 1/10 with water and the number of yeast was determined by measuring A600.

ROGA Powder: Grains were collected from several T strains shown to have active glucoamylase. The seed was ground with Kleco and all powders were collected.

AMYI Powder: Grain from T0 corn expressing AMYI as described above was collected and polished.

Control Powder: Grains of similar genetic background were ground in the same manner as ROGA expressing corn.

The inoculation mixture was prepared in sterile tubes: it contained yeast cells (1 × 10 ⁷ ), yeast extract (8.6 mg), tetracycline (55 μg) per 1.65 ml. 1.65 ml was added per gram of powder to each fermentation tube.

Fermentation formulation: The powder was weighed at 1.8 g / tube with self weighing 17 × 100 mm sterile polypropylene. 50 μL of 0.9MH ₂ SO ₄ was added to reach final pH before fermentation to 5. The inoculation mixture (2.1 ml) was added per tube with RXGA, AMYI-P and amylase desalting buffer as described below. The amount of buffer was adjusted based on the water content of each powder so that the total solids content was constant in each tube. The tubes were mixed throughout, weighed and placed in plastic bags and incubated at 30 ° C.

Fermentation tubes were weighed at regular intervals over the course of 67 hours. The weight loss corresponds to the release of CO ₂ during fermentation. The ethanol content of the samples was determined after 67 hours of fermentation by the DCL ethanol assay method. Kit (Catalog No. 229-29) was purchased from Diagnostic Chemicals Limited, D1E 1B0, Prince Edward Charlottetown, Canada. Samples (10 μl) were extracted three times from each fermentation tube and diluted with 990 μl of water. 10 μl of the diluted sample was mixed with 1.25 ml of a 12.5 / 1 mixture of assay buffer / ADH-NAD reagent. Standards (0, 5, 10, 15 & 20% v / v ETOH) were diluted and assayed in the same manner. The reaction was incubated at 37 ° C. for 10 minutes before reading A340. Standards were prepared twice and samples from each fermentation were made three times (including the initial dilution). The weight of the sample varied over time as detailed in the table below. Weight loss is expressed as a percentage of the initial sample weight at time zero.

These data indicate that ROGA enzyme expressed in maize increases efficiencies compared to the control without enzyme. This also confirms previous data indicating that AMYI expressed in corn kernels is a potential activator of starch fermentation in corn. Ethanol content is detailed below.

These data also demonstrate that the expression of Rifupus duck glucoamylase in corn promotes increased starch fermentation in corn. Similarly, expression of barley amylase in corn makes corn starch more fermentable without the addition of exogenous enzymes.

Example 55

Cellobiohydrolase I

Trichoderma reesei ) Cellobiohydrolase I (CBH I) gene was amplified and cloned by RT-PCR based on published database sequence (Accession No. E00389). cDNA sequences were analyzed for the presence of signal sequences using the SignalP program, which predicted 17 amino acid signal sequences. The DNA sequence encoding the signal sequence was replaced with ATG by PCR as shown by the sequence (SEQ ID NO: 79). Subsequent constructs were prepared using this cDNA sequence. Additional constructs were prepared by substituting the corn optimized form of the gene (SEQ ID NO: 93).

Example 56

Cellobiohydrolase II

Trichoderma Reese Cellobiohydrolase II (CBH II) gene was amplified and cloned by RT-PCR based on published database sequence (Accession No. M55080). cDNA sequences were analyzed for the presence of signal sequences using the SignalP program, which predicted 18 amino acid signal sequences. The DNA sequence encoding the signal sequence was replaced with ATG by PCR as shown by the sequence (SEQ ID NO: 81). Subsequent constructs were prepared using this cDNA sequence. Additional constructs were prepared by substituting the corn optimized form of the gene (SEQ ID NO: 94).

Example 57

Construction of Transformation Vectors for Tricoderma Rishi Cellobiohydrolase I and Cellobiohydrolase II

Cloning of the Tricoderma lishi cellobiohydrolase I ( cbhi ) cDNA of native N-terminal signal sequence Radix is described in Example 55. Expression cassettes were constructed to express Trichoderma reesei cellobiohydrolase I cDNA in corn endosperm with various targeting signals as follows.

Plasmid 12392 contains a Trichoderma lishi cbhi cDNA cloned behind the γ zein promoter for expression in the endocrine for expression in the cytoplasm.

Plasmid 12391 contains a corn γ-agent N-terminal signal sequence (MRVLLVALALLALAASATS (signal sequence 17)) fused to a Trichoderma lishi cbhi cDNA as described above in Example 1 for targeting to cytoplasmic networks and secretion into apoplasts . (See Torrent et al. 1997). This fusion was cloned behind the corn gamma agent promoter for specific expression in endocrine.

Plasmid 12392 contains an N-terminal signal sequence, a corn γ-zein fused to a Trichoderma lishi cbhi cDNA with a C-terminal addition of the sequence KDEL for targeting to and retention within cytoplasmic reticulum (ER) (see, Munro and Pelham, 1987]. This fusion was cloned behind the gamma agent promoter for specific expression in endocrine.

Plasmid 12656 includes waxy starch targeting peptides (Klogen et al., 1986) fused to Trichoderma licci cbhi cDNA for efficacy setting into starch. This fusion was cloned behind the corn γ zein promoter for specific expression in endocrine.

All expression cassettes were inserted into the binary vector (pNOV2117) for transformation into maize via Agrobacterium infection. The binary vector contained a phosphomannose isomerase (PMI) gene that allows the selection of transformed cells with mannose. Transformed corn plants were self-pollinated or crossbred and seeds were harvested for analysis.

Add Preparation of a construct (plasmid 12 652, 12 653, 12 654 and 12 655), the Trichoderma receiver cbhi fine move in the same manner as described with respect to the cDNA Trichoderma receiving cell bio-hydrolase II (cbhii) above targeting fused to cDNA signals It was. These fusions were cloned behind the corn Q protein promoter (50 Kd γ zein) (SEQ ID NO: 98) for specific expression in endocrine and transformed with corn as described above. Transformed corn plants were self-pollinated or crossbred and seeds were harvested for analysis.

Combinations of these enzymes can be made by crossing plants expressing individual enzymes or by cloning several expression cassettes into the same binary vector to enable cotransformation.

Example 58

Expression of Cbhi in Corn

T1 seeds from self-pollinated corn plants transformed with plasmid 12390, 12391 or 12392 were obtained. 12390 construct targets expression of CbhI in endocrine cytoplasmic network, 12391 construct targets expression of CbhI in endocrine apoplasts, and 12392 construct expresses expression of CbhI in endocrine cytoplasm To target.

Extraction and Detection of CbhI from Corn Powder: Polyclonal antibodies against CbhI and CbhII were prepared in goats according to established protocols. Powders from CbhI transgenic seeds were obtained by grinding them in an Autogizer polisher. Approximately 50 mg of powder was resuspended in 0.5 ml of 20 mM NaPO ₄ buffer, pH 7.4, 150 mM NaCl, and then incubated for 15 minutes at room temperature with continued stirring. The incubated mixture was then spun at 10,000 × g for 10 minutes. Supernatant was used as the enzyme source. 30 μl of this extract was loaded on a 4-12% NuPAGE gel (invitrogen) and isolated in NuPAGE MES running buffer (invitrogen). Proteins were blotted onto nitrocellulose membranes and Western blot analysis was performed according to a confirmed protocol using rabbit anti-chlorine IgG (H + L) conjugated with alkaline phosphatase following the specific antibodies described above. Alkaline phosphatase activity was detected by incubating the membrane with a ready-made BCIP / MBT (plus) substrate from Moss Inc.

Western blot analysis was performed on T1 seeds from different results transformed with plasmid 12390. Expression of CbhI protein was compared to non-transformation control and detected in a number of results.

A cracked maize assay was performed essentially as described in Example 49, using transformed seeds expressing Cnhi. Starch recovery from the transgenic seeds was measured and the results are stated in Table 24.

Week 3-Not Expressed Week 4 CBHI Expression Condition Starch (mg) 400 ppm SO2-without bromelain 40.2 78.1 400 ppm SO2-bromelain added 48.1 118.7 2000 ppm SO2-without bromelain 47.5 73.1 2000 ppm SO2-bromelain added 49.2 109

Example 59

Preparation of Endoglucanase I Constructs

The Trichoderma Reese endoglucanase I (EGLI) gene is disclosed in published database sequences (Accession No. M15665; Based on Penttila et al., 1986, it was amplified and cloned by PCR. Since only genomic sequences can be obtained, cDNA was prepared from genomic sequences by removing two introns using overlapping PCR. The cDNA sequences obtained were analyzed for the presence of signal sequences using the SignalP program, which predicted 22 amino acid signal sequences. The DNA sequence encoding the signal sequence was replaced with ATC by PCR as shown by the sequence (SEQ ID NO: 83). This cDNA sequence was used to prepare a continuous construct as described below.

Nested PCR

Nested PCR is a technique used to fuse the complementary ends of two or more PCR products (Ho et al., 1989), adding base pair (bp) changes, adding bp or cutting bp Can be used to At the location of the intended bp change, forward and reverse mutant primers (Mut-F and Mut-R) are prepared comprising the 15 bp sequence on each side of the intended change and change. For example, to remove introns, the primer would have to consist of the last 15 bp of exon 1 fused to the first 15 bp of exon 2. Also prepared are primers that anneal to the ends of the sequences to be amplified (eg, ATG and stop codon primers). PCR amplification of this product proceeds with ATG / Mut-R primer pairs and Mut-F / termination primers in independent reactions. The product is gel purified and fused together in PCR without primer addition. The fusion reaction is separated from the gel and the correct size band is gel purified and cloned. Multiple changes can be achieved simultaneously by adding additional mutagenic primer pairs.

EGLI Plant Expression Constructs

An expression cassette was prepared to express Trichoderma Reese EGLI cDNA in corn endosperm as follows.

13025 was cloned after the γ zein promoter for cytoplasmic localization and specific expression in endocrine. Contains the assay EGLI gene.

13026 is used for targeting to cytoplasmic reticulum and secretion into apoplasts. N-terminal signal peptide (MRLLVALALLALAASATS), a corn γ-agent fused to the Risei EGLI gene. This fusion was cloned behind the corn gamma agent promoter for specific expression in endocrine.

13027 contains a C. terminal addition of the sequence KDEL for targeting to and retention within cytoplasmic reticulum (ER). N-terminal signal sequence, a maize γ-agent fused to the Risei EGLI gene. This fusion was cloned behind the gamma agent promoter for specific expression in endocrine.

13028 is used to establish the efficacy of the starch in the lumen. Corn granules bound starch synthase I (GBSSI) N-terminal signal peptide (N-terminal 77 amino acids) fused to the Risei EGLI gene. This fusion was cloned behind the corn γ zein promoter for specific expression in endocrine.

13029, with the C-terminal addition of the starch binding domain (C-terminal 301 amino acids) of the corn GBSSI gene, for targeting to starch granules. Corn GBSSI N-terminal signal peptide fused to the Risei EGLI gene. This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

Additional expression cassettes are prepared using a corn optimized form of EGLI (SEQ ID NO: 95).

EGLI enzyme assay

EGLI enzyme activity is measured in maize transformants using the Malt beta-glucanase assay kit (Cat. No. K-MBGL) (Megazyme International Ireland Ltd.). Enzymatic activity of the EGLI expresser is examined by a corn fiber hydrolysis assay as described in Example 53.

Example 60

β-glucosidase 2

The Tricoderma Reese β-glucosidase 2 (BGL2) gene was amplified and cloned by RT-PCR based on SEQ ID NO: AB003110 (Takashima et al., 1999).

BGL2 Plant Expression Construct

An expression cassette was prepared to express Trichoderma Reese BGL2 cDNA (SEQ ID NO: 89) in corn endosperm as follows.

13030 was cloned behind the γ zein promoter for cytoplasmic localization and specific expression in endocrine. Contains the assay BGL2 gene.

13031 is used for targeting to cytoplasmic reticulum and secretion into apoplasts. N-terminal signal peptide (MRLLVALALLALAASATS), a corn γ-agent fused to the Reese BGL2 gene. This fusion was cloned behind the corn gamma agent promoter for specific expression in endocrine.

13032 contains a C. terminal addition of the sequence KDEL for targeting to and retention therein. N-terminal signal sequence, a maize γ-agent fused to the Risei BGL2 gene. This fusion was cloned behind the gamma agent promoter for specific expression in endocrine.

13033 is used to establish the efficacy of the starch in the lumen. Corn granules bound starch synthase I (GBSSI) N-terminal signal peptide (N-terminal 77 amino acids) fused to the Reese BGL2 gene. This fusion was cloned behind the corn γ zein promoter for specific expression in endocrine.

13034, with the C-terminal addition of the starch binding domain (C-terminal 301 amino acids) of the corn GBSSI gene, for targeting to starch granules. Corn GBSSI N-terminal signal peptide fused to the Reese BGL2 gene. This fusion was cloned behind the corn γ-zein promoter for specific expression in endocrine.

Additional expression cassettes are prepared by substituting the corn optimized form of BGL2 (SEQ ID NO: 96).

All expression cassettes were inserted into the binary vector (pNOV2117) for transformation into maize via Agrobacterium infection. The binary vector contained a phosphomannose isomerase (PMI) gene that allows the selection of transformed cells with mannose. Transformed corn plants were self-pollinated or crossbred and seeds were harvested for analysis.

BGL2 Enzyme Assay

The BGL2 enzyme assay is a protocol modified by the author Bauer and Kelly (Bauder, MW and Keely, RM 1998. The family 1 β-glucosidases from Pyrococcus furiosus and Agrobacterium faecalis share a common catalytic mechanism. Biochemistry 37: 17170-17178). The protocol can be modified to incubate the sample at 37 ° C. instead of 100 ° C. Enzymatic activity of BGL2-expressors is examined by fiber hydrolysis assay.

Example 61

β-glucosidase D

Trichoderma lysase β-glucosidase D (CEL3D) gene is disclosed in published database sequences (Accession No. AY281378; Amplification was based on Foreman et al., 2003 and cloned by PCR. Since only genomic sequences can be obtained, as described in Example 58, cDNA was prepared from genomic sequences by removing introns using overlapping PCR. The cDNA obtained (SEQ ID NO: 91) can be used for successive constructs. The corn optimized form of the obtained cDNA (SEQ ID NO: 97) can also be used for the construct.

Plant constructs can be prepared and BGL2 replaced with CEL3D to perform the β-glucosidase assay as described for BGL2 in Example 60.

Example 62

Lipase

CDNA encoding lipases are described in SEQ ID NOs: D85895, AF04488 and AF04489 (Tsuchiya et al., 1996; Yu et al., 2003) and sequences 59-60 It is prepared using the methodology.

Lipase enzyme activity can be measured in transgenic maize using a fluorescent lipase assay kit (Cat. No. M0612) (Maker Gene Technologies, Inc.). Lipase activity was also determined in vivo using fluorescent substrate 1, 2-dioleoyl-3- (pyren-1-yl) decanoyl-rac-glycerol (M0258) (also manufactured by Maker Gene Technologies, Inc.). Can be measured at.

Example 63

Expression of phytase in rice

Vectors 11267 and 11268 contain binary vectors encoding Nov9x phytase. Expression of Nov9x phytase in both vectors is under the control of the rice glutelin-1 promoter (SEQ ID NO: 67). Vectors 11267 and 11268 are from pNOV2117.

The Nov9x phytase expression cassette in vector 11267 includes a rice glutelin-1 promoter, a Nov9x phytase gene with an apoplast targeting signal, a PEPC intron and a 35S terminator. The product of the Nov9x phytase coding sequence in vector 11267 is shown by SEQ ID NO: 110.

The Nov9x phytase expression cassette in vector 11268 includes a rice glutelin-1 promoter, a Nov9x phytase gene with ER retention (SEQ ID NO: 111), a PEPC intron and a 35S terminator. The product of the Nov9x phytase coding sequence in vector 11268 is shown as SEQ ID NO: 112.

11267 Nov9x phytase with apoplast targeting DNA sequence (SEQ ID NO: 109). Detoxification start and stop codons are underlined. Sequences encoding the signal sequence of the 27-kD gamma-zein protein are shown in bold.

11267 Nov9x phytase with apoplast targeting gene product (SEQ ID NO: 110). The signal sequence of the 27-kD gamma-zein protein is shown in bold.

11268 Nov9x phytase with ER retention DNA sequence (SEQ ID NO: 111). Sequences encoding the signal sequence of the 27-kD gamma-zein protein are shown in bold. The sequence encoding the SEKDEK hexapeptide ER retention signal is underlined.

11268 Nov9x phytase, gene product with ER retention sequence (SEQ ID NO: 112). The signal sequence of the 27-kD gamma-zein protein is shown in bold. The ER retention signal is underlined.

Preparation of Transgenic Rice Plants

Rice (duck sativa) is used for the production of transformed plants. Various rice cultivars may be used (Hiei et al., 1994, Plant Journal 6: 271-282; Dong et al., 1996, Molecular Breeding 2: 267-276; Hiei et al., 1997, Plant Molecular Biology , 35: 205-218]. In addition, the various media components described below may vary in concentration or may be substituted. Initiate the embryogenic reaction and / or MS-CIM medium (MS base salt, 4.3 g / liter; B5 vitamin (200 ×), 5 ml / liter; sucrose, 30 g / liter; proline, 500 mg / liter; glutamine, 500 mg / liter) ; Casein hydrolysate, 300 mg / liter; 2,4-D (1 mg / ml), 2 ml / liter; adjusted to pH 5.8 with 1N KOH; phytagel, 3 g / liter) to obtain a culture from mature embryos . Mature embryos or obtained culture strains at the initial stage of the culture reaction are inoculated and co-cultured with Agrobacterium strain LBA4404 comprising the desired vector construct. Agrobacterium is incubated at 28 ° C. for ˜2 days from glycerol mother liquor on solid YPC medium (100 mg / L spectinomycin and any other suitable antibiotic). Agrobacterium is resuspended in liquid MS-CIM medium. Agrobacterium culture is diluted with an OD600 of 0.2-0.3 and acetosyringone is added at a final concentration of 200 uM. After Agrobacterium is induced with acetosyringone, the solution is mixed with rice culture. For inoculation, cultures are infiltrated with bacterial suspensions. The liquid bacterial suspension is removed and the inoculated culture is placed on co-culture medium and incubated at 22 ° C. for 2 days. The culture is then transferred to MS-CIM medium with ticarcillin (400 mg / liter) to inhibit the growth of Agrobacterium. For constructs using the PMI selectable marker gene (Reed et al., In Vitro Cell. Dev. Biol.-Plant 37: 127-132), the cultures were subjected to a carbohydrate source (2%) after 7 days. Mannose, MS with 300 mg / liter ticarcillin) is transferred to selection medium containing mannose and incubated in the dark for 3-4 weeks. Resistant colonies were then transferred to regeneration induction medium (MS without 2,4-D, 0.5 mg / liter IAA, 1 mg / liter gelatin, 200 mg / liter ticarcillin, 2% mannose and 3% sorbitol) and dark Incubate for 14 days at. Proliferating colonies are then transferred to another circular regeneration induction medium and transferred to the light source culture chamber. The regenerated shoots are transferred to GA7-1 medium (MS without hormones and with 2% sorbitol) for 2 weeks and then transferred to the greenhouse if they are large enough and have suitable roots. Plants are planted in soil in a greenhouse and incubated until maturity.

Example 64

Analysis of Transgenic Rice Seeds Expressing Nov9X Phytase

ELISA for Quantification of Nov9X Phytase from Rice Seeds

Quantification of phytase expressed in transgenic rice was assayed by ELISA. One (1 g) rice seed was ground to powder in a Kleco seed grinder. 50 mg of powder was resuspended in sodium acetate buffer described in the Example for Nov9X Phytase Activity Assay and diluted as needed for immunoassay. Nov9X immunoassay is a quantitative sandwich assay for the detection of phytase using two polyclonal antibodies. Rabbit antibodies were purified using Protein A, goat antibodies were E. coli. It had purified immunoaffinity against recombinant phytase (Nov9X) protein prepared in E. coli inclusions. Using these highly specific antibodies, the assay can measure picogram levels of phytase in transformed plants. There are three basic parts to the test. The phytase protein in the sample is captured on solid phase microtiter wells using rabbit antibodies. A "sandwich" is then formed between the solid phase antibody, the phytase protein and the second antibody added to the wells. After the washing step, the unbound second antibody is removed and the bound antibody is detected using alkaline phosphatase-labeled antibody. Substrates for enzymes are added and color expression is measured by reading the absorbance of each well. The standard curve uses a four-parameter curve fit to plot concentration versus absorbance.

Phytase activity assay

Determination of phytase activity, based on the estimation of inorganic phosphate released upon hydrolysis of phytic acid, is described by Engelen, A.J. et al., J. AOAC, Inter., 84, 629 (2001)]. One unit of enzyme activity is defined as the amount of enzyme that liberates 1 μmol of inorganic phosphate per minute under assay conditions. For example, 2.0 ml of enzyme preparation can be incubated at 37 ° C. for 60 minutes with 4.0 ml of 9.1 mM sodium phytate in 250 mM sodium acetate buffer at pH 5.5 with 1 mM CaCl 2 added to determine phytase activity. After incubation, the reaction is terminated by addition of 4.0 ml of a color-terminating reagent consisting of an equal portion of 10% (w / v) ammonium molybdate and 0.235% (w / v) ammonium vanadate solution. The precipitate is removed by centrifugation and the released phosphate salt is measured spectrophotometrically against a set of phosphate standards at 415 nm. Phytase activity is calculated by inserting A415 nm absorbance values obtained from phytase containing samples using the calculated phosphate standard curve.

The process can be scaled down and adjusted to the desired container to adjust for smaller volumes. Preferred containers include glass test tubes and plastic microplates. Partial immersion of the reaction vessel (s) in the water bath is essential to maintain a constant temperature during the enzymatic reaction.

Transformation Week Μg phytase / g powder ^* Phytase active units per gram powder ^** Endogenous Inorganic Phosphate (μmol / g g) released during cooking of stripped rice Endogenous Inorganic Phosphate (μmol / g g) released during cooking of peeled, polished rice seeds Wild type 0 0 1.442 0.469 One 510 916 1.934 0.840 2 1518 2800 2.894 1.703 ^* Μg phytase was assayed by sandwich ELISA ^** phytase activity was assayed by phytase activity assay as described above.

Assay of Inorganic Phosphate Release in Cooked Transgenic Rice Expressing Phytase

Two samples of 1 g of seed from selected rice transgenic and control wild-type strains were stripped using a benchtop Kett TR200 automatic rice peeler. One sample was then polished for 30 seconds in a Kett rice mill. Two volumes of H 2 O were added to each sample, and the rice was cooked by infiltrating the tube with a beaker containing water. The water was boiled and kept at total rotary boiling for 10 minutes. The “cooked” rice seeds were then ground with a water-containing paste that brought the total volume of the te slurry to 6 ml. The slurry was centrifuged at 15,000 × g for 10 minutes and the clear supernatant was assayed for released endogenous inorganic phosphate. The assay of the released phosphate is based on color formation as a result of molybdate and vanadate ions complexed with inorganic phosphate and is measured spectrophotometrically at 415 nm as described in the Examples for Phytase Enzyme Activity. The results are shown in Table 24.

All documents, patents, and patent applications are incorporated herein by reference. Although the invention has been described in the foregoing specification with reference to certain preferred embodiments thereof, many details are set forth for the purpose of illustration, and one of ordinary skill in the art will appreciate that the invention has room for additional aspects and the specifics described herein. It will be appreciated that the details may vary considerably without departing from the basic principles of the invention.

Sequence list

<110> SYNGENTA PARTICIPATIONS AG <120> Self-processing plants and plant parts <130> 109846.317 <160> 112 <170> KopatentIn 1.71 <210> 1 <211> 436 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 1 Met Ala Lys Tyr Leu Glu Leu Glu Glu Gly Gly Val Ile Met Gln Ala  1 5 10 15 Phe Tyr Trp Asp Val Pro Ser Gly Gly Ile Trp Trp Asp Thr Ile Arg             20 25 30 Gln Lys Ile Pro Glu Trp Tyr Asp Ala Gly Ile Ser Ala Ile Trp Ile         35 40 45 Pro Pro Ala Ser Lys Gly Met Ser Gly Gly Tyr Ser Met Gly Tyr Asp     50 55 60 Pro Tyr Asp Tyr Phe Asp Leu Gly Glu Tyr Tyr Gln Lys Gly Thr Val 65 70 75 80 Glu Thr Arg Phe Gly Ser Lys Gln Glu Leu Ile Asn Met Ile Asn Thr                 85 90 95 Ala His Ala Tyr Gly Ile Lys Val Ile Ala Asp Ile Val Ile Asn His             100 105 110 Arg Ala Gly Gly Asp Leu Glu Trp Asn Pro Phe Val Gly Asp Tyr Thr         115 120 125 Trp Thr Asp Phe Ser Lys Val Ala Ser Gly Lys Tyr Thr Ala Asn Tyr     130 135 140 Leu Asp Phe His Pro Asn Glu Leu His Ala Gly Asp Ser Gly Thr Phe 145 150 155 160 Gly Gly Tyr Pro Asp Ile Cys His Asp Lys Ser Trp Asp Gln Tyr Trp                 165 170 175 Leu Trp Ala Ser Gln Glu Ser Tyr Ala Ala Tyr Leu Arg Ser Ile Gly             180 185 190 Ile Asp Ala Trp Arg Phe Asp Tyr Val Lys Gly Tyr Gly Ala Trp Val         195 200 205 Val Lys Asp Trp Leu Asn Trp Trp Gly Gly Trp Ala Val Gly Glu Tyr     210 215 220 Trp Asp Thr Asn Val Asp Ala Leu Leu Asn Trp Ala Tyr Ser Ser Gly 225 230 235 240 Ala Lys Val Phe Asp Phe Pro Leu Tyr Tyr Lys Met Asp Ala Ala Phe                 245 250 255 Asp Asn Lys Asn Ile Pro Ala Leu Val Glu Ala Leu Lys Asn Gly Gly              260 265 270 Thr Val Val Ser Arg Asp Pro Phe Lys Ala Val Thr Phe Val Ala Asn         275 280 285 His Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala Phe Ile     290 295 300 Leu Thr Tyr Glu Gly Gln Pro Thr Ile Phe Tyr Arg Asp Tyr Glu Glu 305 310 315 320 Trp Leu Asn Lys Asp Lys Leu Lys Asn Leu Ile Trp Ile His Asp Asn                 325 330 335 Leu Ala Gly Gly Ser Thr Ser Ile Val Tyr Tyr Asp Ser Asp Glu Met             340 345 350 Ile Phe Val Arg Asn Gly Tyr Gly Ser Lys Pro Gly Leu Ile Thr Tyr         355 360 365 Ile Asn Leu Gly Ser Ser Lys Val Gly Arg Trp Val Tyr Val Pro Lys     370 375 380 Phe Ala Gly Ala Cys Ile His Glu Tyr Thr Gly Asn Leu Gly Gly Trp 385 390 395 400 Val Asp Lys Tyr Val Tyr Ser Ser Gly Trp Val Tyr Leu Glu Ala Pro                 405 410 415 Ala Tyr Asp Pro Ala Asn Gly Gln Tyr Gly Tyr Ser Val Trp Ser Tyr             420 425 430 Cys Gly Val Gly         435 <210> 2 <211> 1308 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 2 atggccaagt acctggagct ggaggagggc ggcgtgatca tgcaggcgtt ctactgggac 60 gtcccgagcg gaggcatctg gtgggacacc atccgccaga agatccccga gtggtacgac 120 gccggcatct ccgcgatctg gataccgcca gcttccaagg gcatgtccgg gggctactcg 180 atgggctacg acccgtacga ctacttcgac ctcggcgagt actaccagaa gggcacggtg 240 gagacgcgct tcgggtccaa gcaggagctc atcaacatga tcaacacggc gcacgcctac 300 ggcatcaagg tcatcgcgga catcgtgatc aaccacaggg ccggcggcga cctggagtgg 360 aacccgttcg tcggcgacta cacctggacg gacttctcca aggtcgcctc cggcaagtac 420 accgccaact acctcgactt ccaccccaac gagctgcacg cgggcgactc cggcacgttc 480 ggcggctacc cggacatctg ccacgacaag tcctgggacc agtactggct ctgggcctcg 540 caggagtcct acgcggccta cctgcgctcc atcggcatcg acgcgtggcg cttcgactac 600 gtcaagggct acggggcctg ggtggtcaag gactggctca actggtgggg cggctgggcg 660 gtgggcgagt actgggacac caacgtcgac gcgctgctca actgggccta ctcctccggc 720 gccaaggtgt tcgacttccc cctgtactac aagatggacg cggccttcga caacaagaac 780 atcccggcgc tcgtcgaggc cctgaagaac ggcggcacgg tggtctcccg cgacccgttc 840 aaggccgtga ccttcgtcgc caaccacgac acggacatca tctggaacaa gtacccggcg 900 tacgccttca tcctcaccta cgagggccag cccacgatct tctaccgcga ctacgaggag 960 tggctgaaca aggacaagct caagaacctg atctggattc acgacaacct cgcgggcggc 1020 tccactagta tcgtgtacta cgactccgac gagatgatct tcgtccgcaa cggctacggc 1080 tccaagcccg gcctgatcac gtacatcaac ctgggctcct ccaaggtggg ccgctgggtg 1140 tacgtcccga agttcgccgg cgcgtgcatc cacgagtaca ccggcaacct cggcggctgg 1200 gtggacaagt acgtgtactc ctccggctgg gtctacctgg aggccccggc ctacgacccc 1260 gccaacggcc agtacggcta ctccgtgtgg tcctactgcg gcgtcggc 1308 <210> 3   <211> 800 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 3 Met Gly His Trp Tyr Lys His Gln Arg Ala Tyr Gln Phe Thr Gly Glu  1 5 10 15 Asp Asp Phe Gly Lys Val Ala Val Val Lys Leu Pro Met Asp Leu Thr             20 25 30 Lys Val Gly Ile Ile Val Arg Leu Asn Glu Trp Gln Ala Lys Asp Val         35 40 45 Ala Lys Asp Arg Phe Ile Glu Ile Lys Asp Gly Lys Ala Glu Val Trp     50 55 60 Ile Leu Gln Gly Val Glu Glu Ile Phe Tyr Glu Lys Pro Asp Thr Ser 65 70 75 80 Pro Arg Ile Phe Phe Ala Gln Ala Arg Ser Asn Lys Val Ile Glu Ala                 85 90 95 Phe Leu Thr Asn Pro Val Asp Thr Lys Lys Lys Glu Leu Phe Lys Val             100 105 110 Thr Val Asp Gly Lys Glu Ile Pro Val Ser Arg Val Glu Lys Ala Asp         115 120 125 Pro Thr Asp Ile Asp Val Thr Asn Tyr Val Arg Ile Val Leu Ser Glu     130 135 140 Ser Leu Lys Glu Glu Asp Leu Arg Lys Asp Val Glu Leu Ile Ilu Glu 145 150 155 160 Gly Tyr Lys Pro Ala Arg Val Ile Met Met Glu Ile Leu Asp Asp Tyr                 165 170 175 Tyr Tyr Asp Gly Glu Leu Gly Ala Val Tyr Ser Pro Glu Lys Thr Ile             180 185 190 Phe Arg Val Trp Ser Pro Val Ser Lys Trp Val Lys Val Leu Leu Phe         195 200 205 Lys Asn Gly Glu Asp Thr Glu Pro Tyr Gln Val Val Asn Met Glu Tyr     210 215 220 Lys Gly Asn Gly Val Trp Glu Ala Val Val Glu Gly Asp Leu Asp Gly 225 230 235 240 Val Phe Tyr Leu Tyr Gln Leu Glu Asn Tyr Gly Lys Ile Arg Thr Thr                 245 250 255 Val Asp Pro Tyr Ser Lys Ala Val Tyr Ala Asn Asn Gln Glu Ser Ala             260 265 270 Val Val Asn Leu Ala Arg Thr Asn Pro Glu Gly Trp Glu Asn Asp Arg         275 280 285 Gly Pro Lys Ile Glu Gly Tyr Glu Asp Ala Ile Ile Tyr Glu Ile His     290 295 300 Ile Ala Asp Ile Thr Gly Leu Glu Asn Ser Gly Val Lys Asn Lys Gly 305 310 315 320 Leu Tyr Leu Gly Leu Thr Glu Glu Asn Thr Lys Gly Pro Gly Gly Val                 325 330 335 Thr Thr Gly Leu Ser His Leu Val Glu Leu Gly Val Thr His Val His             340 345 350 Ile Leu Pro Phe Phe Asp Phe Tyr Thr Gly Asp Glu Leu Asp Lys Asp         355 360 365 Phe Glu Lys Tyr Tyr Asn Trp Gly Tyr Asp Pro Tyr Leu Phe Met Val     370 375 380 Pro Glu Gly Arg Tyr Ser Thr Asp Pro Lys Asn Pro His Thr Arg Ile 385 390 395 400  Arg Glu Val Lys Glu Met Val Lys Ala Leu His Lys His Gly Ile Gly                 405 410 415 Val Ile Met Asp Met Val Phe Pro His Thr Tyr Gly Ile Gly Glu Leu             420 425 430 Ser Ala Phe Asp Gln Thr Val Pro Tyr Tyr Phe Tyr Arg Ile Asp Lys         435 440 445 Thr Gly Ala Tyr Leu Asn Glu Ser Gly Cys Gly Asn Val Ile Ala Ser     450 455 460 Glu Arg Pro Met Met Arg Lys Phe Ile Val Asp Thr Val Thr Tyr Trp 465 470 475 480 Val Lys Glu Tyr His Ile Asp Gly Phe Arg Phe Asp Gln Met Gly Leu                 485 490 495 Ile Asp Lys Lys Thr Met Leu Glu Val Glu Arg Ala Leu His Lys Ile             500 505 510 Asp Pro Thr Ile Ile Leu Tyr Gly Glu Pro Trp Gly Gly Trp Gly Ala         515 520 525 Pro Ile Arg Phe Gly Lys Ser Asp Val Ala Gly Thr His Val Ala Ala     530 535 540 Phe Asn Asp Glu Phe Arg Asp Ala Ile Arg Gly Ser Val Phe Asn Pro 545 550 555 560 Ser Val Lys Gly Phe Val Met Gly Gly Tyr Gly Lys Glu Thr Lys Ile                 565 570 575 Lys Arg Gly Val Val Gly Ser Ile Asn Tyr Asp Gly Lys Leu Ile Lys             580 585 590 Ser Phe Ala Leu Asp Pro Glu Glu Thr Ile Asn Tyr Ala Ala Cys His         595 600 605 Asp Asn His Thr Leu Trp Asp Lys Asn Tyr Leu Ala Ala Lys Ala Asp     610 615 620 Lys Lys Lys Glu Trp Thr Glu Glu Glu Leu Lys Asn Ala Gln Lys Leu 625 630 635 640 Ala Gly Ala Ile Leu Leu Thr Ser Gln Gly Val Pro Phe Leu His Gly                 645 650 655 Gly Gln Asp Phe Cys Arg Thr Thr Asn Phe Asn Asp Asn Ser Tyr Asn             660 665 670 Ala Pro Ile Ser Ile Asn Gly Phe Asp Tyr Glu Arg Lys Leu Gln Phe         675 680 685 Ile Asp Val Phe Asn Tyr His Lys Gly Leu Ile Lys Leu Arg Lys Glu     690 695 700 His Pro Ala Phe Arg Leu Lys Asn Ala Glu Glu Ile Lys Lys His Leu 705 710 715 720 Glu Phe Leu Pro Gly Gly Arg Arg Ile Val Ala Phe Met Leu Lys Asp                 725 730 735 His Ala Gly Gly Asp Pro Trp Lys Asp Ile Val Val Ile Tyr Asn Gly             740 745 750 Asn Leu Glu Lys Thr Thr Tyr Lys Leu Pro Glu Gly Lys Trp Asn Val         755 760 765 Val Val Asn Ser Gln Lys Ala Gly Thr Glu Val Ile Glu Thr Val Glu     770 775 780 Gly Thr Ile Glu Leu Asp Pro Leu Ser Ala Tyr Val Leu Tyr Arg Glu 785 790 795 800 <210> 4 <211> 2400 <212> DNA <213> Artificial Sequence <220>   <223> synthetic <400> 4 atgggccact ggtacaagca ccagcgcgcc taccagttca ccggcgagga cgacttcggg 60 aaggtggccg tggtgaagct cccgatggac ctcaccaagg tgggcatcat cgtgcgcctc 120 aacgagtggc aggcgaagga cgtggccaag gaccgcttca tcgagatcaa ggacggcaag 180 gccgaggtgt ggatactcca gggcgtggag gagatcttct acgagaagcc ggacacctcc 240 ccgcgcatct tcttcgccca ggcccgctcc aacaaggtga tcgaggcctt cctcaccaac 300 ccggtggaca ccaagaagaa ggagctgttc aaggtgaccg tcgacggcaa ggagatcccg 360 gtgtcccgcg tggagaaggc cgacccgacc gacatcgacg tgaccaacta cgtgcgcatc 420 gtgctctccg agtccctcaa ggaggaggac ctccgcaagg acgtggagct gatcatcgag 480 ggctacaagc cggcccgcgt gatcatgatg gagatcctcg acgactacta ctacgacggc 540 gagctggggg cggtgtactc cccggagaag accatcttcc gcgtgtggtc cccggtgtcc 600 aagtgggtga aggtgctcct cttcaagaac ggcgaggaca ccgagccgta ccaggtggtg 660 aacatggagt acaagggcaa cggcgtgtgg gaggccgtgg tggagggcga cctcgacggc 720 gtgttctacc tctaccagct ggagaactac ggcaagatcc gcaccaccgt ggacccgtac 780 tccaaggccg tgtacgccaa caaccaggag tctgcagtgg tgaacctcgc ccgcaccaac 840 ccggagggct gggagaacga ccgcggcccg aagatcgagg gctacgagga cgccatcatc 900 tacgagatcc acatcgccga catcaccggc ctggagaact ccggcgtgaa gaacaagggc 960 ctctacctcg gcctcaccga ggagaacacc aaggccccgg gcggcgtgac caccggcctc 1020 tcccacctcg tggagctggg cgtgacccac gtgcacatcc tcccgttctt cgacttctac 1080 accggcgacg agctggacaa ggacttcgag aagtactaca actggggcta cgacccgtac 1140 ctcttcatgg tgccggaggg ccgctactcc accgacccga agaacccgca cacccgaatt 1200 cgcgaggtga aggagatggt gaaggccctc cacaagcacg gcatcggcgt gatcatggac 1260 atggtgttcc cgcacaccta cggcatcggc gagctgtccg ccttcgacca gaccgtgccg 1320 tactacttct accgcatcga caagaccggc gcctacctca acgagtccgg ctgcggcaac 1380 gtgatcgcct ccgagcgccc gatgatgcgc aagttcatcg tggacaccgt gacctactgg 1440 gtgaaggagt accacatcga cggcttccgc ttcgaccaga tgggcctcat cgacaagaag 1500 accatgctgg aggtggagcg cgccctccac aagatcgacc cgaccatcat cctctacggc 1560 gagccgtggg gcggctgggg ggccccgatc cgcttcggca agtccgacgt ggccggcacc 1620 cacgtggccg ccttcaacga cgagttccgc gacgccatcc gcggctccgt gttcaacccg 1680 tccgtgaagg gcttcgtgat gggcggctac ggcaaggaga ccaagatcaa gcgcggcgtg 1740 gtgggctcca tcaactacga cggcaagctc atcaagtcct tcgccctcga cccggaggag 1800 accatcaact acgccgcctg ccacgacaac cacaccctct gggacaagaa ctacctcgcc 1860 gccaaggccg acaagaagaa ggagtggacc gaggaggagc tgaagaacgc ccagaagctc 1920 gccggcgcca tcctcctcac tagtcagggc gtgccgttcc tccacggcgg ccaggacttc 1980 tgccgcacca ccaacttcaa cgacaactcc tacaacgccc cgatctccat caacggcttc 2040 gactacgagc gcaagctcca gttcatcgac gtgttcaact accacaaggg cctcatcaag 2100 ctccgcaagg agcacccggc cttccgcctc aagaacgccg aggagatcaa gaagcacctg 2160 gagttcctcc cgggcgggcg ccgcatcgtg gccttcatgc tcaaggacca cgccggcggc 2220 gacccgtgga aggacatcgt ggtgatctac aacggcaacc tggagaagac cacctacaag 2280 ctcccggagg gcaagtggaa cgtggtggtg aactcccaga aggccggcac cgaggtgatc 2340 gagaccgtgg agggcaccat cgagctggac ccgctctccg cctacgtgct ctaccgcgag 2400 <210> 5 <211> 693 <212> PRT <213> Sulfolobus solfataricus <400> 5 Met Glu Thr Ile Lys Ile Tyr Glu Asn Lys Gly Val Tyr Lys Val Val  1 5 10 15 Ile Gly Glu Pro Phe Pro Pro Ile Glu Phe Pro Leu Glu Gln Lys Ile             20 25 30 Ser Ser Asn Lys Ser Leu Ser Glu Leu Gly Leu Thr Ile Val Gln Gln         35 40 45 Gly Asn Lys Val Ile Val Glu Lys Ser Leu Asp Leu Lys Glu His Ile      50 55 60 Ile Gly Leu Gly Glu Lys Ala Phe Glu Leu Asp Arg Lys Arg Lys Arg 65 70 75 80 Tyr Val Met Tyr Asn Val Asp Ala Gly Ala Tyr Lys Lys Tyr Gln Asp                 85 90 95 Pro Leu Tyr Val Ser Ile Pro Leu Phe Ile Ser Val Lys Asp Gly Val             100 105 110 Ala Thr Gly Tyr Phe Phe Asn Ser Ala Ser Lys Val Ile Phe Asp Val         115 120 125 Gly Leu Glu Glu Tyr Asp Lys Val Ile Val Thr Ile Pro Glu Asp Ser     130 135 140 Val Glu Phe Tyr Val Ile Glu Gly Pro Arg Ile Glu Asp Val Leu Glu 145 150 155 160 Lys Tyr Thr Glu Leu Thr Gly Lys Pro Phe Leu Pro Pro Met Trp Ala                 165 170 175 Phe Gly Tyr Met Ile Ser Arg Tyr Ser Tyr Tyr Pro Gln Asp Lys Val             180 185 190 Val Glu Leu Val Asp Ile Met Gln Lys Glu Gly Phe Arg Val Ala Gly         195 200 205 Val Phe Leu Asp Ile His Tyr Met Asp Ser Tyr Lys Leu Phe Thr Trp     210 215 220 His Pro Tyr Arg Phe Pro Glu Pro Lys Lys Leu Ile Asp Glu Leu His 225 230 235 240 Lys Arg Asn Val Lys Leu Ile Thr Ile Val Asp His Gly Ile Arg Val                 245 250 255 Asp Gln Asn Tyr Ser Pro Phe Leu Ser Gly Met Gly Lys Phe Cys Glu             260 265 270 Ile Glu Ser Gly Glu Leu Phe Val Gly Lys Met Trp Pro Gly Thr Thr         275 280 285 Val Tyr Pro Asp Phe Phe Arg Glu Asp Thr Arg Glu Trp Trp Ala Gly     290 295 300 Leu Ile Ser Glu Trp Leu Ser Gln Gly Val Asp Gly Ile Trp Leu Asp 305 310 315 320 Met Asn Glu Pro Thr Asp Phe Ser Arg Ala Ile Glu Ile Arg Asp Val                 325 330 335 Leu Ser Ser Leu Pro Val Gln Phe Arg Asp Asp Arg Leu Val Thr Thr             340 345 350 Phe Pro Asp Asn Val Val His Tyr Leu Arg Gly Lys Arg Val Lys His         355 360 365 Glu Lys Val Arg Asn Ala Tyr Pro Leu Tyr Glu Ala Met Ala Thr Phe     370 375 380 Lys Gly Phe Arg Thr Ser His Arg Asn Glu Ile Phe Ile Leu Ser Arg 385 390 395 400 Ala Gly Tyr Ala Gly Ile Gln Arg Tyr Ala Phe Ile Trp Thr Gly Asp                 405 410 415 Asn Thr Pro Ser Trp Asp Asp Leu Lys Leu Gln Leu Gln Leu Val Leu             420 425 430 Gly Leu Ser Ile Ser Gly Val Pro Phe Val Gly Cys Asp Ile Gly Gly         435 440 445 Phe Gln Gly Arg Asn Phe Ala Glu Ile Asp Asn Ser Met Asp Leu Leu     450 455 460 Val Lys Tyr Tyr Ala Leu Ala Leu Phe Phe Pro Phe Tyr Arg Ser His 465 470 475 480 Lys Ala Thr Asp Gly Ile Asp Thr Glu Pro Val Phe Leu Pro Asp Tyr                 485 490 495 Tyr Lys Glu Lys Val Lys Glu Ile Val Glu Leu Arg Tyr Lys Phe Leu             500 505 510 Pro Tyr Ile Tyr Ser Leu Ala Leu Glu Ala Ser Glu Lys Gly His Pro          515 520 525 Val Ile Arg Pro Leu Phe Tyr Glu Phe Gln Asp Asp Asp Asp Met Tyr     530 535 540 Arg Ile Glu Asp Glu Tyr Met Val Gly Lys Tyr Leu Leu Tyr Ala Pro 545 550 555 560 Ile Val Ser Lys Glu Glu Ser Arg Leu Val Thr Leu Pro Arg Gly Lys                 565 570 575 Trp Tyr Asn Tyr Trp Asn Gly Glu Ile Ile Asn Gly Lys Ser Val Val             580 585 590 Lys Ser Thr His Glu Leu Pro Ile Tyr Leu Arg Glu Gly Ser Ile Ile         595 600 605 Pro Leu Glu Gly Asp Glu Leu Ile Val Tyr Gly Glu Thr Ser Phe Lys     610 615 620 Arg Tyr Asp Asn Ala Glu Ile Thr Ser Ser Ser Asn Glu Ile Lys Phe 625 630 635 640 Ser Arg Glu Ile Tyr Val Ser Lys Leu Thr Ile Thr Ser Glu Lys Pro                 645 650 655 Val Ser Lys Ile Ile Val Asp Asp Ser Lys Glu Ile Gln Val Glu Lys             660 665 670 Thr Met Gln Asn Thr Tyr Val Ala Lys Ile Asn Gln Lys Ile Arg Gly         675 680 685 Lys Ile Asn Leu Glu     690 <210> 6 <211> 2082 <212> DNA <213> Sulfolobus solfataricus <400> 6 atggagacca tcaagatcta cgagaacaag ggcgtgtaca aggtggtgat cggcgagccg 60 ttcccgccga tcgagttccc gctcgagcag aagatctcct ccaacaagtc cctctccgag 120 ctgggcctca ccatcgtgca gcagggcaac aaggtgatcg tggagaagtc cctcgacctc 180 aaggagcaca tcatcggcct cggcgagaag gccttcgagc tggaccgcaa gcgcaagcgc 240 tacgtgatgt acaacgtgga cgccggcgcc tacaagaagt accaggaccc gctctacgtg 300 tccatcccgc tcttcatctc cgtgaaggac ggcgtggcca ccggctactt cttcaactcc 360 gcctccaagg tgatcttcga cgtgggcctc gaggagtacg acaaggtgat cgtgaccatc 420 ccggaggact ccgtggagtt ctacgtgatc gagggcccgc gcatcgagga cgtgctcgag 480 aagtacaccg agctgaccgg caagccgttc ctcccgccga tgtgggcctt cggctacatg 540 atctcccgct actcctacta cccgcaggac aaggtggtgg agctggtgga catcatgcag 600 aaggagggct tccgcgtggc cggcgtgttc ctcgacatcc actacatgga ctcctacaag 660 ctcttcacct ggcacccgta ccgcttcccg gagccgaaga agctcatcga cgagctgcac 720 aagcgcaacg tgaagctcat caccatcgtg gaccacggca tccgcgtgga ccagaactac 780 tccccgttcc tctccggcat gggcaagttc tgcgagatcg agtccggcga gctgttcgtg 840 ggcaagatgt ggccgggcac caccgtgtac ccggacttct tccgcgagga cacccgcgag 900 tggtgggccg gcctcatctc cgagtggctc tcccagggcg tggacggcat ctggctcgac 960 atgaacgagc cgaccgactt ctcccgcgcc atcgagatcc gcgacgtgct ctcctccctc 1020 ccggtgcagt tccgcgacga ccgcctcgtg accaccttcc cggacaacgt ggtgcactac 1080 ctccgcggca agcgcgtgaa gcacgagaag gtgcgcaacg cctacccgct ctacgaggcg 1140 atggccacct tcaagggctt ccgcacctcc caccgcaacg agatcttcat cctctcccgc 1200 gccggctacg ccggcatcca gcgctacgcc ttcatctgga ccggcgacaa caccccgtcc 1260 tgggacgacc tcaagctcca gctccagctc gtgctcggcc tctccatctc cggcgtgccg 1320 ttcgtgggct gcgacatcgg cggcttccag ggccgcaact tcgccgagat cgacaactcg 1380 atggacctcc tcgtgaagta ctacgccctc gccctcttct tcccgttcta ccgctcccac 1440 aaggccaccg acggcatcga caccgagccg gtgttcctcc cggactacta caaggagaag 1500 gtgaaggaga tcgtggagct gcgctacaag ttcctcccgt acatctactc cctcgccctc 1560 gaggcctccg agaagggcca cccggtgatc cgcccgctct tctacgagtt ccaggacgac 1620  gacgacatgt accgcatcga ggacgagtac atggtgggca agtacctcct ctacgccccg 1680 atcgtgtcca aggaggagtc ccgcctcgtg accctcccgc gcggcaagtg gtacaactac 1740 tggaacggcg agatcatcaa cggcaagtcc gtggtgaagt ccacccacga gctgccgatc 1800 tacctccgcg agggctccat catcccgctc gagggcgacg agctgatcgt gtacggcgag 1860 acctccttca agcgctacga caacgccgag atcacctcct cctccaacga gatcaagttc 1920 tcccgcgaga tctacgtgtc caagctcacc atcacctccg agaagccggt gtccaagatc 1980 atcgtggacg actccaagga gatccaggtg gagaagacca tgcagaacac ctacgtggcc 2040 aagatcaacc agaagatccg cggcaagatc aacctcgagt ga 2082 <210> 7 <211> 1818 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 7 atggcggctc tggccacgtc gcagctcgtc gcaacgcgcg ccggcctggg cgtcccggac 60 gcgtccacgt tccgccgcgg cgccgcgcag ggcctgaggg gggcccgggc gtcggcggcg 120 gcggacacgc tcagcatgcg gaccagcgcg cgcgcggcgc ccaggcacca gcaccagcag 180 gcgcgccgcg gggccaggtt cccgtcgctc gtcgtgtgcg ccagcgccgg catgaacgtc 240 gtcttcgtcg gcgccgagat ggcgccgtgg agcaagaccg gaggcctcgg cgacgtcctc 300 ggcggcctgc cgccggccat ggccgcgaac gggcaccgtg tcatggtcgt ctctccccgc 360 tacgaccagt acaaggacgc ctgggacacc agcgtcgtgt ccgagatcaa gatgggagac 420 gggtacgaga cggtcaggtt cttccactgc tacaagcgcg gagtggaccg cgtgttcgtt 480 gaccacccac tgttcctgga gagggtttgg ggaaagaccg aggagaagat ctacgggcct 540 gtcgctggaa cggactacag ggacaaccag ctgcggttca gcctgctatg ccaggcagca 600 cttgaagctc caaggatcct gagcctcaac aacaacccat acttctccgg accatacggg 660 gaggacgtcg tgttcgtctg caacgactgg cacaccggcc ctctctcgtg ctacctcaag 720 agcaactacc agtcccacgg catctacagg gacgcaaaga ccgctttctg catccacaac 780 atctcctacc agggccggtt cgccttctcc gactacccgg agctgaacct ccccgagaga 840 ttcaagtcgt ccttcgattt catcgacggc tacgagaagc ccgtggaagg ccggaagatc 900 aactggatga aggccgggat cctcgaggcc gacagggtcc tcaccgtcag cccctactac 960 gccgaggagc tcatctccgg catcgccagg ggctgcgagc tcgacaacat catgcgcctc 1020 accggcatca ccggcatcgt caacggcatg gacgtcagcg agtgggaccc cagcagggac 1080 aagtacatcg ccgtgaagta cgacgtgtcg acggccgtgg aggccaaggc gctgaacaag 1140 gaggcgctgc aggcggaggt cgggctcccg gtggaccgga acatcccgct ggtggcgttc 1200 atcggcaggc tggaagagca gaagggcccc gacgtcatgg cggccgccat cccgcagctc 1260 atggagatgg tggaggacgt gcagatcgtt ctgctgggca cgggcaagaa gaagttcgag 1320 cgcatgctca tgagcgccga ggagaagttc ccaggcaagg tgcgcgccgt ggtcaagttc 1380 aacgcggcgc tggcgcacca catcatggcc ggcgccgacg tgctcgccgt caccagccgc 1440 ttcgagccct gcggcctcat ccagctgcag gggatgcgat acggaacgcc ctgcgcctgc 1500 gcgtccaccg gtggactcgt cgacaccatc atcgaaggca agaccgggtt ccacatgggc 1560 cgcctcagcg tcgactgcaa cgtcgtggag ccggcggacg tcaagaaggt ggccaccacc 1620 ttgcagcgcg ccatcaaggt ggtcggcacg ccggcgtacg aggagatggt gaggaactgc 1680 atgatccagg atctctcctg gaagggccct gccaagaact gggagaacgt gctgctcagc 1740 ctcggggtcg ccggcggcga gccaggggtt gaaggcgagg agatcgcgcc gctcgccaag 1800 gagaacgtgg ccgcgccc 1818 <210> 8 <211> 606 <212> PRT <213> Artificial Sequence <220> <223> synthetic   <400> 8 Met Ala Ala Leu Ala Thr Ser Gln Leu Val Ala Thr Arg Ala Gly Leu  1 5 10 15 Gly Val Pro Asp Ala Ser Thr Phe Arg Arg Gly Ala Ala Gln Gly Leu             20 25 30 Arg Gly Ala Arg Ala Ser Ala Ala Ala Asp Thr Leu Ser Met Arg Thr         35 40 45 Ser Ala Arg Ala Ala Pro Arg His Gln His Gln Gln Ala Arg Arg Gly     50 55 60 Ala Arg Phe Pro Ser Leu Val Val Cys Ala Ser Ala Gly Met Asn Val 65 70 75 80 Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser Lys Thr Gly Gly Leu                 85 90 95 Gly Asp Val Leu Gly Gly Leu Pro Pro Ala Met Ala Ala Asn Gly His             100 105 110 Arg Val Met Val Val Ser Pro Arg Tyr Asp Gln Tyr Lys Asp Ala Trp         115 120 125 Asp Thr Ser Val Val Ser Glu Ile Lys Met Gly Asp Gly Tyr Glu Thr     130 135 140 Val Arg Phe Phe His Cys Tyr Lys Arg Gly Val Asp Arg Val Phe Val 145 150 155 160 Asp His Pro Leu Phe Leu Glu Arg Val Trp Gly Lys Thr Glu Glu Lys                 165 170 175 Ile Tyr Gly Pro Val Ala Gly Thr Asp Tyr Arg Asp Asn Gln Leu Arg             180 185 190 Phe Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala Pro Arg Ile Leu Ser         195 200 205 Leu Asn Asn Asn Pro Tyr Phe Ser Gly Pro Tyr Gly Glu Asp Val Val     210 215 220 Phe Val Cys Asn Asp Trp His Thr Gly Pro Leu Ser Cys Tyr Leu Lys 225 230 235 240 Ser Asn Tyr Gln Ser His Gly Ile Tyr Arg Asp Ala Lys Thr Ala Phe                 245 250 255 Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe Ala Phe Ser Asp Tyr             260 265 270 Pro Glu Leu Asn Leu Pro Glu Arg Phe Lys Ser Ser Phe Asp Phe Ile         275 280 285 Asp Gly Tyr Glu Lys Pro Val Glu Gly Arg Lys Ile Asn Trp Met Lys     290 295 300 Ala Gly Ile Leu Glu Ala Asp Arg Val Leu Thr Val Ser Pro Tyr Tyr 305 310 315 320 Ala Glu Glu Leu Ile Ser Gly Ile Ala Arg Gly Cys Glu Leu Asp Asn                 325 330 335 Ile Met Arg Leu Thr Gly Ile Thr Gly Ile Val Asn Gly Met Asp Val             340 345 350 Ser Glu Trp Asp Pro Ser Arg Asp Lys Tyr Ile Ala Val Lys Tyr Asp         355 360 365 Val Ser Thr Ala Val Glu Ala Lys Ala Leu Asn Lys Glu Ala Leu Gln     370 375 380 Ala Glu Val Gly Leu Pro Val Asp Arg Asn Ile Pro Leu Val Ala Phe 385 390 395 400 Ile Gly Arg Leu Glu Glu Gln Lys Gly Pro Asp Val Met Ala Ala Ala                 405 410 415 Ile Pro Gln Leu Met Glu Met Val Glu Asp Val Gln Ile Val Leu Leu             420 425 430 Gly Thr Gly Lys Lys Lys Phe Glu Arg Met Leu Met Ser Ala Glu Glu         435 440 445 Lys Phe Pro Gly Lys Val Arg Ala Val Val Lys Phe Asn Ala Ala Leu      450 455 460 Ala His His Ile Met Ala Gly Ala Asp Val Leu Ala Val Thr Ser Arg 465 470 475 480 Phe Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg Tyr Gly Thr                 485 490 495 Pro Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp Thr Ile Ile Glu             500 505 510 Gly Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp Cys Asn Val         515 520 525 Val Glu Pro Ala Asp Val Lys Lys Val Ala Thr Thr Leu Gln Arg Ala     530 535 540 Ile Lys Val Val Gly Thr Pro Ala Tyr Glu Glu Met Val Arg Asn Cys 545 550 555 560 Met Ile Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn Trp Glu Asn                 565 570 575 Val Leu Leu Ser Leu Gly Val Ala Gly Gly Glu Pro Gly Val Glu Gly             580 585 590 Glu Glu Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala Pro         595 600 605 <210> 9 <211> 2223 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 9 atggccaagt acctggagct ggaggagggc ggcgtgatca tgcaggcgtt ctactgggac 60 gtcccgagcg gaggcatctg gtgggacacc atccgccaga agatccccga gtggtacgac 120 gccggcatct ccgcgatctg gataccgcca gcttccaagg gcatgtccgg gggctactcg 180 atgggctacg acccgtacga ctacttcgac ctcggcgagt actaccagaa gggcacggtg 240 gagacgcgct tcgggtccaa gcaggagctc atcaacatga tcaacacggc gcacgcctac 300 ggcatcaagg tcatcgcgga catcgtgatc aaccacaggg ccggcggcga cctggagtgg 360 aacccgttcg tcggcgacta cacctggacg gacttctcca aggtcgcctc cggcaagtac 420 accgccaact acctcgactt ccaccccaac gagctgcacg cgggcgactc cggcacgttc 480 ggcggctacc cggacatctg ccacgacaag tcctgggacc agtactggct ctgggcctcg 540 caggagtcct acgcggccta cctgcgctcc atcggcatcg acgcgtggcg cttcgactac 600 gtcaagggct acggggcctg ggtggtcaag gactggctca actggtgggg cggctgggcg 660 gtgggcgagt actgggacac caacgtcgac gcgctgctca actgggccta ctcctccggc 720 gccaaggtgt tcgacttccc cctgtactac aagatggacg cggccttcga caacaagaac 780 atcccggcgc tcgtcgaggc cctgaagaac ggcggcacgg tggtctcccg cgacccgttc 840 aaggccgtga ccttcgtcgc caaccacgac acggacatca tctggaacaa gtacccggcg 900 tacgccttca tcctcaccta cgagggccag cccacgatct tctaccgcga ctacgaggag 960 tggctgaaca aggacaagct caagaacctg atctggattc acgacaacct cgcgggcggc 1020 tccactagta tcgtgtacta cgactccgac gagatgatct tcgtccgcaa cggctacggc 1080 tccaagcccg gcctgatcac gtacatcaac ctgggctcct ccaaggtggg ccgctgggtg 1140 tacgtcccga agttcgccgg cgcgtgcatc cacgagtaca ccggcaacct cggcggctgg 1200 gtggacaagt acgtgtactc ctccggctgg gtctacctgg aggccccggc ctacgacccc 1260 gccaacggcc agtacggcta ctccgtgtgg tcctactgcg gcgtcggcac atcgattgct 1320 ggcatcctcg aggccgacag ggtcctcacc gtcagcccct actacgccga ggagctcatc 1380 tccggcatcg ccaggggctg cgagctcgac aacatcatgc gcctcaccgg catcaccggc 1440 atcgtcaacg gcatggacgt cagcgagtgg gaccccagca gggacaagta catcgccgtg 1500 aagtacgacg tgtcgacggc cgtggaggcc aaggcgctga acaaggaggc gctgcaggcg 1560 gaggtcgggc tcccggtgga ccggaacatc ccgctggtgg cgttcatcgg caggctggaa 1620 gagcagaagg gccccgacgt catggcggcc gccatcccgc agctcatgga gatggtggag 1680  gacgtgcaga tcgttctgct gggcacgggc aagaagaagt tcgagcgcat gctcatgagc 1740 gccgaggaga agttcccagg caaggtgcgc gccgtggtca agttcaacgc ggcgctggcg 1800 caccacatca tggccggcgc cgacgtgctc gccgtcacca gccgcttcga gccctgcggc 1860 ctcatccagc tgcaggggat gcgatacgga acgccctgcg cctgcgcgtc caccggtgga 1920 ctcgtcgaca ccatcatcga aggcaagacc gggttccaca tgggccgcct cagcgtcgac 1980 tgcaacgtcg tggagccggc ggacgtcaag aaggtggcca ccaccttgca gcgcgccatc 2040 aaggtggtcg gcacgccggc gtacgaggag atggtgagga actgcatgat ccaggatctc 2100 tcctggaagg gccctgccaa gaactgggag aacgtgctgc tcagcctcgg ggtcgccggc 2160 ggcgagccag gggttgaagg cgaggagatc gcgccgctcg ccaaggagaa cgtggccgcg 2220 ccc 2223 <210> 10 <211> 741 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 10 Met Ala Lys Tyr Leu Glu Leu Glu Glu Gly Gly Val Ile Met Gln Ala  1 5 10 15 Phe Tyr Trp Asp Val Pro Ser Gly Gly Ile Trp Trp Asp Thr Ile Arg             20 25 30 Gln Lys Ile Pro Glu Trp Tyr Asp Ala Gly Ile Ser Ala Ile Trp Ile         35 40 45 Pro Pro Ala Ser Lys Gly Met Ser Gly Gly Tyr Ser Met Gly Tyr Asp     50 55 60 Pro Tyr Asp Tyr Phe Asp Leu Gly Glu Tyr Tyr Gln Lys Gly Thr Val 65 70 75 80 Glu Thr Arg Phe Gly Ser Lys Gln Glu Leu Ile Asn Met Ile Asn Thr                 85 90 95 Ala His Ala Tyr Gly Ile Lys Val Ile Ala Asp Ile Val Ile Asn His             100 105 110 Arg Ala Gly Gly Asp Leu Glu Trp Asn Pro Phe Val Gly Asp Tyr Thr         115 120 125 Trp Thr Asp Phe Ser Lys Val Ala Ser Gly Lys Tyr Thr Ala Asn Tyr     130 135 140 Leu Asp Phe His Pro Asn Glu Leu His Ala Gly Asp Ser Gly Thr Phe 145 150 155 160 Gly Gly Tyr Pro Asp Ile Cys His Asp Lys Ser Trp Asp Gln Tyr Trp                 165 170 175 Leu Trp Ala Ser Gln Glu Ser Tyr Ala Ala Tyr Leu Arg Ser Ile Gly             180 185 190 Ile Asp Ala Trp Arg Phe Asp Tyr Val Lys Gly Tyr Gly Ala Trp Val         195 200 205 Val Lys Asp Trp Leu Asn Trp Trp Gly Gly Trp Ala Val Gly Glu Tyr     210 215 220 Trp Asp Thr Asn Val Asp Ala Leu Leu Asn Trp Ala Tyr Ser Ser Gly 225 230 235 240 Ala Lys Val Phe Asp Phe Pro Leu Tyr Tyr Lys Met Asp Ala Ala Phe                 245 250 255 Asp Asn Lys Asn Ile Pro Ala Leu Val Glu Ala Leu Lys Asn Gly Gly             260 265 270 Thr Val Val Ser Arg Asp Pro Phe Lys Ala Val Thr Phe Val Ala Asn         275 280 285 His Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala Phe Ile     290 295 300  Leu Thr Tyr Glu Gly Gln Pro Thr Ile Phe Tyr Arg Asp Tyr Glu Glu 305 310 315 320 Trp Leu Asn Lys Asp Lys Leu Lys Asn Leu Ile Trp Ile His Asp Asn                 325 330 335 Leu Ala Gly Gly Ser Thr Ser Ile Val Tyr Tyr Asp Ser Asp Glu Met             340 345 350 Ile Phe Val Arg Asn Gly Tyr Gly Ser Lys Pro Gly Leu Ile Thr Tyr         355 360 365 Ile Asn Leu Gly Ser Ser Lys Val Gly Arg Trp Val Tyr Val Pro Lys     370 375 380 Phe Ala Gly Ala Cys Ile His Glu Tyr Thr Gly Asn Leu Gly Gly Trp 385 390 395 400 Val Asp Lys Tyr Val Tyr Ser Ser Gly Trp Val Tyr Leu Glu Ala Pro                 405 410 415 Ala Tyr Asp Pro Ala Asn Gly Gln Tyr Gly Tyr Ser Val Trp Ser Tyr             420 425 430 Cys Gly Val Gly Thr Ser Ile Ala Gly Ile Leu Glu Ala Asp Arg Val         435 440 445 Leu Thr Val Ser Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ile Ala     450 455 460 Arg Gly Cys Glu Leu Asp Asn Ile Met Arg Leu Thr Gly Ile Thr Gly 465 470 475 480 Ile Val Asn Gly Met Asp Val Ser Glu Trp Asp Pro Ser Arg Asp Lys                 485 490 495 Tyr Ile Ala Val Lys Tyr Asp Val Ser Thr Ala Val Glu Ala Lys Ala             500 505 510 Leu Asn Lys Glu Ala Leu Gln Ala Glu Val Gly Leu Pro Val Asp Arg         515 520 525 Asn Ile Pro Leu Val Ala Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly     530 535 540 Pro Asp Val Met Ala Ala Ala Ile Pro Gln Leu Met Glu Met Val Glu 545 550 555 560 Asp Val Gln Ile Val Leu Leu Gly Thr Gly Lys Lys Lys Phe Glu Arg                 565 570 575 Met Leu Met Ser Ala Glu Glu Lys Phe Pro Gly Lys Val Arg Ala Val             580 585 590 Val Lys Phe Asn Ala Ala Leu Ala His His Ile Met Ala Gly Ala Asp         595 600 605 Val Leu Ala Val Thr Ser Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu     610 615 620 Gln Gly Met Arg Tyr Gly Thr Pro Cys Ala Cys Ala Ser Thr Gly Gly 625 630 635 640 Leu Val Asp Thr Ile Ile Glu Gly Lys Thr Gly Phe His Met Gly Arg                 645 650 655 Leu Ser Val Asp Cys Asn Val Val Glu Pro Ala Asp Val Lys Lys Val             660 665 670 Ala Thr Thr Leu Gln Arg Ala Ile Lys Val Val Gly Thr Pro Ala Tyr         675 680 685 Glu Glu Met Val Arg Asn Cys Met Ile Gln Asp Leu Ser Trp Lys Gly     690 695 700 Pro Ala Lys Asn Trp Glu Asn Val Leu Leu Ser Leu Gly Val Ala Gly 705 710 715 720 Gly Glu Pro Gly Val Glu Gly Glu Glu Ile Ala Pro Leu Ala Lys Glu                 725 730 735 Asn Val Ala Ala Pro             740   <210> 11 <211> 1515 <212> DNA <213> Zea mays <400> 11 ggagagctat gagacgtatg tcctcaaagc cactttgcat tgtgtgaaac caatatcgat 60 ctttgttact tcatcatgca tgaacatttg tggaaactac tagcttacaa gcattagtga 120 cagctcagaa aaaagttatc tatgaaaggt ttcatgtgta ccgtgggaaa tgagaaatgt 180 tgccaactca aacaccttca atatgttgtt tgcaggcaaa ctcttctgga agaaaggtgt 240 ctaaaactat gaacgggtta cagaaaggta taaaccacgg ctgtgcattt tggaagtatc 300 atctatagat gtctgttgag gggaaagccg tacgccaacg ttatttactc agaaacagct 360 tcaacacaca gttgtctgct ttatgatggc atctccaccc aggcacccac catcacctat 420 ctctcgtgcc tgtttatttt cttgcccttt ctgatcataa aaaaacatta agagtttgca 480 aacatgcata ggcatatcaa tatgctcatt tattaatttg ctagcagatc atcttcctac 540 tctttacttt atttattgtt tgaaaaatat gtcctgcacc tagggagctc gtatacagta 600 ccaatgcatc ttcattaaat gtgaatttca gaaaggaagt aggaacctat gagagtattt 660 ttcaaaatta attagcggct tctattatgt ttatagcaaa ggccaagggc aaaattggaa 720 cactaatgat ggttggttgc atgagtctgt cgattacttg caagaaatgt gaacctttgt 780 ttctgtgcgt gggcataaaa caaacagctt ctagcctctt ttacggtact tgcacttgca 840 agaaatgtga actccttttc atttctgtat gtggacataa tgccaaagca tccaggcttt 900 ttcatggttg ttgatgtctt tacacagttc atctccacca gtatgccctc ctcatactct 960 atataaacac atcaacagca tcgcaattag ccacaagatc acttcgggag gcaagtgcga 1020 tttcgatctc gcagccacct ttttttgttc tgttgtaagt ataccttccc ttaccatctt 1080 tatctgttag tttaatttgt aattgggaag tattagtgga aagaggatga gatgctatca 1140 tctatgtact ctgcaaatgc atctgacgtt atatgggctg cttcatataa tttgaattgc 1200 tccattcttg ccgacaatat attgcaaggt atatgcctag ttccatcaaa agttctgttt 1260 tttcattcta aaagcatttt agtggcacac aatttttgtc catgagggaa aggaaatctg 1320 ttttggttac tttgcttgag gtgcattctt catatgtcca gttttatgga agtaataaac 1380 ttcagtttgg tcataagatg tcatattaaa gggcaaacat atattcaatg ttcaattcat 1440 cgtaaatgtt ccctttttgt aaaagattgc atactcattt atttgagttg caggtgtatc 1500 tagtagttgg aggag 1515 <210> 12 <211> 673 <212> DNA <213> Zea mays <400> 12 gatcatccag gtgcaaccgt ataagtccta aagtggtgag gaacacgaaa caaccatgca 60 ttggcatgta aagctccaag aatttgttgt atccttaaca actcacagaa catcaaccaa 120 aattgcacgt caagggtatt gggtaagaaa caatcaaaca aatcctctct gtgtgcaaag 180 aaacacggtg agtcatgccg agatcatact catctgatat acatgcttac agctcacaag 240 acattacaaa caactcatat tgcattacaa agatcgtttc atgaaaaata aaataggccg 300 gacaggacaa aaatccttga cgtgtaaagt aaatttacaa caaaaaaaaa gccatatgtc 360 aagctaaatc taattcgttt tacgtagatc aacaacctgt agaaggcaac aaaactgagc 420 cacgcagaag tacagaatga ttccagatga accatcgacg tgctacgtaa agagagtgac 480 gagtcatata catttggcaa gaaaccatga agctgcctac agccgtctcg gtggcataag 540 aacacaagaa attgtgttaa ttaatcaaag ctataaataa cgctcgcatg cctgtgcact 600 tctccatcac caccactggg tcttcagacc attagcttta tctactccag agcgcagaag 660 aacccgatcg aca 673 <210> 13 <211> 454 <212> PRT <213> Artificial Sequence <220>   <223> synthetic <400> 13 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala Thr Ser Ala Lys Tyr Leu Glu Leu Glu Glu Gly Gly Val Ile Met             20 25 30 Gln Ala Phe Tyr Trp Asp Val Pro Ser Gly Gly Ile Trp Trp Asp Thr         35 40 45 Ile Arg Gln Lys Ile Pro Glu Trp Tyr Asp Ala Gly Ile Ser Ala Ile     50 55 60 Trp Ile Pro Pro Ala Ser Lys Gly Met Ser Gly Gly Tyr Ser Met Gly 65 70 75 80 Tyr Asp Pro Tyr Asp Tyr Phe Asp Leu Gly Glu Tyr Tyr Gln Lys Gly                 85 90 95 Thr Val Glu Thr Arg Phe Gly Ser Lys Gln Glu Leu Ile Asn Met Ile             100 105 110 Asn Thr Ala His Ala Tyr Gly Ile Lys Val Ile Ala Asp Ile Val Ile         115 120 125 Asn His Arg Ala Gly Gly Asp Leu Glu Trp Asn Pro Phe Val Gly Asp     130 135 140 Tyr Thr Trp Thr Asp Phe Ser Lys Val Ala Ser Gly Lys Tyr Thr Ala 145 150 155 160 Asn Tyr Leu Asp Phe His Pro Asn Glu Leu His Ala Gly Asp Ser Gly                 165 170 175 Thr Phe Gly Gly Tyr Pro Asp Ile Cys His Asp Lys Ser Trp Asp Gln             180 185 190 Tyr Trp Leu Trp Ala Ser Gln Glu Ser Tyr Ala Ala Tyr Leu Arg Ser         195 200 205 Ile Gly Ile Asp Ala Trp Arg Phe Asp Tyr Val Lys Gly Tyr Gly Ala     210 215 220 Trp Val Val Lys Asp Trp Leu Asn Trp Trp Gly Gly Trp Ala Val Gly 225 230 235 240 Glu Tyr Trp Asp Thr Asn Val Asp Ala Leu Leu Asn Trp Ala Tyr Ser                 245 250 255 Ser Gly Ala Lys Val Phe Asp Phe Pro Leu Tyr Tyr Lys Met Asp Ala             260 265 270 Ala Phe Asp Asn Lys Asn Ile Pro Ala Leu Val Glu Ala Leu Lys Asn         275 280 285 Gly Gly Thr Val Val Ser Arg Asp Pro Phe Lys Ala Val Thr Phe Val     290 295 300 Ala Asn His Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala 305 310 315 320 Phe Ile Leu Thr Tyr Glu Gly Gln Pro Thr Ile Phe Tyr Arg Asp Tyr                 325 330 335 Glu Glu Trp Leu Asn Lys Asp Lys Leu Lys Asn Leu Ile Trp Ile His             340 345 350 Asp Asn Leu Ala Gly Gly Ser Thr Ser Ile Val Tyr Tyr Asp Ser Asp         355 360 365 Glu Met Ile Phe Val Arg Asn Gly Tyr Gly Ser Lys Pro Gly Leu Ile     370 375 380 Thr Tyr Ile Asn Leu Gly Ser Ser Lys Val Gly Arg Trp Val Tyr Val 385 390 395 400 Pro Lys Phe Ala Gly Ala Cys Ile His Glu Tyr Thr Gly Asn Leu Gly                 405 410 415 Gly Trp Val Asp Lys Tyr Val Tyr Ser Ser Gly Trp Val Tyr Leu Glu             420 425 430 Ala Pro Ala Tyr Asp Pro Ala Asn Gly Gln Tyr Gly Tyr Ser Val Trp          435 440 445 Ser Tyr Cys Gly Val Gly     450 <210> 14 <211> 460 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 14 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala Thr Ser Ala Lys Tyr Leu Glu Leu Glu Glu Gly Gly Val Ile Met             20 25 30 Gln Ala Phe Tyr Trp Asp Val Pro Ser Gly Gly Ile Trp Trp Asp Thr         35 40 45 Ile Arg Gln Lys Ile Pro Glu Trp Tyr Asp Ala Gly Ile Ser Ala Ile     50 55 60 Trp Ile Pro Pro Ala Ser Lys Gly Met Ser Gly Gly Tyr Ser Met Gly 65 70 75 80 Tyr Asp Pro Tyr Asp Tyr Phe Asp Leu Gly Glu Tyr Tyr Gln Lys Gly                 85 90 95 Thr Val Glu Thr Arg Phe Gly Ser Lys Gln Glu Leu Ile Asn Met Ile             100 105 110 Asn Thr Ala His Ala Tyr Gly Ile Lys Val Ile Ala Asp Ile Val Ile         115 120 125 Asn His Arg Ala Gly Gly Asp Leu Glu Trp Asn Pro Phe Val Gly Asp     130 135 140 Tyr Thr Trp Thr Asp Phe Ser Lys Val Ala Ser Gly Lys Tyr Thr Ala 145 150 155 160 Asn Tyr Leu Asp Phe His Pro Asn Glu Leu His Ala Gly Asp Ser Gly                 165 170 175 Thr Phe Gly Gly Tyr Pro Asp Ile Cys His Asp Lys Ser Trp Asp Gln             180 185 190 Tyr Trp Leu Trp Ala Ser Gln Glu Ser Tyr Ala Ala Tyr Leu Arg Ser         195 200 205 Ile Gly Ile Asp Ala Trp Arg Phe Asp Tyr Val Lys Gly Tyr Gly Ala     210 215 220 Trp Val Val Lys Asp Trp Leu Asn Trp Trp Gly Gly Trp Ala Val Gly 225 230 235 240 Glu Tyr Trp Asp Thr Asn Val Asp Ala Leu Leu Asn Trp Ala Tyr Ser                 245 250 255 Ser Gly Ala Lys Val Phe Asp Phe Pro Leu Tyr Tyr Lys Met Asp Ala             260 265 270 Ala Phe Asp Asn Lys Asn Ile Pro Ala Leu Val Glu Ala Leu Lys Asn         275 280 285 Gly Gly Thr Val Val Ser Arg Asp Pro Phe Lys Ala Val Thr Phe Val     290 295 300 Ala Asn His Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala 305 310 315 320 Phe Ile Leu Thr Tyr Glu Gly Gln Pro Thr Ile Phe Tyr Arg Asp Tyr                 325 330 335 Glu Glu Trp Leu Asn Lys Asp Lys Leu Lys Asn Leu Ile Trp Ile His             340 345 350  Asp Asn Leu Ala Gly Gly Ser Thr Ser Ile Val Tyr Tyr Asp Ser Asp         355 360 365 Glu Met Ile Phe Val Arg Asn Gly Tyr Gly Ser Lys Pro Gly Leu Ile     370 375 380 Thr Tyr Ile Asn Leu Gly Ser Ser Lys Val Gly Arg Trp Val Tyr Val 385 390 395 400 Pro Lys Phe Ala Gly Ala Cys Ile His Glu Tyr Thr Gly Asn Leu Gly                 405 410 415 Gly Trp Val Asp Lys Tyr Val Tyr Ser Ser Gly Trp Val Tyr Leu Glu             420 425 430 Ala Pro Ala Tyr Asp Pro Ala Asn Gly Gln Tyr Gly Tyr Ser Val Trp         435 440 445 Ser Tyr Cys Gly Val Gly Ser Glu Lys Asp Glu Leu     450 455 460 <210> 15 <211> 518 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 15 Met Leu Ala Ala Leu Ala Thr Ser Gln Leu Val Ala Thr Arg Ala Gly  1 5 10 15 Leu Gly Val Pro Asp Ala Ser Thr Phe Arg Arg Gly Ala Ala Gln Gly             20 25 30 Leu Arg Gly Ala Arg Ala Ser Ala Ala Ala Asp Thr Leu Ser Met Arg         35 40 45 Thr Ser Ala Arg Ala Ala Pro Arg His Gln His Gln Gln Ala Arg Arg     50 55 60 Gly Ala Arg Phe Pro Ser Leu Val Val Cys Ala Ser Ala Gly Ala Met 65 70 75 80 Ala Lys Tyr Leu Glu Leu Glu Glu Gly Gly Val Ile Met Gln Ala Phe                 85 90 95 Tyr Trp Asp Val Pro Ser Gly Gly Ile Trp Trp Asp Thr Ile Arg Gln             100 105 110 Lys Ile Pro Glu Trp Tyr Asp Ala Gly Ile Ser Ala Ile Trp Ile Pro         115 120 125 Pro Ala Ser Lys Gly Met Ser Gly Gyr Tyr Ser Met Gly Tyr Asp Pro     130 135 140 Tyr Asp Tyr Phe Asp Leu Gly Glu Tyr Tyr Gln Lys Gly Thr Val Glu 145 150 155 160 Thr Arg Phe Gly Ser Lys Gln Glu Leu Ile Asn Met Ile Asn Thr Ala                 165 170 175 His Ala Tyr Gly Ile Lys Val Ile Ala Asp Ile Val Ile Asn His Arg             180 185 190 Ala Gly Gly Asp Leu Glu Trp Asn Pro Phe Val Gly Asp Tyr Thr Trp         195 200 205 Thr Asp Phe Ser Lys Val Ala Ser Gly Lys Tyr Thr Ala Asn Tyr Leu     210 215 220 Asp Phe His Pro Asn Glu Leu His Ala Gly Asp Ser Gly Thr Phe Gly 225 230 235 240 Gly Tyr Pro Asp Ile Cys His Asp Lys Ser Trp Asp Gln Tyr Trp Leu                 245 250 255 Trp Ala Ser Gln Glu Ser Tyr Ala Ala Tyr Leu Arg Ser Ile Gly Ile              260 265 270 Asp Ala Trp Arg Phe Asp Tyr Val Lys Gly Tyr Gly Ala Trp Val Val         275 280 285 Lys Asp Trp Leu Asn Trp Trp Gly Gly Trp Ala Val Gly Glu Tyr Trp     290 295 300 Asp Thr Asn Val Asp Ala Leu Leu Asn Trp Ala Tyr Ser Ser Gly Ala 305 310 315 320 Lys Val Phe Asp Phe Pro Leu Tyr Tyr Lys Met Asp Ala Ala Phe Asp                 325 330 335 Asn Lys Asn Ile Pro Ala Leu Val Glu Ala Leu Lys Asn Gly Gly Thr             340 345 350 Val Val Ser Arg Asp Pro Phe Lys Ala Val Thr Phe Val Ala Asn His         355 360 365 Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala Phe Ile Leu     370 375 380 Thr Tyr Glu Gly Gln Pro Thr Ile Phe Tyr Arg Asp Tyr Glu Glu Trp 385 390 395 400 Leu Asn Lys Asp Lys Leu Lys Asn Leu Ile Trp Ile His Asp Asn Leu                 405 410 415 Ala Gly Gly Ser Thr Ser Ile Val Tyr Tyr Asp Ser Asp Glu Met Ile             420 425 430 Phe Val Arg Asn Gly Tyr Gly Ser Lys Pro Gly Leu Ile Thr Tyr Ile         435 440 445 Asn Leu Gly Ser Ser Lys Val Gly Arg Trp Val Tyr Val Pro Lys Phe     450 455 460 Ala Gly Ala Cys Ile His Glu Tyr Thr Gly Asn Leu Gly Gly Trp Val 465 470 475 480 Asp Lys Tyr Val Tyr Ser Ser Gly Trp Val Tyr Leu Glu Ala Pro Ala                 485 490 495 Tyr Asp Pro Ala Asn Gly Gln Tyr Gly Tyr Ser Val Trp Ser Tyr Cys             500 505 510 Gly Val Gly Thr Ser Ile         515 <210> 16 <211> 820 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 16 Met Leu Ala Ala Leu Ala Thr Ser Gln Leu Val Ala Thr Arg Ala Gly  1 5 10 15 Leu Gly Val Pro Asp Ala Ser Thr Phe Arg Arg Gly Ala Ala Gln Gly             20 25 30 Leu Arg Gly Ala Arg Ala Ser Ala Ala Ala Asp Thr Leu Ser Met Arg         35 40 45 Thr Ser Ala Arg Ala Ala Pro Arg His Gln His Gln Gln Ala Arg Arg     50 55 60 Gly Ala Arg Phe Pro Ser Leu Val Val Cys Ala Ser Ala Gly Ala Met 65 70 75 80 Ala Lys Tyr Leu Glu Leu Glu Glu Gly Gly Val Ile Met Gln Ala Phe                 85 90 95 Tyr Trp Asp Val Pro Ser Gly Gly Ile Trp Trp Asp Thr Ile Arg Gln             100 105 110  Lys Ile Pro Glu Trp Tyr Asp Ala Gly Ile Ser Ala Ile Trp Ile Pro         115 120 125 Pro Ala Ser Lys Gly Met Ser Gly Gyr Tyr Ser Met Gly Tyr Asp Pro     130 135 140 Tyr Asp Tyr Phe Asp Leu Gly Glu Tyr Tyr Gln Lys Gly Thr Val Glu 145 150 155 160 Thr Arg Phe Gly Ser Lys Gln Glu Leu Ile Asn Met Ile Asn Thr Ala                 165 170 175 His Ala Tyr Gly Ile Lys Val Ile Ala Asp Ile Val Ile Asn His Arg             180 185 190 Ala Gly Gly Asp Leu Glu Trp Asn Pro Phe Val Gly Asp Tyr Thr Trp         195 200 205 Thr Asp Phe Ser Lys Val Ala Ser Gly Lys Tyr Thr Ala Asn Tyr Leu     210 215 220 Asp Phe His Pro Asn Glu Leu His Ala Gly Asp Ser Gly Thr Phe Gly 225 230 235 240 Gly Tyr Pro Asp Ile Cys His Asp Lys Ser Trp Asp Gln Tyr Trp Leu                 245 250 255 Trp Ala Ser Gln Glu Ser Tyr Ala Ala Tyr Leu Arg Ser Ile Gly Ile             260 265 270 Asp Ala Trp Arg Phe Asp Tyr Val Lys Gly Tyr Gly Ala Trp Val Val         275 280 285 Lys Asp Trp Leu Asn Trp Trp Gly Gly Trp Ala Val Gly Glu Tyr Trp     290 295 300 Asp Thr Asn Val Asp Ala Leu Leu Asn Trp Ala Tyr Ser Ser Gly Ala 305 310 315 320 Lys Val Phe Asp Phe Pro Leu Tyr Tyr Lys Met Asp Ala Ala Phe Asp                 325 330 335 Asn Lys Asn Ile Pro Ala Leu Val Glu Ala Leu Lys Asn Gly Gly Thr             340 345 350 Val Val Ser Arg Asp Pro Phe Lys Ala Val Thr Phe Val Ala Asn His         355 360 365 Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala Phe Ile Leu     370 375 380 Thr Tyr Glu Gly Gln Pro Thr Ile Phe Tyr Arg Asp Tyr Glu Glu Trp 385 390 395 400 Leu Asn Lys Asp Lys Leu Lys Asn Leu Ile Trp Ile His Asp Asn Leu                 405 410 415 Ala Gly Gly Ser Thr Ser Ile Val Tyr Tyr Asp Ser Asp Glu Met Ile             420 425 430 Phe Val Arg Asn Gly Tyr Gly Ser Lys Pro Gly Leu Ile Thr Tyr Ile         435 440 445 Asn Leu Gly Ser Ser Lys Val Gly Arg Trp Val Tyr Val Pro Lys Phe     450 455 460 Ala Gly Ala Cys Ile His Glu Tyr Thr Gly Asn Leu Gly Gly Trp Val 465 470 475 480 Asp Lys Tyr Val Tyr Ser Ser Gly Trp Val Tyr Leu Glu Ala Pro Ala                 485 490 495 Tyr Asp Pro Ala Asn Gly Gln Tyr Gly Tyr Ser Val Trp Ser Tyr Cys             500 505 510 Gly Val Gly Thr Ser Ile Ala Gly Ile Leu Glu Ala Asp Arg Val Leu         515 520 525 Thr Val Ser Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ile Ala Arg     530 535 540 Gly Cys Glu Leu Asp Asn Ile Met Arg Leu Thr Gly Ile Thr Gly Ile 545 550 555 560 Val Asn Gly Met Asp Val Ser Glu Trp Asp Pro Ser Arg Asp Lys Tyr                 565 570 575  Ile Ala Val Lys Tyr Asp Val Ser Thr Ala Val Glu Ala Lys Ala Leu             580 585 590 Asn Lys Glu Ala Leu Gln Ala Glu Val Gly Leu Pro Val Asp Arg Asn         595 600 605 Ile Pro Leu Val Ala Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Pro     610 615 620 Asp Val Met Ala Ala Ala Ile Pro Gln Leu Met Glu Met Val Glu Asp 625 630 635 640 Val Gln Ile Val Leu Leu Gly Thr Gly Lys Lys Lys Phe Glu Arg Met                 645 650 655 Leu Met Ser Ala Glu Glu Lys Phe Pro Gly Lys Val Arg Ala Val Val             660 665 670 Lys Phe Asn Ala Ala Leu Ala His His Ile Met Ala Gly Ala Asp Val         675 680 685 Leu Ala Val Thr Ser Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu Gln     690 695 700 Gly Met Arg Tyr Gly Thr Pro Cys Ala Cys Ala Ser Thr Gly Gly Leu 705 710 715 720 Val Asp Thr Ile Ile Glu Gly Lys Thr Gly Phe His Met Gly Arg Leu                 725 730 735 Ser Val Asp Cys Asn Val Val Glu Pro Ala Asp Val Lys Lys Val Ala             740 745 750 Thr Thr Leu Gln Arg Ala Ile Lys Val Val Gly Thr Pro Ala Tyr Glu         755 760 765 Glu Met Val Arg Asn Cys Met Ile Gln Asp Leu Ser Trp Lys Gly Pro     770 775 780 Ala Lys Asn Trp Glu Asn Val Leu Leu Ser Leu Gly Val Ala Gly Gly 785 790 795 800 Glu Pro Gly Val Glu Gly Glu Glu Ile Ala Pro Leu Ala Lys Glu Asn                 805 810 815 Val Ala Ala Pro             820 <210> 17 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 17 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala thr ser              <210> 18 <211> 444 <212> PRT <213> Thermotoga maritima <400> 18 Met Ala Glu Phe Phe Pro Glu Ile Pro Lys Ile Gln Phe Glu Gly Lys  1 5 10 15 Glu Ser Thr Asn Pro Leu Ala Phe Arg Phe Tyr Asp Pro Asn Glu Val              20 25 30 Ile Asp Gly Lys Pro Leu Lys Asp His Leu Lys Phe Ser Val Ala Phe         35 40 45 Trp His Thr Phe Val Asn Glu Gly Arg Asp Pro Phe Gly Asp Pro Thr     50 55 60 Ala Glu Arg Pro Trp Asn Arg Phe Ser Asp Pro Met Asp Lys Ala Phe 65 70 75 80 Ala Arg Val Asp Ala Leu Phe Glu Phe Cys Glu Lys Leu Asn Ile Glu                 85 90 95 Tyr Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Lys Thr Leu             100 105 110 Arg Glu Thr Asn Lys Ile Leu Asp Lys Val Val Glu Arg Ile Lys Glu         115 120 125 Arg Met Lys Asp Ser Asn Val Lys Leu Leu Trp Gly Thr Ala Asn Leu     130 135 140 Phe Ser His Pro Arg Tyr Met His Gly Ala Ala Thr Thr Cys Ser Ala 145 150 155 160 Asp Val Phe Ala Tyr Ala Ala Ala Gln Val Lys Lys Ala Leu Glu Ile                 165 170 175 Thr Lys Glu Leu Gly Gly Glu Gly Tyr Val Phe Trp Gly Gly Arg Glu             180 185 190 Gly Tyr Glu Thr Leu Leu Asn Thr Asp Leu Gly Leu Glu Leu Glu Asn         195 200 205 Leu Ala Arg Phe Leu Arg Met Ala Val Glu Tyr Ala Lys Lys Ile Gly     210 215 220 Phe Thr Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys 225 230 235 240 His Gln Tyr Asp Phe Asp Val Ala Thr Ala Tyr Ala Phe Leu Lys Asn                 245 250 255 His Gly Leu Asp Glu Tyr Phe Lys Phe Asn Ile Glu Ala Asn His Ala             260 265 270 Thr Leu Ala Gly His Thr Phe Gln His Glu Leu Arg Met Ala Arg Ile         275 280 285 Leu Gly Lys Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Leu Leu Leu     290 295 300 Gly Trp Asp Thr Asp Gln Phe Pro Thr Asn Ile Tyr Asp Thr Thr Leu 305 310 315 320 Ala Met Tyr Glu Val Ile Lys Ala Gly Gly Phe Thr Lys Gly Gly Leu                 325 330 335 Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Tyr Lys Val Glu Asp Leu             340 345 350 Phe Ile Gly His Ile Ala Gly Met Asp Thr Phe Ala Leu Gly Phe Lys         355 360 365 Ile Ala Tyr Lys Leu Ala Lys Asp Gly Val Phe Asp Lys Phe Ile Glu     370 375 380 Glu Lys Tyr Arg Ser Phe Lys Glu Gly Ile Gly Lys Glu Ile Val Glu 385 390 395 400 Gly Lys Thr Asp Phe Glu Lys Leu Glu Glu Tyr Ile Ile Asp Lys Glu                 405 410 415 Asp Ile Glu Leu Pro Ser Gly Lys Gln Glu Tyr Leu Glu Ser Leu Leu             420 425 430 Asn Ser Tyr Ile Val Lys Thr Ile Ala Glu Leu Arg         435 440 <210> 19 <211> 1335 <212> DNA   <213> Thermotoga maritima <400> 19 atggccgagt tcttcccgga gatcccgaag atccagttcg agggcaagga gtccaccaac 60 ccgctcgcct tccgcttcta cgacccgaac gaggtgatcg acggcaagcc gctcaaggac 120 cacctcaagt tctccgtggc cttctggcac accttcgtga acgagggccg cgacccgttc 180 ggcgacccga ccgccgagcg cccgtggaac cgcttctccg acccgatgga caaggccttc 240 gcccgcgtgg acgccctctt cgagttctgc gagaagctca acatcgagta cttctgcttc 300 cacgaccgcg acatcgcccc ggagggcaag accctccgcg agaccaacaa gatcctcgac 360 aaggtggtgg agcgcatcaa ggagcgcatg aaggactcca acgtgaagct cctctggggc 420 accgccaacc tcttctccca cccgcgctac atgcacggcg ccgccaccac ctgctccgcc 480 gacgtgttcg cctacgccgc cgcccaggtg aagaaggccc tggagatcac caaggagctg 540 ggcggcgagg gctacgtgtt ctggggcggc cgcgagggct acgagaccct cctcaacacc 600 gacctcggcc tggagctgga gaacctcgcc cgcttcctcc gcatggccgt ggagtacgcc 660 aagaagatcg gcttcaccgg ccagttcctc atcgagccga agccgaagga gccgaccaag 720 caccagtacg acttcgacgt ggccaccgcc tacgccttcc tcaagaacca cggcctcgac 780 gagtacttca agttcaacat cgaggccaac cacgccaccc tcgccggcca caccttccag 840 cacgagctgc gcatggcccg catcctcggc aagctcggct ccatcgacgc caaccagggc 900 gacctcctcc tcggctggga caccgaccag ttcccgacca acatctacga caccaccctc 960 gccatgtacg aggtgatcaa ggccggcggc ttcaccaagg gcggcctcaa cttcgacgcc 1020 aaggtgcgcc gcgcctccta caaggtggag gacctcttca tcggccacat cgccggcatg 1080 gacaccttcg ccctcggctt caagatcgcc tacaagctcg ccaaggacgg cgtgttcgac 1140 aagttcatcg aggagaagta ccgctccttc aaggagggca tcggcaagga gatcgtggag 1200 ggcaagaccg acttcgagaa gctggaggag tacatcatcg acaaggagga catcgagctg 1260 ccgtccggca agcaggagta cctggagtcc ctcctcaact cctacatcgt gaagaccatc 1320 gccgagctgc gctga 1335 <210> 20 <211> 444 <212> PRT <213> Thermotoga neapolitana <400> 20 Met Ala Glu Phe Phe Pro Glu Ile Pro Lys Val Gln Phe Glu Gly Lys  1 5 10 15 Glu Ser Thr Asn Pro Leu Ala Phe Lys Phe Tyr Asp Pro Glu Glu Ile             20 25 30 Ile Asp Gly Lys Pro Leu Lys Asp His Leu Lys Phe Ser Val Ala Phe         35 40 45 Trp His Thr Phe Val Asn Glu Gly Arg Asp Pro Phe Gly Asp Pro Thr     50 55 60 Ala Asp Arg Pro Trp Asn Arg Tyr Thr Asp Pro Met Asp Lys Ala Phe 65 70 75 80 Ala Arg Val Asp Ala Leu Phe Glu Phe Cys Glu Lys Leu Asn Ile Glu                 85 90 95 Tyr Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Lys Thr Leu             100 105 110 Arg Glu Thr Asn Lys Ile Leu Asp Lys Val Val Glu Arg Ile Lys Glu         115 120 125 Arg Met Lys Asp Ser Asn Val Lys Leu Leu Trp Gly Thr Ala Asn Leu     130 135 140 Phe Ser His Pro Arg Tyr Met His Gly Ala Ala Thr Thr Cys Ser Ala 145 150 155 160 Asp Val Phe Ala Tyr Ala Ala Ala Gln Val Lys Lys Ala Leu Glu Ile                 165 170 175 Thr Lys Glu Leu Gly Gly Glu Gly Tyr Val Phe Trp Gly Gly Arg Glu             180 185 190 Gly Tyr Glu Thr Leu Leu Asn Thr Asp Leu Gly Phe Glu Leu Glu Asn          195 200 205 Leu Ala Arg Phe Leu Arg Met Ala Val Asp Tyr Ala Lys Arg Ile Gly     210 215 220 Phe Thr Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys 225 230 235 240 His Gln Tyr Asp Phe Asp Val Ala Thr Ala Tyr Ala Phe Leu Lys Ser                 245 250 255 His Gly Leu Asp Glu Tyr Phe Lys Phe Asn Ile Glu Ala Asn His Ala             260 265 270 Thr Leu Ala Gly His Thr Phe Gln His Glu Leu Arg Met Ala Arg Ile         275 280 285 Leu Gly Lys Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Leu Leu Leu     290 295 300 Gly Trp Asp Thr Asp Gln Phe Pro Thr Asn Val Tyr Asp Thr Thr Leu 305 310 315 320 Ala Met Tyr Glu Val Ile Lys Ala Gly Gly Phe Thr Lys Gly Gly Leu                 325 330 335 Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Tyr Lys Val Glu Asp Leu             340 345 350 Phe Ile Gly His Ile Ala Gly Met Asp Thr Phe Ala Leu Gly Phe Lys         355 360 365 Val Ala Tyr Lys Leu Val Lys Asp Gly Val Leu Asp Lys Phe Ile Glu     370 375 380 Glu Lys Tyr Arg Ser Phe Arg Glu Gly Ile Gly Arg Asp Ile Val Glu 385 390 395 400 Gly Lys Val Asp Phe Glu Lys Leu Glu Glu Tyr Ile Ile Asp Lys Glu                 405 410 415 Thr Ile Glu Leu Pro Ser Gly Lys Gln Glu Tyr Leu Glu Ser Leu Ile             420 425 430 Asn Ser Tyr Ile Val Lys Thr Ile Leu Glu Leu Arg         435 440 <210> 21 <211> 1335 <212> DNA <213> Thermotoga neapolitana <400> 21 atggccgagt tcttcccgga gatcccgaag gtgcagttcg agggcaagga gtccaccaac 60 ccgctcgcct tcaagttcta cgacccggag gagatcatcg acggcaagcc gctcaaggac 120 cacctcaagt tctccgtggc cttctggcac accttcgtga acgagggccg cgacccgttc 180 ggcgacccga ccgccgaccg cccgtggaac cgctacaccg acccgatgga caaggccttc 240 gcccgcgtgg acgccctctt cgagttctgc gagaagctca acatcgagta cttctgcttc 300 cacgaccgcg acatcgcccc ggagggcaag accctccgcg agaccaacaa gatcctcgac 360 aaggtggtgg agcgcatcaa ggagcgcatg aaggactcca acgtgaagct cctctggggc 420 accgccaacc tcttctccca cccgcgctac atgcacggcg ccgccaccac ctgctccgcc 480 gacgtgttcg cctacgccgc cgcccaggtg aagaaggccc tggagatcac caaggagctg 540 ggcggcgagg gctacgtgtt ctggggcggc cgcgagggct acgagaccct cctcaacacc 600 gacctcggct tcgagctgga gaacctcgcc cgcttcctcc gcatggccgt ggactacgcc 660 aagcgcatcg gcttcaccgg ccagttcctc atcgagccga agccgaagga gccgaccaag 720 caccagtacg acttcgacgt ggccaccgcc tacgccttcc tcaagtccca cggcctcgac 780 gagtacttca agttcaacat cgaggccaac cacgccaccc tcgccggcca caccttccag 840 cacgagctgc gcatggcccg catcctcggc aagctcggct ccatcgacgc caaccagggc 900 gacctcctcc tcggctggga caccgaccag ttcccgacca acgtgtacga caccaccctc 960 gccatgtacg aggtgatcaa ggccggcggc ttcaccaagg gcggcctcaa cttcgacgcc 1020 aaggtgcgcc gcgcctccta caaggtggag gacctcttca tcggccacat cgccggcatg 1080 gacaccttcg ccctcggctt caaggtggcc tacaagctcg tgaaggacgg cgtgctcgac 1140  aagttcatcg aggagaagta ccgctccttc cgcgagggca tcggccgcga catcgtggag 1200 ggcaaggtgg acttcgagaa gctggaggag tacatcatcg acaaggagac catcgagctg 1260 ccgtccggca agcaggagta cctggagtcc ctcatcaact cctacatcgt gaagaccatc 1320 ctggagctgc gctga 1335 <210> 22 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 22 agcgaattca tggcggctct ggccacgt 28 <210> 23 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 23 agctaagctt cagggcgcgg ccacgttct 29 <210> 24 <211> 825 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 24 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala Thr Ser Ala Gly His Trp Tyr Lys His Gln Arg Ala Tyr Gln Phe             20 25 30 Thr Gly Glu Asp Asp Phe Gly Lys Val Ala Val Val Lys Leu Pro Met         35 40 45 Asp Leu Thr Lys Val Gly Ile Ile Val Arg Leu Asn Glu Trp Gln Ala     50 55 60 Lys Asp Val Ala Lys Asp Arg Phe Ile Glu Ile Lys Asp Gly Lys Ala 65 70 75 80 Glu Val Trp Ile Leu Gln Gly Val Glu Glu Ile Phe Tyr Glu Lys Pro                 85 90 95 Asp Thr Ser Pro Arg Ile Phe Phe Ala Gln Ala Arg Ser Asn Lys Val             100 105 110 Ile Glu Ala Phe Leu Thr Asn Pro Val Asp Thr Lys Lys Lys Glu Leu         115 120 125 Phe Lys Val Thr Val Asp Gly Lys Glu Ile Pro Val Ser Arg Val Glu     130 135 140 Lys Ala Asp Pro Thr Asp Ile Asp Val Thr Asn Tyr Val Arg Ile Val 145 150 155 160 Leu Ser Glu Ser Leu Lys Glu Glu Asp Leu Arg Lys Asp Val Glu Leu                 165 170 175  Ile Ile Glu Gly Tyr Lys Pro Ala Arg Val Ile Met Met Glu Ile Leu             180 185 190 Asp Asp Tyr Tyr Tyr Asp Gly Glu Leu Gly Ala Val Tyr Ser Pro Glu         195 200 205 Lys Thr Ile Phe Arg Val Trp Ser Pro Val Ser Lys Trp Val Lys Val     210 215 220 Leu Leu Phe Lys Asn Gly Glu Asp Thr Glu Pro Tyr Gln Val Val Asn 225 230 235 240 Met Glu Tyr Lys Gly Asn Gly Val Trp Glu Ala Val Val Glu Gly Asp                 245 250 255 Leu Asp Gly Val Phe Tyr Leu Tyr Gln Leu Glu Asn Tyr Gly Lys Ile             260 265 270 Arg Thr Thr Val Asp Pro Tyr Ser Lys Ala Val Tyr Ala Asn Asn Gln         275 280 285 Glu Ser Ala Val Val Asn Leu Ala Arg Thr Asn Pro Glu Gly Trp Glu     290 295 300 Asn Asp Arg Gly Pro Lys Ile Glu Gly Tyr Glu Asp Ala Ile Ile Tyr 305 310 315 320 Glu Ile His Ile Ala Asp Ile Thr Gly Leu Glu Asn Ser Gly Val Lys                 325 330 335 Asn Lys Gly Leu Tyr Leu Gly Leu Thr Glu Glu Asn Thr Lys Ala Pro             340 345 350 Gly Gly Val Thr Thr Gly Leu Ser His Leu Val Glu Leu Gly Val Thr         355 360 365 His Val His Ile Leu Pro Phe Phe Asp Phe Tyr Thr Gly Asp Glu Leu     370 375 380 Asp Lys Asp Phe Glu Lys Tyr Tyr Asn Trp Gly Tyr Asp Pro Tyr Leu 385 390 395 400 Phe Met Val Pro Glu Gly Arg Tyr Ser Thr Asp Pro Lys Asn Pro His                 405 410 415 Thr Arg Ile Arg Glu Val Lys Glu Met Val Lys Ala Leu His Lys His             420 425 430 Gly Ile Gly Val Ile Met Asp Met Val Phe Pro His Thr Tyr Gly Ile         435 440 445 Gly Glu Leu Ser Ala Phe Asp Gln Thr Val Pro Tyr Tyr Phe Tyr Arg     450 455 460 Ile Asp Lys Thr Gly Ala Tyr Leu Asn Glu Ser Gly Cys Gly Asn Val 465 470 475 480 Ile Ala Ser Glu Arg Pro Met Met Arg Lys Phe Ile Val Asp Thr Val                 485 490 495 Thr Tyr Trp Val Lys Glu Tyr His Ile Asp Gly Phe Arg Phe Asp Gln             500 505 510 Met Gly Leu Ile Asp Lys Lys Thr Met Leu Glu Val Glu Arg Ala Leu         515 520 525 His Lys Ile Asp Pro Thr Ile Ile Leu Tyr Gly Glu Pro Trp Gly Gly     530 535 540 Trp Gly Ala Pro Ile Arg Phe Gly Lys Ser Asp Val Ala Gly Thr His 545 550 555 560 Val Ala Ala Phe Asn Asp Glu Phe Arg Asp Ala Ile Arg Gly Ser Val                 565 570 575 Phe Asn Pro Ser Val Lys Gly Phe Val Met Gly Gly Tyr Gly Lys Glu             580 585 590 Thr Lys Ile Lys Arg Gly Val Val Gly Ser Ile Asn Tyr Asp Gly Lys         595 600 605 Leu Ile Lys Ser Phe Ala Leu Asp Pro Glu Glu Thr Ile Asn Tyr Ala     610 615 620 Ala Cys His Asp Asn His Thr Leu Trp Asp Lys Asn Tyr Leu Ala Ala 625 630 635 640  Lys Ala Asp Lys Lys Lys Glu Trp Thr Glu Glu Glu Leu Lys Asn Ala                 645 650 655 Gln Lys Leu Ala Gly Ala Ile Leu Leu Thr Ser Gln Gly Val Pro Phe             660 665 670 Leu His Gly Gly Gln Asp Phe Cys Arg Thr Thr Asn Phe Asn Asp Asn         675 680 685 Ser Tyr Asn Ala Pro Ile Ser Ile Asn Gly Phe Asp Tyr Glu Arg Lys     690 695 700 Leu Gln Phe Ile Asp Val Phe Asn Tyr His Lys Gly Leu Ile Lys Leu 705 710 715 720 Arg Lys Glu His Pro Ala Phe Arg Leu Lys Asn Ala Glu Glu Ile Lys                 725 730 735 Lys His Leu Glu Phe Leu Pro Gly Gly Arg Arg Ile Val Ala Phe Met             740 745 750 Leu Lys Asp His Ala Gly Gly Asp Pro Trp Lys Asp Ile Val Val Ile         755 760 765 Tyr Asn Gly Asn Leu Glu Lys Thr Thr Tyr Lys Leu Pro Glu Gly Lys     770 775 780 Trp Asn Val Val Val Asn Ser Gln Lys Ala Gly Thr Glu Val Ile Glu 785 790 795 800 Thr Val Glu Gly Thr Ile Glu Leu Asp Pro Leu Ser Ala Tyr Val Leu                 805 810 815 Tyr Arg Glu Ser Glu Lys Asp Glu Leu             820 825 <210> 25 <211> 2478 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 25 atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc caccagcgct 60 ggccactggt acaagcacca gcgcgcctac cagttcaccg gcgaggacga cttcgggaag 120 gtggccgtgg tgaagctccc gatggacctc accaaggtgg gcatcatcgt gcgcctcaac 180 gagtggcagg cgaaggacgt ggccaaggac cgcttcatcg agatcaagga cggcaaggcc 240 gaggtgtgga tactccaggg cgtggaggag atcttctacg agaagccgga cacctccccg 300 cgcatcttct tcgcccaggc ccgctccaac aaggtgatcg aggccttcct caccaacccg 360 gtggacacca agaagaagga gctgttcaag gtgaccgtcg acggcaagga gatcccggtg 420 tcccgcgtgg agaaggccga cccgaccgac atcgacgtga ccaactacgt gcgcatcgtg 480 ctctccgagt ccctcaagga ggaggacctc cgcaaggacg tggagctgat catcgagggc 540 tacaagccgg cccgcgtgat catgatggag atcctcgacg actactacta cgacggcgag 600 ctgggggcgg tgtactcccc ggagaagacc atcttccgcg tgtggtcccc ggtgtccaag 660 tgggtgaagg tgctcctctt caagaacggc gaggacaccg agccgtacca ggtggtgaac 720 atggagtaca agggcaacgg cgtgtgggag gccgtggtgg agggcgacct cgacggcgtg 780 ttctacctct accagctgga gaactacggc aagatccgca ccaccgtgga cccgtactcc 840 aaggccgtgt acgccaacaa ccaggagtct gcagtggtga acctcgcccg caccaacccg 900 gagggctggg agaacgaccg cggcccgaag atcgagggct acgaggacgc catcatctac 960 gagatccaca tcgccgacat caccggcctg gagaactccg gcgtgaagaa caagggcctc 1020 tacctcggcc tcaccgagga gaacaccaag gccccgggcg gcgtgaccac cggcctctcc 1080 cacctcgtgg agctgggcgt gacccacgtg cacatcctcc cgttcttcga cttctacacc 1140 ggcgacgagc tggacaagga cttcgagaag tactacaact ggggctacga cccgtacctc 1200 ttcatggtgc cggagggccg ctactccacc gacccgaaga acccgcacac ccgaattcgc 1260 gaggtgaagg agatggtgaa ggccctccac aagcacggca tcggcgtgat catggacatg 1320 gtgttcccgc acacctacgg catcggcgag ctgtccgcct tcgaccagac cgtgccgtac 1380  tacttctacc gcatcgacaa gaccggcgcc tacctcaacg agtccggctg cggcaacgtg 1440 atcgcctccg agcgcccgat gatgcgcaag ttcatcgtgg acaccgtgac ctactgggtg 1500 aaggagtacc acatcgacgg cttccgcttc gaccagatgg gcctcatcga caagaagacc 1560 atgctggagg tggagcgcgc cctccacaag atcgacccga ccatcatcct ctacggcgag 1620 ccgtggggcg gctggggggc cccgatccgc ttcggcaagt ccgacgtggc cggcacccac 1680 gtggccgcct tcaacgacga gttccgcgac gccatccgcg gctccgtgtt caacccgtcc 1740 gtgaagggct tcgtgatggg cggctacggc aaggagacca agatcaagcg cggcgtggtg 1800 ggctccatca actacgacgg caagctcatc aagtccttcg ccctcgaccc ggaggagacc 1860 atcaactacg ccgcctgcca cgacaaccac accctctggg acaagaacta cctcgccgcc 1920 aaggccgaca agaagaagga gtggaccgag gaggagctga agaacgccca gaagctcgcc 1980 ggcgccatcc tcctcactag tcagggcgtg ccgttcctcc acggcggcca ggacttctgc 2040 cgcaccacca acttcaacga caactcctac aacgccccga tctccatcaa cggcttcgac 2100 tacgagcgca agctccagtt catcgacgtg ttcaactacc acaagggcct catcaagctc 2160 cgcaaggagc acccggcctt ccgcctcaag aacgccgagg agatcaagaa gcacctggag 2220 ttcctcccgg gcgggcgccg catcgtggcc ttcatgctca aggaccacgc cggcggcgac 2280 ccgtggaagg acatcgtggt gatctacaac ggcaacctgg agaagaccac ctacaagctc 2340 ccggagggca agtggaacgt ggtggtgaac tcccagaagg ccggcaccga ggtgatcgag 2400 accgtggagg gcaccatcga gctggacccg ctctccgcct acgtgctcta ccgcgagtcc 2460 gagaaggacg agctgtga 2478 <210> 26 <211> 718 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 26 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala Thr Ser Met Glu Thr Ile Lys Ile Tyr Glu Asn Lys Gly Val Tyr             20 25 30 Lys Val Val Ile Gly Glu Pro Phe Pro Pro Ile Glu Phe Pro Leu Glu         35 40 45 Gln Lys Ile Ser Ser Asn Lys Ser Leu Ser Glu Leu Gly Leu Thr Ile     50 55 60 Val Gln Gln Gly Asn Lys Val Ile Val Glu Lys Ser Leu Asp Leu Lys 65 70 75 80 Glu His Ile Ile Gly Leu Gly Glu Lys Ala Phe Glu Leu Asp Arg Lys                 85 90 95 Arg Lys Arg Tyr Val Met Tyr Asn Val Asp Ala Gly Ala Tyr Lys Lys             100 105 110 Tyr Gln Asp Pro Leu Tyr Val Ser Ile Pro Leu Phe Ile Ser Val Lys         115 120 125 Asp Gly Val Ala Thr Gly Tyr Phe Phe Asn Ser Ala Ser Lys Val Ile     130 135 140 Phe Asp Val Gly Leu Glu Glu Tyr Asp Lys Val Ile Val Thr Ile Pro 145 150 155 160 Glu Asp Ser Val Glu Phe Tyr Val Ile Glu Gly Pro Arg Ile Glu Asp                 165 170 175 Val Leu Glu Lys Tyr Thr Glu Leu Thr Gly Lys Pro Phe Leu Pro Pro             180 185 190 Met Trp Ala Phe Gly Tyr Met Ile Ser Arg Tyr Ser Tyr Tyr Pro Gln         195 200 205 Asp Lys Val Val Glu Leu Val Asp Ile Met Gln Lys Glu Gly Phe Arg     210 215 220 Val Ala Gly Val Phe Leu Asp Ile His Tyr Met Asp Ser Tyr Lys Leu  225 230 235 240 Phe Thr Trp His Pro Tyr Arg Phe Pro Glu Pro Lys Lys Leu Ile Asp                 245 250 255 Glu Leu His Lys Arg Asn Val Lys Leu Ile Thr Ile Val Asp His Gly             260 265 270 Ile Arg Val Asp Gln Asn Tyr Ser Pro Phe Leu Ser Gly Met Gly Lys         275 280 285 Phe Cys Glu Ile Glu Ser Gly Glu Leu Phe Val Gly Lys Met Trp Pro     290 295 300 Gly Thr Thr Val Tyr Pro Asp Phe Phe Arg Glu Asp Thr Arg Glu Trp 305 310 315 320 Trp Ala Gly Leu Ile Ser Glu Trp Leu Ser Gln Gly Val Asp Gly Ile                 325 330 335 Trp Leu Asp Met Asn Glu Pro Thr Asp Phe Ser Arg Ala Ile Glu Ile             340 345 350 Arg Asp Val Leu Ser Ser Leu Pro Val Gln Phe Arg Asp Asp Arg Leu         355 360 365 Val Thr Thr Phe Pro Asp Asn Val Val His Tyr Leu Arg Gly Lys Arg     370 375 380 Val Lys His Glu Lys Val Arg Asn Ala Tyr Pro Leu Tyr Glu Ala Met 385 390 395 400 Ala Thr Phe Lys Gly Phe Arg Thr Ser His Arg Asn Glu Ile Phe Ile                 405 410 415 Leu Ser Arg Ala Gly Tyr Ala Gly Ile Gln Arg Tyr Ala Phe Ile Trp             420 425 430 Thr Gly Asp Asn Thr Pro Ser Trp Asp Asp Leu Lys Leu Gln Leu Gln         435 440 445 Leu Val Leu Gly Leu Ser Ile Ser Gly Val Pro Phe Val Gly Cys Asp     450 455 460 Ile Gly Gly Phe Gln Gly Arg Asn Phe Ala Glu Ile Asp Asn Ser Met 465 470 475 480 Asp Leu Leu Val Lys Tyr Tyr Ala Leu Ala Leu Phe Phe Pro Phe Tyr                 485 490 495 Arg Ser His Lys Ala Thr Asp Gly Ile Asp Thr Glu Pro Val Phe Leu             500 505 510 Pro Asp Tyr Tyr Lys Glu Lys Val Lys Glu Ile Val Glu Leu Arg Tyr         515 520 525 Lys Phe Leu Pro Tyr Ile Tyr Ser Leu Ala Leu Glu Ala Ser Glu Lys     530 535 540 Gly His Pro Val Ile Arg Pro Leu Phe Tyr Glu Phe Gln Asp Asp Asp 545 550 555 560 Asp Met Tyr Arg Ile Glu Asp Glu Tyr Met Val Gly Lys Tyr Leu Leu                 565 570 575 Tyr Ala Pro Ile Val Ser Lys Glu Glu Ser Arg Leu Val Thr Leu Pro             580 585 590 Arg Gly Lys Trp Tyr Asn Tyr Trp Asn Gly Glu Ile Ile Asn Gly Lys         595 600 605 Ser Val Val Lys Ser Thr His Glu Leu Pro Ile Tyr Leu Arg Glu Gly     610 615 620 Ser Ile Ile Pro Leu Glu Gly Asp Glu Leu Ile Val Tyr Gly Glu Thr 625 630 635 640 Ser Phe Lys Arg Tyr Asp Asn Ala Glu Ile Thr Ser Ser Ser Asn Glu                 645 650 655 Ile Lys Phe Ser Arg Glu Ile Tyr Val Ser Lys Leu Thr Ile Thr Ser             660 665 670 Glu Lys Pro Val Ser Lys Ile Ile Val Asp Asp Ser Lys Glu Ile Gln         675 680 685 Val Glu Lys Thr Met Gln Asn Thr Tyr Val Ala Lys Ile Asn Gln Lys      690 695 700 Ile Arg Gly Lys Ile Asn Leu Glu Ser Glu Lys Asp Glu Leu 705 710 715 <210> 27 <211> 712 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 27 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala Thr Ser Met Glu Thr Ile Lys Ile Tyr Glu Asn Lys Gly Val Tyr             20 25 30 Lys Val Val Ile Gly Glu Pro Phe Pro Pro Ile Glu Phe Pro Leu Glu         35 40 45 Gln Lys Ile Ser Ser Asn Lys Ser Leu Ser Glu Leu Gly Leu Thr Ile     50 55 60 Val Gln Gln Gly Asn Lys Val Ile Val Glu Lys Ser Leu Asp Leu Lys 65 70 75 80 Glu His Ile Ile Gly Leu Gly Glu Lys Ala Phe Glu Leu Asp Arg Lys                 85 90 95 Arg Lys Arg Tyr Val Met Tyr Asn Val Asp Ala Gly Ala Tyr Lys Lys             100 105 110 Tyr Gln Asp Pro Leu Tyr Val Ser Ile Pro Leu Phe Ile Ser Val Lys         115 120 125 Asp Gly Val Ala Thr Gly Tyr Phe Phe Asn Ser Ala Ser Lys Val Ile     130 135 140 Phe Asp Val Gly Leu Glu Glu Tyr Asp Lys Val Ile Val Thr Ile Pro 145 150 155 160 Glu Asp Ser Val Glu Phe Tyr Val Ile Glu Gly Pro Arg Ile Glu Asp                 165 170 175 Val Leu Glu Lys Tyr Thr Glu Leu Thr Gly Lys Pro Phe Leu Pro Pro             180 185 190 Met Trp Ala Phe Gly Tyr Met Ile Ser Arg Tyr Ser Tyr Tyr Pro Gln         195 200 205 Asp Lys Val Val Glu Leu Val Asp Ile Met Gln Lys Glu Gly Phe Arg     210 215 220 Val Ala Gly Val Phe Leu Asp Ile His Tyr Met Asp Ser Tyr Lys Leu 225 230 235 240 Phe Thr Trp His Pro Tyr Arg Phe Pro Glu Pro Lys Lys Leu Ile Asp                 245 250 255 Glu Leu His Lys Arg Asn Val Lys Leu Ile Thr Ile Val Asp His Gly             260 265 270 Ile Arg Val Asp Gln Asn Tyr Ser Pro Phe Leu Ser Gly Met Gly Lys         275 280 285 Phe Cys Glu Ile Glu Ser Gly Glu Leu Phe Val Gly Lys Met Trp Pro     290 295 300 Gly Thr Thr Val Tyr Pro Asp Phe Phe Arg Glu Asp Thr Arg Glu Trp 305 310 315 320 Trp Ala Gly Leu Ile Ser Glu Trp Leu Ser Gln Gly Val Asp Gly Ile                 325 330 335 Trp Leu Asp Met Asn Glu Pro Thr Asp Phe Ser Arg Ala Ile Glu Ile             340 345 350  Arg Asp Val Leu Ser Ser Leu Pro Val Gln Phe Arg Asp Asp Arg Leu         355 360 365 Val Thr Thr Phe Pro Asp Asn Val Val His Tyr Leu Arg Gly Lys Arg     370 375 380 Val Lys His Glu Lys Val Arg Asn Ala Tyr Pro Leu Tyr Glu Ala Met 385 390 395 400 Ala Thr Phe Lys Gly Phe Arg Thr Ser His Arg Asn Glu Ile Phe Ile                 405 410 415 Leu Ser Arg Ala Gly Tyr Ala Gly Ile Gln Arg Tyr Ala Phe Ile Trp             420 425 430 Thr Gly Asp Asn Thr Pro Ser Trp Asp Asp Leu Lys Leu Gln Leu Gln         435 440 445 Leu Val Leu Gly Leu Ser Ile Ser Gly Val Pro Phe Val Gly Cys Asp     450 455 460 Ile Gly Gly Phe Gln Gly Arg Asn Phe Ala Glu Ile Asp Asn Ser Met 465 470 475 480 Asp Leu Leu Val Lys Tyr Tyr Ala Leu Ala Leu Phe Phe Pro Phe Tyr                 485 490 495 Arg Ser His Lys Ala Thr Asp Gly Ile Asp Thr Glu Pro Val Phe Leu             500 505 510 Pro Asp Tyr Tyr Lys Glu Lys Val Lys Glu Ile Val Glu Leu Arg Tyr         515 520 525 Lys Phe Leu Pro Tyr Ile Tyr Ser Leu Ala Leu Glu Ala Ser Glu Lys     530 535 540 Gly His Pro Val Ile Arg Pro Leu Phe Tyr Glu Phe Gln Asp Asp Asp 545 550 555 560 Asp Met Tyr Arg Ile Glu Asp Glu Tyr Met Val Gly Lys Tyr Leu Leu                 565 570 575 Tyr Ala Pro Ile Val Ser Lys Glu Glu Ser Arg Leu Val Thr Leu Pro             580 585 590 Arg Gly Lys Trp Tyr Asn Tyr Trp Asn Gly Glu Ile Ile Asn Gly Lys         595 600 605 Ser Val Val Lys Ser Thr His Glu Leu Pro Ile Tyr Leu Arg Glu Gly     610 615 620 Ser Ile Ile Pro Leu Glu Gly Asp Glu Leu Ile Val Tyr Gly Glu Thr 625 630 635 640 Ser Phe Lys Arg Tyr Asp Asn Ala Glu Ile Thr Ser Ser Ser Asn Glu                 645 650 655 Ile Lys Phe Ser Arg Glu Ile Tyr Val Ser Lys Leu Thr Ile Thr Ser             660 665 670 Glu Lys Pro Val Ser Lys Ile Ile Val Asp Asp Ser Lys Glu Ile Gln         675 680 685 Val Glu Lys Thr Met Gln Asn Thr Tyr Val Ala Lys Ile Asn Gln Lys     690 695 700 Ile Arg Gly Lys Ile Asn Leu Glu 705 710 <210> 28 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 28 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser   1 5 10 15 Ala Thr Ser Met Ala Glu Phe Phe Pro Glu Ile Pro Lys Ile Gln Phe             20 25 30 Glu Gly Lys Glu Ser Thr Asn Pro Leu Ala Phe Arg Phe Tyr Asp Pro         35 40 45 Asn Glu Val Ile Asp Gly Lys Pro Leu Lys Asp His Leu Lys Phe Ser     50 55 60 Val Ala Phe Trp His Thr Phe Val Asn Glu Gly Arg Asp Pro Phe Gly 65 70 75 80 Asp Pro Thr Ala Glu Arg Pro Trp Asn Arg Phe Ser Asp Pro Met Asp                 85 90 95 Lys Ala Phe Ala Arg Val Asp Ala Leu Phe Glu Phe Cys Glu Lys Leu             100 105 110 Asn Ile Glu Tyr Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly         115 120 125 Lys Thr Leu Arg Glu Thr Asn Lys Ile Leu Asp Lys Val Val Glu Arg     130 135 140 Ile Lys Glu Arg Met Lys Asp Ser Asn Val Lys Leu Leu Trp Gly Thr 145 150 155 160 Ala Asn Leu Phe Ser His Pro Arg Tyr Met His Gly Ala Ala Thr Thr                 165 170 175 Cys Ser Ala Asp Val Phe Ala Tyr Ala Ala Ala Gln Val Lys Lys Ala             180 185 190 Leu Glu Ile Thr Lys Glu Leu Gly Gly Glu Gly Tyr Val Phe Trp Gly         195 200 205 Gly Arg Glu Gly Tyr Glu Thr Leu Leu Asn Thr Asp Leu Gly Leu Glu     210 215 220 Leu Glu Asn Leu Ala Arg Phe Leu Arg Met Ala Val Glu Tyr Ala Lys 225 230 235 240 Lys Ile Gly Phe Thr Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu                 245 250 255 Pro Thr Lys His Gln Tyr Asp Phe Asp Val Ala Thr Ala Tyr Ala Phe             260 265 270 Leu Lys Asn His Gly Leu Asp Glu Tyr Phe Lys Phe Asn Ile Glu Ala         275 280 285 Asn His Ala Thr Leu Ala Gly His Thr Phe Gln His Glu Leu Arg Met     290 295 300 Ala Arg Ile Leu Gly Lys Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp 305 310 315 320 Leu Leu Leu Gly Trp Asp Thr Asp Gln Phe Pro Thr Asn Ile Tyr Asp                 325 330 335 Thr Thr Leu Ala Met Tyr Glu Val Ile Lys Ala Gly Gly Phe Thr Lys             340 345 350 Gly Gly Leu Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Tyr Lys Val         355 360 365 Glu Asp Leu Phe Ile Gly His Ile Ala Gly Met Asp Thr Phe Ala Leu     370 375 380 Gly Phe Lys Ile Ala Tyr Lys Leu Ala Lys Asp Gly Val Phe Asp Lys 385 390 395 400 Phe Ile Glu Glu Lys Tyr Arg Ser Phe Lys Glu Gly Ile Gly Lys Glu                 405 410 415 Ile Val Glu Gly Lys Thr Asp Phe Glu Lys Leu Glu Glu Tyr Ile Ile             420 425 430 Asp Lys Glu Asp Ile Glu Leu Pro Ser Gly Lys Gln Glu Tyr Leu Glu         435 440 445 Ser Leu Leu Asn Ser Tyr Ile Val Lys Thr Ile Ala Glu Leu Arg Ser     450 455 460 Glu Lys Asp Glu Leu  465 <210> 29 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 29 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala Thr Ser Met Ala Glu Phe Phe Pro Glu Ile Pro Lys Val Gln Phe             20 25 30 Glu Gly Lys Glu Ser Thr Asn Pro Leu Ala Phe Lys Phe Tyr Asp Pro         35 40 45 Glu Glu Ile Ile Asp Gly Lys Pro Leu Lys Asp His Leu Lys Phe Ser     50 55 60 Val Ala Phe Trp His Thr Phe Val Asn Glu Gly Arg Asp Pro Phe Gly 65 70 75 80 Asp Pro Thr Ala Asp Arg Pro Trp Asn Arg Tyr Thr Asp Pro Met Asp                 85 90 95 Lys Ala Phe Ala Arg Val Asp Ala Leu Phe Glu Phe Cys Glu Lys Leu             100 105 110 Asn Ile Glu Tyr Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly         115 120 125 Lys Thr Leu Arg Glu Thr Asn Lys Ile Leu Asp Lys Val Val Glu Arg     130 135 140 Ile Lys Glu Arg Met Lys Asp Ser Asn Val Lys Leu Leu Trp Gly Thr 145 150 155 160 Ala Asn Leu Phe Ser His Pro Arg Tyr Met His Gly Ala Ala Thr Thr                 165 170 175 Cys Ser Ala Asp Val Phe Ala Tyr Ala Ala Ala Gln Val Lys Lys Ala             180 185 190 Leu Glu Ile Thr Lys Glu Leu Gly Gly Glu Gly Tyr Val Phe Trp Gly         195 200 205 Gly Arg Glu Gly Tyr Glu Thr Leu Leu Asn Thr Asp Leu Gly Phe Glu     210 215 220 Leu Glu Asn Leu Ala Arg Phe Leu Arg Met Ala Val Asp Tyr Ala Lys 225 230 235 240 Arg Ile Gly Phe Thr Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu                 245 250 255 Pro Thr Lys His Gln Tyr Asp Phe Asp Val Ala Thr Ala Tyr Ala Phe             260 265 270 Leu Lys Ser His Gly Leu Asp Glu Tyr Phe Lys Phe Asn Ile Glu Ala         275 280 285 Asn His Ala Thr Leu Ala Gly His Thr Phe Gln His Glu Leu Arg Met     290 295 300 Ala Arg Ile Leu Gly Lys Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp 305 310 315 320 Leu Leu Leu Gly Trp Asp Thr Asp Gln Phe Pro Thr Asn Val Tyr Asp                 325 330 335 Thr Thr Leu Ala Met Tyr Glu Val Ile Lys Ala Gly Gly Phe Thr Lys             340 345 350 Gly Gly Leu Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Tyr Lys Val         355 360 365  Glu Asp Leu Phe Ile Gly His Ile Ala Gly Met Asp Thr Phe Ala Leu     370 375 380 Gly Phe Lys Val Ala Tyr Lys Leu Val Lys Asp Gly Val Leu Asp Lys 385 390 395 400 Phe Ile Glu Glu Lys Tyr Arg Ser Phe Arg Glu Gly Ile Gly Arg Asp                 405 410 415 Ile Val Glu Gly Lys Val Asp Phe Glu Lys Leu Glu Glu Tyr Ile Ile             420 425 430 Asp Lys Glu Thr Ile Glu Leu Pro Ser Gly Lys Gln Glu Tyr Leu Glu         435 440 445 Ser Leu Ile Asn Ser Tyr Ile Val Lys Thr Ile Leu Glu Leu Arg Ser     450 455 460 Glu Lys Asp Glu Leu 465 <210> 30 <211> 463 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 30 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala Thr Ser Met Ala Glu Phe Phe Pro Glu Ile Pro Lys Val Gln Phe             20 25 30 Glu Gly Lys Glu Ser Thr Asn Pro Leu Ala Phe Lys Phe Tyr Asp Pro         35 40 45 Glu Glu Ile Ile Asp Gly Lys Pro Leu Lys Asp His Leu Lys Phe Ser     50 55 60 Val Ala Phe Trp His Thr Phe Val Asn Glu Gly Arg Asp Pro Phe Gly 65 70 75 80 Asp Pro Thr Ala Asp Arg Pro Trp Asn Arg Tyr Thr Asp Pro Met Asp                 85 90 95 Lys Ala Phe Ala Arg Val Asp Ala Leu Phe Glu Phe Cys Glu Lys Leu             100 105 110 Asn Ile Glu Tyr Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly         115 120 125 Lys Thr Leu Arg Glu Thr Asn Lys Ile Leu Asp Lys Val Val Glu Arg     130 135 140 Ile Lys Glu Arg Met Lys Asp Ser Asn Val Lys Leu Leu Trp Gly Thr 145 150 155 160 Ala Asn Leu Phe Ser His Pro Arg Tyr Met His Gly Ala Ala Thr Thr                 165 170 175 Cys Ser Ala Asp Val Phe Ala Tyr Ala Ala Ala Gln Val Lys Lys Ala             180 185 190 Leu Glu Ile Thr Lys Glu Leu Gly Gly Glu Gly Tyr Val Phe Trp Gly         195 200 205 Gly Arg Glu Gly Tyr Glu Thr Leu Leu Asn Thr Asp Leu Gly Phe Glu     210 215 220 Leu Glu Asn Leu Ala Arg Phe Leu Arg Met Ala Val Asp Tyr Ala Lys 225 230 235 240 Arg Ile Gly Phe Thr Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu                 245 250 255 Pro Thr Lys His Gln Tyr Asp Phe Asp Val Ala Thr Ala Tyr Ala Phe              260 265 270 Leu Lys Ser His Gly Leu Asp Glu Tyr Phe Lys Phe Asn Ile Glu Ala         275 280 285 Asn His Ala Thr Leu Ala Gly His Thr Phe Gln His Glu Leu Arg Met     290 295 300 Ala Arg Ile Leu Gly Lys Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp 305 310 315 320 Leu Leu Leu Gly Trp Asp Thr Asp Gln Phe Pro Thr Asn Val Tyr Asp                 325 330 335 Thr Thr Leu Ala Met Tyr Glu Val Ile Lys Ala Gly Gly Phe Thr Lys             340 345 350 Gly Gly Leu Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Tyr Lys Val         355 360 365 Glu Asp Leu Phe Ile Gly His Ile Ala Gly Met Asp Thr Phe Ala Leu     370 375 380 Gly Phe Lys Val Ala Tyr Lys Leu Val Lys Asp Gly Val Leu Asp Lys 385 390 395 400 Phe Ile Glu Glu Lys Tyr Arg Ser Phe Arg Glu Gly Ile Gly Arg Asp                 405 410 415 Ile Val Glu Gly Lys Val Asp Phe Glu Lys Leu Glu Glu Tyr Ile Ile             420 425 430 Asp Lys Glu Thr Ile Glu Leu Pro Ser Gly Lys Gln Glu Tyr Leu Glu         435 440 445 Ser Leu Ile Asn Ser Tyr Ile Val Lys Thr Ile Leu Glu Leu Arg     450 455 460 <210> 31 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 31 Met Gly Lys Asn Gly Asn Leu Cys Cys Phe Ser Leu Leu Leu Leu Leu  1 5 10 15 Leu Ala Gly Leu Ala Ser Gly His Gln             20 25 <210> 32 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 32 Met Gly Phe Val Leu Phe Ser Gln Leu Pro Ser Phe Leu Leu Val Ser  1 5 10 15 Thr Leu Leu Leu Phe Leu Val Ile Ser His Ser Cys Arg Ala             20 25 30 <210> 33   <211> 460 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 33 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala Thr Ser Ala Lys Tyr Leu Glu Leu Glu Glu Gly Gly Val Ile Met             20 25 30 Gln Ala Phe Tyr Trp Asp Val Pro Ser Gly Gly Ile Trp Trp Asp Thr         35 40 45 Ile Arg Gln Lys Ile Pro Glu Trp Tyr Asp Ala Gly Ile Ser Ala Ile     50 55 60 Trp Ile Pro Pro Ala Ser Lys Gly Met Ser Gly Gly Tyr Ser Met Gly 65 70 75 80 Tyr Asp Pro Tyr Asp Tyr Phe Asp Leu Gly Glu Tyr Tyr Gln Lys Gly                 85 90 95 Thr Val Glu Thr Arg Phe Gly Ser Lys Gln Glu Leu Ile Asn Met Ile             100 105 110 Asn Thr Ala His Ala Tyr Gly Ile Lys Val Ile Ala Asp Ile Val Ile         115 120 125 Asn His Arg Ala Gly Gly Asp Leu Glu Trp Asn Pro Phe Val Gly Asp     130 135 140 Tyr Thr Trp Thr Asp Phe Ser Lys Val Ala Ser Gly Lys Tyr Thr Ala 145 150 155 160 Asn Tyr Leu Asp Phe His Pro Asn Glu Leu His Ala Gly Asp Ser Gly                 165 170 175 Thr Phe Gly Gly Tyr Pro Asp Ile Cys His Asp Lys Ser Trp Asp Gln             180 185 190 Tyr Trp Leu Trp Ala Ser Gln Glu Ser Tyr Ala Ala Tyr Leu Arg Ser         195 200 205 Ile Gly Ile Asp Ala Trp Arg Phe Asp Tyr Val Lys Gly Tyr Gly Ala     210 215 220 Trp Val Val Lys Asp Trp Leu Asn Trp Trp Gly Gly Trp Ala Val Gly 225 230 235 240 Glu Tyr Trp Asp Thr Asn Val Asp Ala Leu Leu Asn Trp Ala Tyr Ser                 245 250 255 Ser Gly Ala Lys Val Phe Asp Phe Pro Leu Tyr Tyr Lys Met Asp Ala             260 265 270 Ala Phe Asp Asn Lys Asn Ile Pro Ala Leu Val Glu Ala Leu Lys Asn         275 280 285 Gly Gly Thr Val Val Ser Arg Asp Pro Phe Lys Ala Val Thr Phe Val     290 295 300 Ala Asn His Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala 305 310 315 320 Phe Ile Leu Thr Tyr Glu Gly Gln Pro Thr Ile Phe Tyr Arg Asp Tyr                 325 330 335 Glu Glu Trp Leu Asn Lys Asp Lys Leu Lys Asn Leu Ile Trp Ile His             340 345 350 Asp Asn Leu Ala Gly Gly Ser Thr Ser Ile Val Tyr Tyr Asp Ser Asp         355 360 365 Glu Met Ile Phe Val Arg Asn Gly Tyr Gly Ser Lys Pro Gly Leu Ile     370 375 380 Thr Tyr Ile Asn Leu Gly Ser Ser Lys Val Gly Arg Trp Val Tyr Val 385 390 395 400  Pro Lys Phe Ala Gly Ala Cys Ile His Glu Tyr Thr Gly Asn Leu Gly                 405 410 415 Gly Trp Val Asp Lys Tyr Val Tyr Ser Ser Gly Trp Val Tyr Leu Glu             420 425 430 Ala Pro Ala Tyr Asp Pro Ala Asn Gly Gln Tyr Gly Tyr Ser Val Trp         435 440 445 Ser Tyr Cys Gly Val Gly Ser Glu Lys Asp Glu Leu     450 455 460 <210> 34 <211> 825 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 34 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala Thr Ser Ala Gly His Trp Tyr Lys His Gln Arg Ala Tyr Gln Phe             20 25 30 Thr Gly Glu Asp Asp Phe Gly Lys Val Ala Val Val Lys Leu Pro Met         35 40 45 Asp Leu Thr Lys Val Gly Ile Ile Val Arg Leu Asn Glu Trp Gln Ala     50 55 60 Lys Asp Val Ala Lys Asp Arg Phe Ile Glu Ile Lys Asp Gly Lys Ala 65 70 75 80 Glu Val Trp Ile Leu Gln Gly Val Glu Glu Ile Phe Tyr Glu Lys Pro                 85 90 95 Asp Thr Ser Pro Arg Ile Phe Phe Ala Gln Ala Arg Ser Asn Lys Val             100 105 110 Ile Glu Ala Phe Leu Thr Asn Pro Val Asp Thr Lys Lys Lys Glu Leu         115 120 125 Phe Lys Val Thr Val Asp Gly Lys Glu Ile Pro Val Ser Arg Val Glu     130 135 140 Lys Ala Asp Pro Thr Asp Ile Asp Val Thr Asn Tyr Val Arg Ile Val 145 150 155 160 Leu Ser Glu Ser Leu Lys Glu Glu Asp Leu Arg Lys Asp Val Glu Leu                 165 170 175 Ile Ile Glu Gly Tyr Lys Pro Ala Arg Val Ile Met Met Glu Ile Leu             180 185 190 Asp Asp Tyr Tyr Tyr Asp Gly Glu Leu Gly Ala Val Tyr Ser Pro Glu         195 200 205 Lys Thr Ile Phe Arg Val Trp Ser Pro Val Ser Lys Trp Val Lys Val     210 215 220 Leu Leu Phe Lys Asn Gly Glu Asp Thr Glu Pro Tyr Gln Val Val Asn 225 230 235 240 Met Glu Tyr Lys Gly Asn Gly Val Trp Glu Ala Val Val Glu Gly Asp                 245 250 255 Leu Asp Gly Val Phe Tyr Leu Tyr Gln Leu Glu Asn Tyr Gly Lys Ile             260 265 270 Arg Thr Thr Val Asp Pro Tyr Ser Lys Ala Val Tyr Ala Asn Asn Gln         275 280 285 Glu Ser Ala Val Val Asn Leu Ala Arg Thr Asn Pro Glu Gly Trp Glu     290 295 300 Asn Asp Arg Gly Pro Lys Ile Glu Gly Tyr Glu Asp Ala Ile Ile Tyr  305 310 315 320 Glu Ile His Ile Ala Asp Ile Thr Gly Leu Glu Asn Ser Gly Val Lys                 325 330 335 Asn Lys Gly Leu Tyr Leu Gly Leu Thr Glu Glu Asn Thr Lys Ala Pro             340 345 350 Gly Gly Val Thr Thr Gly Leu Ser His Leu Val Glu Leu Gly Val Thr         355 360 365 His Val His Ile Leu Pro Phe Phe Asp Phe Tyr Thr Gly Asp Glu Leu     370 375 380 Asp Lys Asp Phe Glu Lys Tyr Tyr Asn Trp Gly Tyr Asp Pro Tyr Leu 385 390 395 400 Phe Met Val Pro Glu Gly Arg Tyr Ser Thr Asp Pro Lys Asn Pro His                 405 410 415 Thr Arg Ile Arg Glu Val Lys Glu Met Val Lys Ala Leu His Lys His             420 425 430 Gly Ile Gly Val Ile Met Asp Met Val Phe Pro His Thr Tyr Gly Ile         435 440 445 Gly Glu Leu Ser Ala Phe Asp Gln Thr Val Pro Tyr Tyr Phe Tyr Arg     450 455 460 Ile Asp Lys Thr Gly Ala Tyr Leu Asn Glu Ser Gly Cys Gly Asn Val 465 470 475 480 Ile Ala Ser Glu Arg Pro Met Met Arg Lys Phe Ile Val Asp Thr Val                 485 490 495 Thr Tyr Trp Val Lys Glu Tyr His Ile Asp Gly Phe Arg Phe Asp Gln             500 505 510 Met Gly Leu Ile Asp Lys Lys Thr Met Leu Glu Val Glu Arg Ala Leu         515 520 525 His Lys Ile Asp Pro Thr Ile Ile Leu Tyr Gly Glu Pro Trp Gly Gly     530 535 540 Trp Gly Ala Pro Ile Arg Phe Gly Lys Ser Asp Val Ala Gly Thr His 545 550 555 560 Val Ala Ala Phe Asn Asp Glu Phe Arg Asp Ala Ile Arg Gly Ser Val                 565 570 575 Phe Asn Pro Ser Val Lys Gly Phe Val Met Gly Gly Tyr Gly Lys Glu             580 585 590 Thr Lys Ile Lys Arg Gly Val Val Gly Ser Ile Asn Tyr Asp Gly Lys         595 600 605 Leu Ile Lys Ser Phe Ala Leu Asp Pro Glu Glu Thr Ile Asn Tyr Ala     610 615 620 Ala Cys His Asp Asn His Thr Leu Trp Asp Lys Asn Tyr Leu Ala Ala 625 630 635 640 Lys Ala Asp Lys Lys Lys Glu Trp Thr Glu Glu Glu Leu Lys Asn Ala                 645 650 655 Gln Lys Leu Ala Gly Ala Ile Leu Leu Thr Ser Gln Gly Val Pro Phe             660 665 670 Leu His Gly Gly Gln Asp Phe Cys Arg Thr Thr Asn Phe Asn Asp Asn         675 680 685 Ser Tyr Asn Ala Pro Ile Ser Ile Asn Gly Phe Asp Tyr Glu Arg Lys     690 695 700 Leu Gln Phe Ile Asp Val Phe Asn Tyr His Lys Gly Leu Ile Lys Leu 705 710 715 720 Arg Lys Glu His Pro Ala Phe Arg Leu Lys Asn Ala Glu Glu Ile Lys                 725 730 735 Lys His Leu Glu Phe Leu Pro Gly Gly Arg Arg Ile Val Ala Phe Met             740 745 750 Leu Lys Asp His Ala Gly Gly Asp Pro Trp Lys Asp Ile Val Val Ile         755 760 765 Tyr Asn Gly Asn Leu Glu Lys Thr Thr Tyr Lys Leu Pro Glu Gly Lys      770 775 780 Trp Asn Val Val Val Asn Ser Gln Lys Ala Gly Thr Glu Val Ile Glu 785 790 795 800 Thr Val Glu Gly Thr Ile Glu Leu Asp Pro Leu Ser Ala Tyr Val Leu                 805 810 815 Tyr Arg Glu Ser Glu Lys Asp Glu Leu             820 825 <210> 35 <211> 460 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 35 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala Thr Ser Ala Lys Tyr Leu Glu Leu Glu Glu Gly Gly Val Ile Met             20 25 30 Gln Ala Phe Tyr Trp Asp Val Pro Ser Gly Gly Ile Trp Trp Asp Thr         35 40 45 Ile Arg Gln Lys Ile Pro Glu Trp Tyr Asp Ala Gly Ile Ser Ala Ile     50 55 60 Trp Ile Pro Pro Ala Ser Lys Gly Met Ser Gly Gly Tyr Ser Met Gly 65 70 75 80 Tyr Asp Pro Tyr Asp Tyr Phe Asp Leu Gly Glu Tyr Tyr Gln Lys Gly                 85 90 95 Thr Val Glu Thr Arg Phe Gly Ser Lys Gln Glu Leu Ile Asn Met Ile             100 105 110 Asn Thr Ala His Ala Tyr Gly Ile Lys Val Ile Ala Asp Ile Val Ile         115 120 125 Asn His Arg Ala Gly Gly Asp Leu Glu Trp Asn Pro Phe Val Gly Asp     130 135 140 Tyr Thr Trp Thr Asp Phe Ser Lys Val Ala Ser Gly Lys Tyr Thr Ala 145 150 155 160 Asn Tyr Leu Asp Phe His Pro Asn Glu Leu His Ala Gly Asp Ser Gly                 165 170 175 Thr Phe Gly Gly Tyr Pro Asp Ile Cys His Asp Lys Ser Trp Asp Gln             180 185 190 Tyr Trp Leu Trp Ala Ser Gln Glu Ser Tyr Ala Ala Tyr Leu Arg Ser         195 200 205 Ile Gly Ile Asp Ala Trp Arg Phe Asp Tyr Val Lys Gly Tyr Gly Ala     210 215 220 Trp Val Val Lys Asp Trp Leu Asn Trp Trp Gly Gly Trp Ala Val Gly 225 230 235 240 Glu Tyr Trp Asp Thr Asn Val Asp Ala Leu Leu Asn Trp Ala Tyr Ser                 245 250 255 Ser Gly Ala Lys Val Phe Asp Phe Pro Leu Tyr Tyr Lys Met Asp Ala             260 265 270 Ala Phe Asp Asn Lys Asn Ile Pro Ala Leu Val Glu Ala Leu Lys Asn         275 280 285 Gly Gly Thr Val Val Ser Arg Asp Pro Phe Lys Ala Val Thr Phe Val     290 295 300 Ala Asn His Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala 305 310 315 320  Phe Ile Leu Thr Tyr Glu Gly Gln Pro Thr Ile Phe Tyr Arg Asp Tyr                 325 330 335 Glu Glu Trp Leu Asn Lys Asp Lys Leu Lys Asn Leu Ile Trp Ile His             340 345 350 Asp Asn Leu Ala Gly Gly Ser Thr Ser Ile Val Tyr Tyr Asp Ser Asp         355 360 365 Glu Met Ile Phe Val Arg Asn Gly Tyr Gly Ser Lys Pro Gly Leu Ile     370 375 380 Thr Tyr Ile Asn Leu Gly Ser Ser Lys Val Gly Arg Trp Val Tyr Val 385 390 395 400 Pro Lys Phe Ala Gly Ala Cys Ile His Glu Tyr Thr Gly Asn Leu Gly                 405 410 415 Gly Trp Val Asp Lys Tyr Val Tyr Ser Ser Gly Trp Val Tyr Leu Glu             420 425 430 Ala Pro Ala Tyr Asp Pro Ala Asn Gly Gln Tyr Gly Tyr Ser Val Trp         435 440 445 Ser Tyr Cys Gly Val Gly Ser Glu Lys Asp Glu Leu     450 455 460 <210> 36 <211> 718 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 36 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser  1 5 10 15 Ala Thr Ser Met Glu Thr Ile Lys Ile Tyr Glu Asn Lys Gly Val Tyr             20 25 30 Lys Val Val Ile Gly Glu Pro Phe Pro Pro Ile Glu Phe Pro Leu Glu         35 40 45 Gln Lys Ile Ser Ser Asn Lys Ser Leu Ser Glu Leu Gly Leu Thr Ile     50 55 60 Val Gln Gln Gly Asn Lys Val Ile Val Glu Lys Ser Leu Asp Leu Lys 65 70 75 80 Glu His Ile Ile Gly Leu Gly Glu Lys Ala Phe Glu Leu Asp Arg Lys                 85 90 95 Arg Lys Arg Tyr Val Met Tyr Asn Val Asp Ala Gly Ala Tyr Lys Lys             100 105 110 Tyr Gln Asp Pro Leu Tyr Val Ser Ile Pro Leu Phe Ile Ser Val Lys         115 120 125 Asp Gly Val Ala Thr Gly Tyr Phe Phe Asn Ser Ala Ser Lys Val Ile     130 135 140 Phe Asp Val Gly Leu Glu Glu Tyr Asp Lys Val Ile Val Thr Ile Pro 145 150 155 160 Glu Asp Ser Val Glu Phe Tyr Val Ile Glu Gly Pro Arg Ile Glu Asp                 165 170 175 Val Leu Glu Lys Tyr Thr Glu Leu Thr Gly Lys Pro Phe Leu Pro Pro             180 185 190 Met Trp Ala Phe Gly Tyr Met Ile Ser Arg Tyr Ser Tyr Tyr Pro Gln         195 200 205 Asp Lys Val Val Glu Leu Val Asp Ile Met Gln Lys Glu Gly Phe Arg     210 215 220 Val Ala Gly Val Phe Leu Asp Ile His Tyr Met Asp Ser Tyr Lys Leu  225 230 235 240 Phe Thr Trp His Pro Tyr Arg Phe Pro Glu Pro Lys Lys Leu Ile Asp                 245 250 255 Glu Leu His Lys Arg Asn Val Lys Leu Ile Thr Ile Val Asp His Gly             260 265 270 Ile Arg Val Asp Gln Asn Tyr Ser Pro Phe Leu Ser Gly Met Gly Lys         275 280 285 Phe Cys Glu Ile Glu Ser Gly Glu Leu Phe Val Gly Lys Met Trp Pro     290 295 300 Gly Thr Thr Val Tyr Pro Asp Phe Phe Arg Glu Asp Thr Arg Glu Trp 305 310 315 320 Trp Ala Gly Leu Ile Ser Glu Trp Leu Ser Gln Gly Val Asp Gly Ile                 325 330 335 Trp Leu Asp Met Asn Glu Pro Thr Asp Phe Ser Arg Ala Ile Glu Ile             340 345 350 Arg Asp Val Leu Ser Ser Leu Pro Val Gln Phe Arg Asp Asp Arg Leu         355 360 365 Val Thr Thr Phe Pro Asp Asn Val Val His Tyr Leu Arg Gly Lys Arg     370 375 380 Val Lys His Glu Lys Val Arg Asn Ala Tyr Pro Leu Tyr Glu Ala Met 385 390 395 400 Ala Thr Phe Lys Gly Phe Arg Thr Ser His Arg Asn Glu Ile Phe Ile                 405 410 415 Leu Ser Arg Ala Gly Tyr Ala Gly Ile Gln Arg Tyr Ala Phe Ile Trp             420 425 430 Thr Gly Asp Asn Thr Pro Ser Trp Asp Asp Leu Lys Leu Gln Leu Gln         435 440 445 Leu Val Leu Gly Leu Ser Ile Ser Gly Val Pro Phe Val Gly Cys Asp     450 455 460 Ile Gly Gly Phe Gln Gly Arg Asn Phe Ala Glu Ile Asp Asn Ser Met 465 470 475 480 Asp Leu Leu Val Lys Tyr Tyr Ala Leu Ala Leu Phe Phe Pro Phe Tyr                 485 490 495 Arg Ser His Lys Ala Thr Asp Gly Ile Asp Thr Glu Pro Val Phe Leu             500 505 510 Pro Asp Tyr Tyr Lys Glu Lys Val Lys Glu Ile Val Glu Leu Arg Tyr         515 520 525 Lys Phe Leu Pro Tyr Ile Tyr Ser Leu Ala Leu Glu Ala Ser Glu Lys     530 535 540 Gly His Pro Val Ile Arg Pro Leu Phe Tyr Glu Phe Gln Asp Asp Asp 545 550 555 560 Asp Met Tyr Arg Ile Glu Asp Glu Tyr Met Val Gly Lys Tyr Leu Leu                 565 570 575 Tyr Ala Pro Ile Val Ser Lys Glu Glu Ser Arg Leu Val Thr Leu Pro             580 585 590 Arg Gly Lys Trp Tyr Asn Tyr Trp Asn Gly Glu Ile Ile Asn Gly Lys         595 600 605 Ser Val Val Lys Ser Thr His Glu Leu Pro Ile Tyr Leu Arg Glu Gly     610 615 620 Ser Ile Ile Pro Leu Glu Gly Asp Glu Leu Ile Val Tyr Gly Glu Thr 625 630 635 640 Ser Phe Lys Arg Tyr Asp Asn Ala Glu Ile Thr Ser Ser Ser Asn Glu                 645 650 655 Ile Lys Phe Ser Arg Glu Ile Tyr Val Ser Lys Leu Thr Ile Thr Ser             660 665 670 Glu Lys Pro Val Ser Lys Ile Ile Val Asp Asp Ser Lys Glu Ile Gln         675 680 685 Val Glu Lys Thr Met Gln Asn Thr Tyr Val Ala Lys Ile Asn Gln Lys      690 695 700 Ile Arg Gly Lys Ile Asn Leu Glu Ser Glu Lys Asp Glu Leu 705 710 715 <210> 37 <211> 1434 <212> DNA <213> Thermotoga maritima <400> 37 atgaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagccc agatctgggt 60 accctggtgc cacgcggttc catggccgag ttcttcccgg agatcccgaa gatccagttc 120 gagggcaagg agtccaccaa cccgctcgcc ttccgcttct acgacccgaa cgaggtgatc 180 gacggcaagc cgctcaagga ccacctcaag ttctccgtgg ccttctggca caccttcgtg 240 aacgagggcc gcgacccgtt cggcgacccg accgccgagc gcccgtggaa ccgcttctcc 300 gacccgatgg acaaggcctt cgcccgcgtg gacgccctct tcgagttctg cgagaagctc 360 aacatcgagt acttctgctt ccacgaccgc gacatcgccc cggagggcaa gaccctccgc 420 gagaccaaca agatcctcga caaggtggtg gagcgcatca aggagcgcat gaaggactcc 480 aacgtgaagc tcctctgggg caccgccaac ctcttctccc acccgcgcta catgcacggc 540 gccgccacca cctgctccgc cgacgtgttc gcctacgccg ccgcccaggt gaagaaggcc 600 ctggagatca ccaaggagct gggcggcgag ggctacgtgt tctggggcgg ccgcgagggc 660 tacgagaccc tcctcaacac cgacctcggc ctggagctgg agaacctcgc ccgcttcctc 720 cgcatggccg tggagtacgc caagaagatc ggcttcaccg gccagttcct catcgagccg 780 aagccgaagg agccgaccaa gcaccagtac gacttcgacg tggccaccgc ctacgccttc 840 ctcaagaacc acggcctcga cgagtacttc aagttcaaca tcgaggccaa ccacgccacc 900 ctcgccggcc acaccttcca gcacgagctg cgcatggccc gcatcctcgg caagctcggc 960 tccatcgacg ccaaccaggg cgacctcctc ctcggctggg acaccgacca gttcccgacc 1020 aacatctacg acaccaccct cgccatgtac gaggtgatca aggccggcgg cttcaccaag 1080 ggcggcctca acttcgacgc caaggtgcgc cgcgcctcct acaaggtgga ggacctcttc 1140 atcggccaca tcgccggcat ggacaccttc gccctcggct tcaagatcgc ctacaagctc 1200 gccaaggacg gcgtgttcga caagttcatc gaggagaagt accgctcctt caaggagggc 1260 atcggcaagg agatcgtgga gggcaagacc gacttcgaga agctggagga gtacatcatc 1320 gacaaggagg acatcgagct gccgtccggc aagcaggagt acctggagtc cctcctcaac 1380 tcctacatcg tgaagaccat cgccgagctg cgctccgaga aggacgagct gtga 1434 <210> 38 <211> 477 <212> PRT <213> Thermotoga maritima <400> 38 Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser  1 5 10 15 Pro Asp Leu Gly Thr Leu Val Pro Arg Gly Ser Met Ala Glu Phe Phe             20 25 30 Pro Glu Ile Pro Lys Ile Gln Phe Glu Gly Lys Glu Ser Thr Asn Pro         35 40 45 Leu Ala Phe Arg Phe Tyr Asp Pro Asn Glu Val Ile Asp Gly Lys Pro     50 55 60 Leu Lys Asp His Leu Lys Phe Ser Val Ala Phe Trp His Thr Phe Val 65 70 75 80 Asn Glu Gly Arg Asp Pro Phe Gly Asp Pro Thr Ala Glu Arg Pro Trp                 85 90 95 Asn Arg Phe Ser Asp Pro Met Asp Lys Ala Phe Ala Arg Val Asp Ala             100 105 110 Leu Phe Glu Phe Cys Glu Lys Leu Asn Ile Glu Tyr Phe Cys Phe His         115 120 125  Asp Arg Asp Ile Ala Pro Glu Gly Lys Thr Leu Arg Glu Thr Asn Lys     130 135 140 Ile Leu Asp Lys Val Val Glu Arg Ile Lys Glu Arg Met Lys Asp Ser 145 150 155 160 Asn Val Lys Leu Leu Trp Gly Thr Ala Asn Leu Phe Ser His Pro Arg                 165 170 175 Tyr Met His Gly Ala Ala Thr Thr Cys Ser Ala Asp Val Phe Ala Tyr             180 185 190 Ala Ala Ala Gln Val Lys Lys Ala Leu Glu Ile Thr Lys Glu Leu Gly         195 200 205 Gly Glu Gly Tyr Val Phe Trp Gly Gly Arg Glu Gly Tyr Glu Thr Leu     210 215 220 Leu Asn Thr Asp Leu Gly Leu Glu Leu Glu Asn Leu Ala Arg Phe Leu 225 230 235 240 Arg Met Ala Val Glu Tyr Ala Lys Lys Ile Gly Phe Thr Gly Gln Phe                 245 250 255 Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys His Gln Tyr Asp Phe             260 265 270 Asp Val Ala Thr Ala Tyr Ala Phe Leu Lys Asn His Gly Leu Asp Glu         275 280 285 Tyr Phe Lys Phe Asn Ile Glu Ala Asn His Ala Thr Leu Ala Gly His     290 295 300 Thr Phe Gln His Glu Leu Arg Met Ala Arg Ile Leu Gly Lys Leu Gly 305 310 315 320 Ser Ile Asp Ala Asn Gln Gly Asp Leu Leu Leu Gly Trp Asp Thr Asp                 325 330 335 Gln Phe Pro Thr Asn Ile Tyr Asp Thr Thr Leu Ala Met Tyr Glu Val             340 345 350 Ile Lys Ala Gly Gly Phe Thr Lys Gly Gly Leu Asn Phe Asp Ala Lys         355 360 365 Val Arg Arg Ala Ser Tyr Lys Val Glu Asp Leu Phe Ile Gly His Ile     370 375 380 Ala Gly Met Asp Thr Phe Ala Leu Gly Phe Lys Ile Ala Tyr Lys Leu 385 390 395 400 Ala Lys Asp Gly Val Phe Asp Lys Phe Ile Glu Glu Lys Tyr Arg Ser                 405 410 415 Phe Lys Glu Gly Ile Gly Lys Glu Ile Val Glu Gly Lys Thr Asp Phe             420 425 430 Glu Lys Leu Glu Glu Tyr Ile Ile Asp Lys Glu Asp Ile Glu Leu Pro         435 440 445 Ser Gly Lys Gln Glu Tyr Leu Glu Ser Leu Leu Asn Ser Tyr Ile Val     450 455 460 Lys Thr Ile Ala Glu Leu Arg Ser Glu Lys Asp Glu Leu 465 470 475 <210> 39 <211> 1434 <212> DNA <213> Thermotoga neapolitana <400> 39 atgaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagccc agatctgggt 60 accctggtgc cacgcggttc catggccgag ttcttcccgg agatcccgaa ggtgcagttc 120 gagggcaagg agtccaccaa cccgctcgcc ttcaagttct acgacccgga ggagatcatc 180 gacggcaagc cgctcaagga ccacctcaag ttctccgtgg ccttctggca caccttcgtg 240 aacgagggcc gcgacccgtt cggcgacccg accgccgacc gcccgtggaa ccgctacacc 300 gacccgatgg acaaggcctt cgcccgcgtg gacgccctct tcgagttctg cgagaagctc 360  aacatcgagt acttctgctt ccacgaccgc gacatcgccc cggagggcaa gaccctccgc 420 gagaccaaca agatcctcga caaggtggtg gagcgcatca aggagcgcat gaaggactcc 480 aacgtgaagc tcctctgggg caccgccaac ctcttctccc acccgcgcta catgcacggc 540 gccgccacca cctgctccgc cgacgtgttc gcctacgccg ccgcccaggt gaagaaggcc 600 ctggagatca ccaaggagct gggcggcgag ggctacgtgt tctggggcgg ccgcgagggc 660 tacgagaccc tcctcaacac cgacctcggc ttcgagctgg agaacctcgc ccgcttcctc 720 cgcatggccg tggactacgc caagcgcatc ggcttcaccg gccagttcct catcgagccg 780 aagccgaagg agccgaccaa gcaccagtac gacttcgacg tggccaccgc ctacgccttc 840 ctcaagtccc acggcctcga cgagtacttc aagttcaaca tcgaggccaa ccacgccacc 900 ctcgccggcc acaccttcca gcacgagctg cgcatggccc gcatcctcgg caagctcggc 960 tccatcgacg ccaaccaggg cgacctcctc ctcggctggg acaccgacca gttcccgacc 1020 aacgtgtacg acaccaccct cgccatgtac gaggtgatca aggccggcgg cttcaccaag 1080 ggcggcctca acttcgacgc caaggtgcgc cgcgcctcct acaaggtgga ggacctcttc 1140 atcggccaca tcgccggcat ggacaccttc gccctcggct tcaaggtggc ctacaagctc 1200 gtgaaggacg gcgtgctcga caagttcatc gaggagaagt accgctcctt ccgcgagggc 1260 atcggccgcg acatcgtgga gggcaaggtg gacttcgaga agctggagga gtacatcatc 1320 gacaaggaga ccatcgagct gccgtccggc aagcaggagt acctggagtc cctcatcaac 1380 tcctacatcg tgaagaccat cctggagctg cgctccgaga aggacgagct gtga 1434 <210> 40 <211> 477 <212> PRT <213> Thermotoga neapolitana <400> 40 Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser  1 5 10 15 Pro Asp Leu Gly Thr Leu Val Pro Arg Gly Ser Met Ala Glu Phe Phe             20 25 30 Pro Glu Ile Pro Lys Val Gln Phe Glu Gly Lys Glu Ser Thr Asn Pro         35 40 45 Leu Ala Phe Lys Phe Tyr Asp Pro Glu Glu Ile Ile Asp Gly Lys Pro     50 55 60 Leu Lys Asp His Leu Lys Phe Ser Val Ala Phe Trp His Thr Phe Val 65 70 75 80 Asn Glu Gly Arg Asp Pro Phe Gly Asp Pro Thr Ala Asp Arg Pro Trp                 85 90 95 Asn Arg Tyr Thr Asp Pro Met Asp Lys Ala Phe Ala Arg Val Asp Ala             100 105 110 Leu Phe Glu Phe Cys Glu Lys Leu Asn Ile Glu Tyr Phe Cys Phe His         115 120 125 Asp Arg Asp Ile Ala Pro Glu Gly Lys Thr Leu Arg Glu Thr Asn Lys     130 135 140 Ile Leu Asp Lys Val Val Glu Arg Ile Lys Glu Arg Met Lys Asp Ser 145 150 155 160 Asn Val Lys Leu Leu Trp Gly Thr Ala Asn Leu Phe Ser His Pro Arg                 165 170 175 Tyr Met His Gly Ala Ala Thr Thr Cys Ser Ala Asp Val Phe Ala Tyr             180 185 190 Ala Ala Ala Gln Val Lys Lys Ala Leu Glu Ile Thr Lys Glu Leu Gly         195 200 205 Gly Glu Gly Tyr Val Phe Trp Gly Gly Arg Glu Gly Tyr Glu Thr Leu     210 215 220 Leu Asn Thr Asp Leu Gly Phe Glu Leu Glu Asn Leu Ala Arg Phe Leu 225 230 235 240 Arg Met Ala Val Asp Tyr Ala Lys Arg Ile Gly Phe Thr Gly Gln Phe                 245 250 255 Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys His Gln Tyr Asp Phe              260 265 270 Asp Val Ala Thr Ala Tyr Ala Phe Leu Lys Ser His Gly Leu Asp Glu         275 280 285 Tyr Phe Lys Phe Asn Ile Glu Ala Asn His Ala Thr Leu Ala Gly His     290 295 300 Thr Phe Gln His Glu Leu Arg Met Ala Arg Ile Leu Gly Lys Leu Gly 305 310 315 320 Ser Ile Asp Ala Asn Gln Gly Asp Leu Leu Leu Gly Trp Asp Thr Asp                 325 330 335 Gln Phe Pro Thr Asn Val Tyr Asp Thr Thr Leu Ala Met Tyr Glu Val             340 345 350 Ile Lys Ala Gly Gly Phe Thr Lys Gly Gly Leu Asn Phe Asp Ala Lys         355 360 365 Val Arg Arg Ala Ser Tyr Lys Val Glu Asp Leu Phe Ile Gly His Ile     370 375 380 Ala Gly Met Asp Thr Phe Ala Leu Gly Phe Lys Val Ala Tyr Lys Leu 385 390 395 400 Val Lys Asp Gly Val Leu Asp Lys Phe Ile Glu Glu Lys Tyr Arg Ser                 405 410 415 Phe Arg Glu Gly Ile Gly Arg Asp Ile Val Glu Gly Lys Val Asp Phe             420 425 430 Glu Lys Leu Glu Glu Tyr Ile Ile Asp Lys Glu Thr Ile Glu Leu Pro         435 440 445 Ser Gly Lys Gln Glu Tyr Leu Glu Ser Leu Ile Asn Ser Tyr Ile Val     450 455 460 Lys Thr Ile Leu Glu Leu Arg Ser Glu Lys Asp Glu Leu 465 470 475 <210> 41 <211> 1435 <212> DNA <213> Thermotoga maritima <400> 41 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcggatcc ccatggccga gttcttcccg 120 gagatcccga agatccagtt cgagggcaag gagtccacca acccgctcgc cttccgcttc 180 tacgacccga acgaggtgat cgacggcaag ccgctcaagg accacctcaa gttctccgtg 240 gccttctggc acaccttcgt gaacgagggc cgcgacccgt tcggcgaccc gaccgccgag 300 cgcccgtgga accgcttctc cgacccgatg gacaaggcct tcgcccgcgt ggacgccctc 360 ttcgagttct gcgagaagct caacatcgag tacttctgct tccacgaccg cgacatcccc 420 cggagggcaa gaccctccgc gagaccaaca agatcctcga caaggtggtg gagcgcatca 480 aggagcgcat gaaggactcc aacgtgaagc tcctctgggg caccgccaac ctcttctccc 540 acccgcgcta catgcacggc gccgccacca cctgctccgc cgacgtgttc gcctacgccg 600 ccgcccaggt gaagaaggcc ctggagatca ccaaggagct gggcggcgag ggctacgtgt 660 tctggggcgg ccgcgagggc tacgagaccc tcctcaacac cgacctcggc ctggagctgg 720 agaacctcgc ccgcttcctc cgcatggccg tggagtacgc caagaagatc ggcttcaccg 780 gccagttcct catcgagccg aagccgaagg agccgaccaa gcaccagtac gcttcgacgt 840 ggccaccgcc tacgccttcc tcaagaacca cggcctcgac gagtacttca agttcaacat 900 cgaggccaac cacgccaccc tcgccggcca caccttccag cacgagctgc gcatggcccg 960 catcctcggc aagctcggct ccatcgacgc caaccagggc gacctcctcc tcggctggga 1020 caccgaccag ttcccgacca acatctacga caccaccctc gccatgtacg aggtgatcaa 1080 ggccggcggc ttcaccaagg gcggcctcaa cttcgacgcc aaggtgcgcc gcgcctccta 1140 caaggtggag gacctcttca tcggccacat cgccggcatg gacaccttcg ccctcggctt 1200 caagatcgcc tacaagctcg ccaaggacgg cgtgttcgac aagttcatcg aggagaagta 1260 ccgctccttc aaggagggca tcggcaagga gatcgtggag ggcaagaccg acttcgagaa 1320 gctggaggag tacatcatcg acaaggagga catcgagctg ccgtccggca agcaggagta 1380  cctggagtcc ctcctcaact cctacatcgt gaagaccatc gccgagctgc gctga 1435 <210> 42 <211> 478 <212> PRT <213> Thermotoga maritima <400> 42 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro  1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg             20 25 30 Ile Pro Met Ala Glu Phe Phe Pro Glu Ile Pro Lys Ile Gln Phe Glu         35 40 45 Gly Lys Glu Ser Thr Asn Pro Leu Ala Phe Arg Phe Tyr Asp Pro Asn     50 55 60 Glu Val Ile Asp Gly Lys Pro Leu Lys Asp His Leu Lys Phe Ser Val 65 70 75 80 Ala Phe Trp His Thr Phe Val Asn Glu Gly Arg Asp Pro Phe Gly Asp                 85 90 95 Pro Thr Ala Glu Arg Pro Trp Asn Arg Phe Ser Asp Pro Met Asp Lys             100 105 110 Ala Phe Ala Arg Val Asp Ala Leu Phe Glu Phe Cys Glu Lys Leu Asn         115 120 125 Ile Glu Tyr Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Lys     130 135 140 Thr Leu Arg Glu Thr Asn Lys Ile Leu Asp Lys Val Val Glu Arg Ile 145 150 155 160 Lys Glu Arg Met Lys Asp Ser Asn Val Lys Leu Leu Trp Gly Thr Ala                 165 170 175 Asn Leu Phe Ser His Pro Arg Tyr Met His Gly Ala Ala Thr Thr Cys             180 185 190 Ser Ala Asp Val Phe Ala Tyr Ala Ala Ala Gln Val Lys Lys Ala Leu         195 200 205 Glu Ile Thr Lys Glu Leu Gly Gly Glu Gly Tyr Val Phe Trp Gly Gly     210 215 220 Arg Glu Gly Tyr Glu Thr Leu Leu Asn Thr Asp Leu Gly Leu Glu Leu 225 230 235 240 Glu Asn Leu Ala Arg Phe Leu Arg Met Ala Val Glu Tyr Ala Lys Lys                 245 250 255 Ile Gly Phe Thr Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro             260 265 270 Thr Lys His Gln Tyr Asp Phe Asp Val Ala Thr Ala Tyr Ala Phe Leu         275 280 285 Lys Asn His Gly Leu Asp Glu Tyr Phe Lys Phe Asn Ile Glu Ala Asn     290 295 300 His Ala Thr Leu Ala Gly His Thr Phe Gln His Glu Leu Arg Met Ala 305 310 315 320 Arg Ile Leu Gly Lys Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Leu                 325 330 335 Leu Leu Gly Trp Asp Thr Asp Gln Phe Pro Thr Asn Ile Tyr Asp Thr             340 345 350 Thr Leu Ala Met Tyr Glu Val Ile Lys Ala Gly Gly Phe Thr Lys Gly         355 360 365 Gly Leu Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Tyr Lys Val Glu     370 375 380 Asp Leu Phe Ile Gly His Ile Ala Gly Met Asp Thr Phe Ala Leu Gly 385 390 395 400  Phe Lys Ile Ala Tyr Lys Leu Ala Lys Asp Gly Val Phe Asp Lys Phe                 405 410 415 Ile Glu Glu Lys Tyr Arg Ser Phe Lys Glu Gly Ile Gly Lys Glu Ile             420 425 430 Val Glu Gly Lys Thr Asp Phe Glu Lys Leu Glu Glu Tyr Ile Ile Asp         435 440 445 Lys Glu Asp Ile Glu Leu Pro Ser Gly Lys Gln Glu Tyr Leu Glu Ser     450 455 460 Leu Leu Asn Ser Tyr Ile Val Lys Thr Ile Ala Glu Leu Arg 465 470 475 <210> 43 <211> 1436 <212> DNA <213> Thermotoga neapolitana <400> 43 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcggatcc ccatggccga gttcttcccg 120 gagatcccga aggtgcagtt cgagggcaag gagtccacca acccgctcgc cttcaagttc 180 tacgacccgg aggagatcat cgacggcaag ccgctcaagg accacctcaa gttctccgtg 240 gccttctggc acaccttcgt gaacgagggc cgcgacccgt tcggcgaccc gaccgccgac 300 cgcccgtgga accgctacac cgacccgatg gacaaggcct tcgcccgcgt ggacgccctc 360 ttcgagttct gcgagaagct caacatcgag tacttctgct tccacgaccg cgacatcccc 420 cggagggcaa gaccctccgc gagaccaaca agatcctcga caaggtggtg gagcgcatca 480 aggagcgcat gaaggactcc aacgtgaagc tcctctgggg caccgccaac ctcttctccc 540 acccgcgcta catgcacggc gccgccacca cctgctccgc cgacgtgttc gcctacgccg 600 ccgcccaggt gaagaaggcc ctggagatca ccaaggagct gggcggcgag ggctacgtgt 660 tctggggcgg ccgcgagggc tacgagaccc tcctcaacac cgacctcggc ttcgagctgg 720 agaacctcgc ccgcttcctc cgcatggccg tggactacgc caagcgcatc ggcttcaccg 780 gccagttcct catcgagccg aagccgaagg agccgaccaa gcaccagtac gacttcgacg 840 tggccaccgc ctacgccttc ctcaagtccc acggcctcga cgagtacttc aagttcaaca 900 tcgaggccaa ccacgccacc ctcgccggcc acaccttcca gcacgagctg cgcatggccc 960 gcatcctcgg caagctcggc tccatcgacg ccaaccaggg cgacctcctc ctcggctggg 1020 acaccgacca gttcccgacc aacgtgtacg acaccaccct cgccatgtac gaggtgatca 1080 aggccggcgg cttcaccaag ggcggcctca acttcgacgc caaggtgcgc cgcgcctcct 1140 acaaggtgga ggacctcttc atcggccaca tcgccggcat ggacaccttc gccctcggct 1200 tcaaggtggc ctacaagctc gtgaaggacg gcgtgctcga caagttcatc gaggagaagt 1260 accgctcctt ccgcgagggc atcggccgcg acatcgtgga gggcaaggtg gacttcgaga 1320 agctggagga gtacatcatc gacaaggaga ccatcgagct gccgtccggc aagcaggagt 1380 acctggagtc cctcatcaac tcctacatcg tgaagaccat cctggagctg cgctga 1436 <210> 44 <211> 478 <212> PRT <213> Thermotoga neapolitana <400> 44 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro  1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg             20 25 30 Ile Pro Met Ala Glu Phe Phe Pro Glu Ile Pro Lys Val Gln Phe Glu         35 40 45 Gly Lys Glu Ser Thr Asn Pro Leu Ala Phe Lys Phe Tyr Asp Pro Glu     50 55 60 Glu Ile Ile Asp Gly Lys Pro Leu Lys Asp His Leu Lys Phe Ser Val  65 70 75 80 Ala Phe Trp His Thr Phe Val Asn Glu Gly Arg Asp Pro Phe Gly Asp                 85 90 95 Pro Thr Ala Asp Arg Pro Trp Asn Arg Tyr Thr Asp Pro Met Asp Lys             100 105 110 Ala Phe Ala Arg Val Asp Ala Leu Phe Glu Phe Cys Glu Lys Leu Asn         115 120 125 Ile Glu Tyr Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Lys     130 135 140 Thr Leu Arg Glu Thr Asn Lys Ile Leu Asp Lys Val Val Glu Arg Ile 145 150 155 160 Lys Glu Arg Met Lys Asp Ser Asn Val Lys Leu Leu Trp Gly Thr Ala                 165 170 175 Asn Leu Phe Ser His Pro Arg Tyr Met His Gly Ala Ala Thr Thr Cys             180 185 190 Ser Ala Asp Val Phe Ala Tyr Ala Ala Ala Gln Val Lys Lys Ala Leu         195 200 205 Glu Ile Thr Lys Glu Leu Gly Gly Glu Gly Tyr Val Phe Trp Gly Gly     210 215 220 Arg Glu Gly Tyr Glu Thr Leu Leu Asn Thr Asp Leu Gly Phe Glu Leu 225 230 235 240 Glu Asn Leu Ala Arg Phe Leu Arg Met Ala Val Asp Tyr Ala Lys Arg                 245 250 255 Ile Gly Phe Thr Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro             260 265 270 Thr Lys His Gln Tyr Asp Phe Asp Val Ala Thr Ala Tyr Ala Phe Leu         275 280 285 Lys Ser His Gly Leu Asp Glu Tyr Phe Lys Phe Asn Ile Glu Ala Asn     290 295 300 His Ala Thr Leu Ala Gly His Thr Phe Gln His Glu Leu Arg Met Ala 305 310 315 320 Arg Ile Leu Gly Lys Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Leu                 325 330 335 Leu Leu Gly Trp Asp Thr Asp Gln Phe Pro Thr Asn Val Tyr Asp Thr             340 345 350 Thr Leu Ala Met Tyr Glu Val Ile Lys Ala Gly Gly Phe Thr Lys Gly         355 360 365 Gly Leu Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Tyr Lys Val Glu     370 375 380 Asp Leu Phe Ile Gly His Ile Ala Gly Met Asp Thr Phe Ala Leu Gly 385 390 395 400 Phe Lys Val Ala Tyr Lys Leu Val Lys Asp Gly Val Leu Asp Lys Phe                 405 410 415 Ile Glu Glu Lys Tyr Arg Ser Phe Arg Glu Gly Ile Gly Arg Asp Ile             420 425 430 Val Glu Gly Lys Val Asp Phe Glu Lys Leu Glu Glu Tyr Ile Ile Asp         435 440 445 Lys Glu Thr Ile Glu Leu Pro Ser Gly Lys Gln Glu Tyr Leu Glu Ser     450 455 460 Leu Ile Asn Ser Tyr Ile Val Lys Thr Ile Leu Glu Leu Arg 465 470 475 <210> 45 <211> 1095 <212> PRT <213> Aspergillus shirousami   <400> 45 Ala Thr Pro Ala Asp Trp Arg Ser Gln Ser Ile Tyr Phe Leu Leu Thr  1 5 10 15 Asp Arg Phe Ala Arg Thr Asp Gly Ser Thr Thr Ala Thr Cys Asn Thr             20 25 30 Ala Asp Gln Lys Tyr Cys Gly Gly Thr Trp Gln Gly Ile Ile Asp Lys         35 40 45 Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Thr Pro     50 55 60 Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly Asp Ala Tyr His 65 70 75 80 Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu Asn Tyr Gly Thr                 85 90 95 Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His Glu Arg Gly Met             100 105 110 Tyr Leu Met Val Asp Val Val Ala Asn His Met Gly Tyr Asp Gly Ala         115 120 125 Gly Ser Ser Val Asp Tyr Ser Val Phe Lys Pro Phe Ser Ser Gln Asp     130 135 140 Tyr Phe His Pro Phe Cys Phe Ile Gln Asn Tyr Glu Asp Gln Thr Gln 145 150 155 160 Val Glu Asp Cys Trp Leu Gly Asp Asn Thr Val Ser Leu Pro Asp Leu                 165 170 175 Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr Asp Trp Val Gly             180 185 190 Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg Ile Asp Thr Val         195 200 205 Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn Lys Ala Ala Gly     210 215 220 Val Tyr Cys Ile Gly Glu Val Leu Asp Val Asp Pro Ala Tyr Thr Cys 225 230 235 240 Pro Tyr Gln Asn Val Met Asp Gly Val Leu Asn Tyr Pro Ile Tyr Tyr                 245 250 255 Pro Leu Leu Asn Ala Phe Lys Ser Thr Ser Gly Ser Met Asp Asp Leu             260 265 270 Tyr Asn Met Ile Asn Thr Val Lys Ser Asp Cys Pro Asp Ser Thr Leu         275 280 285 Leu Gly Thr Phe Val Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr     290 295 300 Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala Phe Ile Ile Leu 305 310 315 320 Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu Gln His Tyr Ala                 325 330 335 Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr             340 345 350 Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala Ser Ala Asn Ala Ile         355 360 365 Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly Phe Val Thr Tyr Lys Asn     370 375 380 Trp Pro Ile Tyr Lys Asp Asp Thr Thr Ile Ala Met Arg Lys Gly Thr 385 390 395 400 Asp Gly Ser Gln Ile Val Thr Ile Leu Ser Asn Lys Gly Ala Ser Gly                 405 410 415 Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly Tyr Thr Ala Gly Gln             420 425 430 Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val Val Val Gly Ser Asp         435 440 445 Gly Asn Val Pro Val Pro Met Ala Gly Gly Leu Pro Arg Val Leu Tyr      450 455 460 Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser Ser Ser Lys Pro 465 470 475 480 Ala Thr Leu Asp Ser Trp Leu Ser Asn Glu Ala Thr Val Ala Arg Thr                 485 490 495 Ala Ile Leu Asn Asn Ile Gly Ala Asp Gly Ala Trp Val Ser Gly Ala             500 505 510 Asp Ser Gly Ile Val Val Ala Ser Pro Ser Thr Asp Asn Pro Asp Tyr         515 520 525 Phe Tyr Thr Trp Thr Arg Asp Ser Gly Ile Val Leu Lys Thr Leu Val     530 535 540 Asp Leu Phe Arg Asn Gly Asp Thr Asp Leu Leu Ser Thr Ile Glu His 545 550 555 560 Tyr Ile Ser Ser Gln Ala Ile Ile Gln Gly Val Ser Asn Pro Ser Gly                 565 570 575 Asp Leu Ser Ser Gly Gly Leu Gly Glu Pro Lys Phe Asn Val Asp Glu             580 585 590 Thr Ala Tyr Ala Gly Ser Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala         595 600 605 Leu Arg Ala Thr Ala Met Ile Gly Phe Gly Gln Trp Leu Leu Asp Asn     610 615 620 Gly Tyr Thr Ser Ala Ala Thr Glu Ile Val Trp Pro Leu Val Arg Asn 625 630 635 640 Asp Leu Ser Tyr Val Ala Gln Tyr Trp Asn Gln Thr Gly Tyr Asp Leu                 645 650 655 Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr Ile Ala Val Gln His             660 665 670 Arg Ala Leu Val Glu Gly Ser Ala Phe Ala Thr Ala Val Gly Ser Ser         675 680 685 Cys Ser Trp Cys Asp Ser Gln Ala Pro Gln Ile Leu Cys Tyr Leu Gln     690 695 700 Ser Phe Trp Thr Gly Ser Tyr Ile Leu Ala Asn Phe Asp Ser Ser Arg 705 710 715 720 Ser Gly Lys Asp Thr Asn Thr Leu Leu Gly Ser Ile His Thr Phe Asp                 725 730 735 Pro Glu Ala Gly Cys Asp Asp Ser Thr Phe Gln Pro Cys Ser Pro Arg             740 745 750 Ala Leu Ala Asn His Lys Glu Val Val Asp Ser Phe Arg Ser Ile Tyr         755 760 765 Thr Leu Asn Asp Gly Leu Ser Asp Ser Glu Ala Val Ala Val Gly Arg     770 775 780 Tyr Pro Glu Asp Ser Tyr Tyr Asn Gly Asn Pro Trp Phe Leu Cys Thr 785 790 795 800 Leu Ala Ala Ala Glu Gln Leu Tyr Asp Ala Leu Tyr Gln Trp Asp Lys                 805 810 815 Gln Gly Ser Leu Glu Ile Thr Asp Val Ser Leu Asp Phe Phe Lys Ala             820 825 830 Leu Tyr Ser Gly Ala Ala Thr Gly Thr Tyr Ser Ser Ser Ser Ser Thr         835 840 845 Tyr Ser Ser Ile Val Ser Ala Val Lys Thr Phe Ala Asp Gly Phe Val     850 855 860 Ser Ile Val Glu Thr His Ala Ala Ser Asn Gly Ser Leu Ser Glu Gln 865 870 875 880 Phe Asp Lys Ser Asp Gly Asp Glu Leu Ser Ala Arg Asp Leu Thr Trp                 885 890 895 Ser Tyr Ala Ala Leu Leu Thr Ala Asn Asn Arg Arg Asn Ser Val Val             900 905 910 Pro Pro Ser Trp Gly Glu Thr Ser Ala Ser Ser Val Pro Gly Thr Cys          915 920 925 Ala Ala Thr Ser Ala Ser Gly Thr Tyr Ser Ser Val Thr Val Thr Ser     930 935 940 Trp Pro Ser Ile Val Ala Thr Gly Gly Thr Thr Thr Thr Ala Thr Thr 945 950 955 960 Thr Gly Ser Gly Gly Val Thr Ser Thr Ser Lys Thr Thr Thr Thr Ala                 965 970 975 Ser Lys Thr Ser Thr Thr Thr Ser Ser Thr Ser Cys Thr Thr Pro Thr             980 985 990 Ala Val Ala Val Thr Phe Asp Leu Thr Ala Thr Thr Thr Tyr Gly Glu         995 1000 1005 Asn Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu Gly Asp Trp Glu Thr     1010 1015 1020 Ser Asp Gly Ile Ala Leu Ser Ala Asp Lys Tyr Thr Ser Ser Asn Pro 1025 1030 1035 1040 Pro Trp Tyr Val Thr Val Thr Leu Pro Ala Gly Glu Ser Phe Glu Tyr                 1045 1050 1055 Lys Phe Ile Arg Val Glu Ser Asp Asp Ser Val Glu Trp Glu Ser Asp             1060 1065 1070 Pro Asn Arg Glu Tyr Thr Val Pro Gln Ala Cys Gly Glu Ser Thr Ala         1075 1080 1085 Thr Val Thr Asp Thr Trp Arg     1090 1095 <210> 46 <211> 3285 <212> DNA <213> Aspergillus shirousami <400> 46 gccaccccgg ccgactggcg ctcccagtcc atctacttcc tcctcaccga ccgcttcgcc 60 cgcaccgacg gctccaccac cgccacctgc aacaccgccg accagaagta ctgcggcggc 120 acctggcagg gcatcatcga caagctcgac tacatccagg gcatgggctt caccgccatc 180 tggatcaccc cggtgaccgc ccagctcccg cagaccaccg cctacggcga cgcctaccac 240 ggctactggc agcaggacat ctactccctc aacgagaact acggcaccgc cgacgacctc 300 aaggccctct cctccgccct ccacgagcgc ggcatgtacc tcatggtgga cgtggtggcc 360 aaccacatgg gctacgacgg cgccggctcc tccgtggact actccgtgtt caagccgttc 420 tcctcccagg actacttcca cccgttctgc ttcatccaga actacgagga ccagacccag 480 gtggaggact gctggctcgg cgacaacacc gtgtccctcc cggacctcga caccaccaag 540 gacgtggtga agaacgagtg gtacgactgg gtgggctccc tcgtgtccaa ctactccatc 600 gacggcctcc gcatcgacac cgtgaagcac gtgcagaagg acttctggcc gggctacaac 660 aaggccgccg gcgtgtactg catcggcgag gtgctcgacg tggacccggc ctacacctgc 720 ccgtaccaga acgtgatgga cggcgtgctc aactacccga tctactaccc gctcctcaac 780 gccttcaagt ccacctccgg ctcgatggac gacctctaca acatgatcaa caccgtgaag 840 tccgactgcc cggactccac cctcctcggc accttcgtgg agaaccacga caacccgcgc 900 ttcgcctcct acaccaacga catcgccctc gccaagaacg tggccgcctt catcatcctc 960 aacgacggca tcccgatcat ctacgccggc caggagcagc actacgccgg cggcaacgac 1020 ccggccaacc gcgaggccac ctggctctcc ggctacccga ccgactccga gctgtacaag 1080 ctcatcgcct ccgccaacgc catccgcaac tacgccatct ccaaggacac cggcttcgtg 1140 acctacaaga actggccgat ctacaaggac gacaccacca tcgccatgcg caagggcacc 1200 gacggctccc agatcgtgac catcctctcc aacaagggcg cctccggcga ctcctacacc 1260 ctctccctct ccggcgccgg ctacaccgcc ggccagcagc tcaccgaggt gatcggctgc 1320 accaccgtga ccgtgggctc cgacggcaac gtgccggtgc cgatggccgg cggcctcccg 1380 cgcgtgctct acccgaccga gaagctcgcc ggctccaaga tatgctcctc ctccaagccg 1440 gccaccctcg actcctggct ctccaacgag gccaccgtgg cccgcaccgc catcctcaac 1500 aacatcggcg ccgacggcgc ctgggtgtcc ggcgccgact ccggcatcgt ggtggcctcc 1560 ccgtccaccg acaacccgga ctacttctac acctggaccc gcgactccgg catcgtgctc 1620  aagaccctcg tggacctctt ccgcaacggc gacaccgacc tcctctccac catcgagcac 1680 tacatctcct cccaggccat catccagggc gtgtccaacc cgtccggcga cctctcctcc 1740 ggcggcctcg gcgagccgaa gttcaacgtg gacgagaccg cctacgccgg ctcctggggc 1800 cgcccgcagc gcgacggccc ggccctccgc gccaccgcca tgatcggctt cggccagtgg 1860 ctcctcgaca acggctacac ctccgccgcc accgagatcg tgtggccgct cgtgcgcaac 1920 gacctctcct acgtggccca gtactggaac cagaccggct acgacctctg ggaggaggtg 1980 aacggctcct ccttcttcac catcgccgtg cagcaccgcg ccctcgtgga gggctccgcc 2040 ttcgccaccg ccgtgggctc ctcctgctcc tggtgcgact cccaggcccc gcagatcctc 2100 tgctacctcc agtccttctg gaccggctcc tacatcctcg ccaacttcga ctcctcccgc 2160 tccggcaagg acaccaacac cctcctcggc tccatccaca ccttcgaccc ggaggccggc 2220 tgcgacgact ccaccttcca gccgtgctcc ccgcgcgccc tcgccaacca caaggaggtg 2280 gtggactcct tccgctccat ctacaccctc aacgacggcc tctccgactc cgaggccgtg 2340 gccgtgggcc gctacccgga ggactcctac tacaacggca acccgtggtt cctctgcacc 2400 ctcgccgccg ccgagcagct ctacgacgcc ctctaccagt gggacaagca gggctccctg 2460 gagatcaccg acgtgtccct cgacttcttc aaggccctct actccggcgc cgccaccggc 2520 acctactcct cctcctcctc cacctactcc tccatcgtgt ccgccgtgaa gaccttcgcc 2580 gacggcttcg tgtccatcgt ggagacccac gccgcctcca acggctccct ctccgagcag 2640 ttcgacaagt ccgacggcga cgagctgtcc gcccgcgacc tcacctggtc ctacgccgcc 2700 ctcctcaccg ccaacaaccg ccgcaactcc gtggtgccgc cgtcctgggg cgagacctcc 2760 gcctcctccg tgccgggcac ctgcgccgcc acctccgcct ccggcaccta ctcctccgtg 2820 accgtgacct cctggccgtc catcgtggcc accggcggca ccaccaccac cgccaccacc 2880 accggctccg gcggcgtgac ctccacctcc aagaccacca ccaccgcctc caagacctcc 2940 accaccacct cctccacctc ctgcaccacc ccgaccgccg tggccgtgac cttcgacctc 3000 accgccacca ccacctacgg cgagaacatc tacctcgtgg gctccatctc ccagctcggc 3060 gactgggaga cctccgacgg catcgccctc tccgccgaca agtacacctc ctccaacccg 3120 ccgtggtacg tgaccgtgac cctcccggcc ggcgagtcct tcgagtacaa gttcatccgc 3180 gtggagtccg acgactccgt ggagtgggag tccgacccga accgcgagta caccgtgccg 3240 caggcctgcg gcgagtccac cgccaccgtg accgacacct ggcgc 3285 <210> 47 <211> 679 <212> PRT <213> Thermoanaerobacterium thermosaccharolyticum <400> 47 Val Leu Ser Gly Cys Ser Asn Asn Val Ser Ser Ile Lys Ile Asp Arg  1 5 10 15 Phe Asn Asn Ile Ser Ala Val Asn Gly Pro Gly Glu Glu Asp Thr Trp             20 25 30 Ala Ser Ala Gln Lys Gln Gly Val Gly Thr Ala Asn Asn Tyr Val Ser         35 40 45 Arg Val Trp Phe Thr Leu Ala Asn Gly Ala Ile Ser Glu Val Tyr Tyr     50 55 60 Pro Thr Ile Asp Thr Ala Asp Val Lys Glu Ile Lys Phe Ile Val Thr 65 70 75 80 Asp Gly Lys Ser Phe Val Ser Asp Glu Thr Lys Asp Ala Ile Ser Lys                 85 90 95 Val Glu Lys Phe Thr Asp Lys Ser Leu Gly Tyr Lys Leu Val Asn Thr             100 105 110 Asp Lys Lys Gly Arg Tyr Arg Ile Thr Lys Glu Ile Phe Thr Asp Val         115 120 125 Lys Arg Asn Ser Leu Ile Met Lys Ala Lys Phe Glu Ala Leu Glu Gly     130 135 140 Ser Ile His Asp Tyr Lys Leu Tyr Leu Ala Tyr Asp Pro His Ile Lys 145 150 155 160 Asn Gln Gly Ser Tyr Asn Glu Gly Tyr Val Ile Lys Ala Asn Asn Asn                 165 170 175 Glu Met Leu Met Ala Lys Arg Asp Asn Val Tyr Thr Ala Leu Ser Ser              180 185 190 Asn Ile Gly Trp Lys Gly Tyr Ser Ile Gly Tyr Tyr Lys Val Asn Asp         195 200 205 Ile Met Thr Asp Leu Asp Glu Asn Lys Gln Met Thr Lys His Tyr Asp     210 215 220 Ser Ala Arg Gly Asn Ile Ile Glu Gly Ala Glu Ile Asp Leu Thr Lys 225 230 235 240 Asn Ser Glu Phe Glu Ile Val Leu Ser Phe Gly Gly Ser Asp Ser Glu                 245 250 255 Ala Ala Lys Thr Ala Leu Glu Thr Leu Gly Glu Asp Tyr Asn Asn Leu             260 265 270 Lys Asn Asn Tyr Ile Asp Glu Trp Thr Lys Tyr Cys Asn Thr Leu Asn         275 280 285 Asn Phe Asn Gly Lys Ala Asn Ser Leu Tyr Tyr Asn Ser Met Met Ile     290 295 300 Leu Lys Ala Ser Glu Asp Lys Thr Asn Lys Gly Ala Tyr Ile Ala Ser 305 310 315 320 Leu Ser Ile Pro Trp Gly Asp Gly Gln Arg Asp Asp Asn Thr Gly Gly                 325 330 335 Tyr His Leu Val Trp Ser Arg Asp Leu Tyr His Val Ala Asn Ala Phe             340 345 350 Ile Ala Ala Gly Asp Val Asp Ser Ala Asn Arg Ser Leu Asp Tyr Leu         355 360 365 Ala Lys Val Val Lys Asp Asn Gly Met Ile Pro Gln Asn Thr Trp Ile     370 375 380 Ser Gly Lys Pro Tyr Trp Thr Ser Ile Gln Leu Asp Glu Gln Ala Asp 385 390 395 400 Pro Ile Ile Leu Ser Tyr Arg Leu Lys Arg Tyr Asp Leu Tyr Asp Ser                 405 410 415 Leu Val Lys Pro Leu Ala Asp Phe Ile Ile Lys Ile Gly Pro Lys Thr             420 425 430 Gly Gln Glu Arg Trp Glu Glu Ile Gly Gly Tyr Ser Pro Ala Thr Met         435 440 445 Ala Ala Glu Val Ala Gly Leu Thr Cys Ala Ala Tyr Ile Ala Glu Gln     450 455 460 Asn Lys Asp Tyr Glu Ser Ala Gln Lys Tyr Gln Glu Lys Ala Asp Asn 465 470 475 480 Trp Gln Lys Leu Ile Asp Asn Leu Thr Tyr Thr Glu Asn Gly Pro Leu                 485 490 495 Gly Asn Gly Gln Tyr Tyr Ile Arg Ile Ala Gly Leu Ser Asp Pro Asn             500 505 510 Ala Asp Phe Met Ile Asn Ile Ala Asn Gly Gly Gly Val Tyr Asp Gln         515 520 525 Lys Glu Ile Val Asp Pro Ser Phe Leu Glu Leu Val Arg Leu Gly Val     530 535 540 Lys Ser Ala Asp Asp Pro Lys Ile Leu Asn Thr Leu Lys Val Val Asp 545 550 555 560 Ser Thr Ile Lys Val Asp Thr Pro Lys Gly Pro Ser Trp Tyr Arg Tyr                 565 570 575 Asn His Asp Gly Tyr Gly Glu Pro Ser Lys Thr Glu Leu Tyr His Gly             580 585 590 Ala Gly Lys Gly Arg Leu Trp Pro Leu Leu Thr Gly Glu Arg Gly Met         595 600 605 Tyr Glu Ile Ala Ala Gly Lys Asp Ala Thr Pro Tyr Val Lys Ala Met     610 615 620 Glu Lys Phe Ala Asn Glu Gly Gly Ile Ile Ser Glu Gln Val Trp Glu 625 630 635 640 Asp Thr Gly Leu Pro Thr Asp Ser Ala Ser Pro Leu Asn Trp Ala His                  645 650 655 Ala Glu Tyr Val Ile Leu Phe Ala Ser Asn Ile Glu His Lys Val Leu             660 665 670 Asp Met Pro Asp Ile Val Tyr         675 <210> 48 <211> 2037 <212> DNA <213> Thermoanaerobacterium thermosaccharolyticum <220> <223> synthetic <400> 48 gtgctctccg gctgctccaa caacgtgtcc tccatcaaga tcgaccgctt caacaacatc 60 tccgccgtga acggcccggg cgaggaggac acctgggcct ccgcccagaa gcagggcgtg 120 ggcaccgcca acaactacgt gtcccgcgtg tggttcaccc tcgccaacgg cgccatctcc 180 gaggtgtact acccgaccat cgacaccgcc gacgtgaagg agatcaagtt catcgtgacc 240 gacggcaagt ccttcgtgtc cgacgagacc aaggacgcca tctccaaggt ggagaagttc 300 accgacaagt ccctcggcta caagctcgtg aacaccgaca agaagggccg ctaccgcatc 360 accaaggaaa tcttcaccga cgtgaagcgc aactccctca tcatgaaggc caagttcgag 420 gccctcgagg gctccatcca cgactacaag ctctacctcg cctacgaccc gcacatcaag 480 aaccagggct cctacaacga gggctacgtg atcaaggcca acaacaacga gatgctcatg 540 gccaagcgcg acaacgtgta caccgccctc tcctccaaca tcggctggaa gggctactcc 600 atcggctact acaaggtgaa cgacatcatg accgacctcg acgagaacaa gcagatgacc 660 aagcactacg actccgcccg cggcaacatc atcgagggcg ccgagatcga cctcaccaag 720 aactccgagt tcgagatcgt gctctccttc ggcggctccg actccgaggc cgccaagacc 780 gccctcgaga ccctcggcga ggactacaac aacctcaaga acaactacat cgacgagtgg 840 accaagtact gcaacaccct caacaacttc aacggcaagg ccaactccct ctactacaac 900 tccatgatga tcctcaaggc ctccgaggac aagaccaaca agggcgccta catcgcctcc 960 ctctccatcc cgtggggcga cggccagcgc gacgacaaca ccggcggcta ccacctcgtg 1020 tggtcccgcg acctctacca cgtggccaac gccttcatcg ccgccggcga cgtggactcc 1080 gccaaccgct ccctcgacta cctcgccaag gtggtgaagg acaacggcat gatcccgcag 1140 aacacctgga tctccggcaa gccgtactgg acctccatcc agctcgacga gcaggccgac 1200 ccgatcatcc tctcctaccg cctcaagcgc tacgacctct acgactccct cgtgaagccg 1260 ctcgccgact tcatcatcaa gatcggcccg aagaccggcc aggagcgctg ggaggagatc 1320 ggcggctact ccccggccac gatggccgcc gaggtggccg gcctcacctg cgccgcctac 1380 atcgccgagc agaacaagga ctacgagtcc gcccagaagt accaggagaa ggccgacaac 1440 tggcagaagc tcatcgacaa cctcacctac accgagaacg gcccgctcgg caacggccag 1500 tactacatcc gcatcgccgg cctctccgac ccgaacgccg acttcatgat caacatcgcc 1560 aacggcggcg gcgtgtacga ccagaaggag atcgtggacc cgtccttcct cgagctggtg 1620 cgcctcggcg tgaagtccgc cgacgacccg aagatcctca acaccctcaa ggtggtggac 1680 tccaccatca aggtggacac cccgaagggc ccgtcctggt atcgctacaa ccacgacggc 1740 tacggcgagc cgtccaagac cgagctgtac cacggcgccg gcaagggccg cctctggccg 1800 ctcctcaccg gcgagcgcgg catgtacgag atcgccgccg gcaaggacgc caccccgtac 1860 gtgaaggcga tggagaagtt cgccaacgag ggcggcatca tctccgagca ggtgtgggag 1920 gacaccggcc tcccgaccga ctccgcctcc ccgctcaact gggcccacgc cgagtacgtg 1980 atcctcttcg cctccaacat cgagcacaag gtgctcgaca tgccggacat cgtgtac 2037 <210> 49 <211> 579 <212> PRT <213> Rhizopus oryzae <400> 49 Ala Ser Ile Pro Ser Ser Ala Ser Val Gln Leu Asp Ser Tyr Asn Tyr   1 5 10 15 Asp Gly Ser Thr Phe Ser Gly Lys Ile Tyr Val Lys Asn Ile Ala Tyr             20 25 30 Ser Lys Lys Val Thr Val Ile Tyr Ala Asp Gly Ser Asp Asn Trp Asn         35 40 45 Asn Asn Gly Asn Thr Ile Ala Ala Ser Tyr Ser Ala Pro Ile Ser Gly     50 55 60 Ser Asn Tyr Glu Tyr Trp Thr Phe Ser Ala Ser Ile Asn Gly Ile Lys 65 70 75 80 Glu Phe Tyr Ile Lys Tyr Glu Val Ser Gly Lys Thr Tyr Tyr Asp Asn                 85 90 95 Asn Asn Ser Ala Asn Tyr Gln Val Ser Thr Ser Lys Pro Thr Thr Thr             100 105 110 Thr Ala Thr Ala Thr Thr Thr Thr Ala Pro Ser Thr Ser Thr Thr Thr         115 120 125 Pro Pro Ser Arg Ser Glu Pro Ala Thr Phe Pro Thr Gly Asn Ser Thr     130 135 140 Ile Ser Ser Trp Ile Lys Lys Gln Glu Gly Ile Ser Arg Phe Ala Met 145 150 155 160 Leu Arg Asn Ile Asn Pro Pro Gly Ser Ala Thr Gly Phe Ile Ala Ala                 165 170 175 Ser Leu Ser Thr Ala Gly Pro Asp Tyr Tyr Tyr Ala Trp Thr Arg Asp             180 185 190 Ala Ala Leu Thr Ser Asn Val Ile Val Tyr Glu Tyr Asn Thr Thr Leu         195 200 205 Ser Gly Asn Lys Thr Ile Leu Asn Val Leu Lys Asp Tyr Val Thr Phe     210 215 220 Ser Val Lys Thr Gln Ser Thr Ser Thr Val Cys Asn Cys Leu Gly Glu 225 230 235 240 Pro Lys Phe Asn Pro Asp Ala Ser Gly Tyr Thr Gly Ala Trp Gly Arg                 245 250 255 Pro Gln Asn Asp Gly Pro Ala Glu Arg Ala Thr Thr Phe Ile Leu Phe             260 265 270 Ala Asp Ser Tyr Leu Thr Gln Thr Lys Asp Ala Ser Tyr Val Thr Gly         275 280 285 Thr Leu Lys Pro Ala Ile Phe Lys Asp Leu Asp Tyr Val Val Asn Val     290 295 300 Trp Ser Asn Gly Cys Phe Asp Leu Trp Glu Glu Val Asn Gly Val His 305 310 315 320 Phe Tyr Thr Leu Met Val Met Arg Lys Gly Leu Leu Leu Gly Ala Asp                 325 330 335 Phe Ala Lys Arg Asn Gly Asp Ser Thr Arg Ala Ser Thr Tyr Ser Ser             340 345 350 Thr Ala Ser Thr Ile Ala Asn Lys Ile Ser Ser Phe Trp Val Ser Ser         355 360 365 Asn Asn Trp Ile Gln Val Ser Gln Ser Val Thr Gly Gly Val Ser Lys     370 375 380 Lys Gly Leu Asp Val Ser Thr Leu Leu Ala Ala Asn Leu Gly Ser Val 385 390 395 400 Asp Asp Gly Phe Phe Thr Pro Gly Ser Glu Lys Ile Leu Ala Thr Ala                 405 410 415 Val Ala Val Glu Asp Ser Phe Ala Ser Leu Tyr Pro Ile Asn Lys Asn             420 425 430 Leu Pro Ser Tyr Leu Gly Asn Ser Ile Gly Arg Tyr Pro Glu Asp Thr         435 440 445 Tyr Asn Gly Asn Gly Asn Ser Gln Gly Asn Ser Trp Phe Leu Ala Val     450 455 460 Thr Gly Tyr Ala Glu Leu Tyr Tyr Arg Ala Ile Lys Glu Trp Ile Gly  465 470 475 480 Asn Gly Gly Val Thr Val Ser Ser Ile Ser Leu Pro Phe Phe Lys Lys                 485 490 495 Phe Asp Ser Ser Ala Thr Ser Gly Lys Lys Tyr Thr Val Gly Thr Ser             500 505 510 Asp Phe Asn Asn Leu Ala Gln Asn Ile Ala Leu Ala Ala Asp Arg Phe         515 520 525 Leu Ser Thr Val Gln Leu His Ala His Asn Asn Gly Ser Leu Ala Glu     530 535 540 Glu Phe Asp Arg Thr Thr Gly Leu Ser Thr Gly Ala Arg Asp Leu Thr 545 550 555 560 Trp Ser His Ala Ser Leu Ile Thr Ala Ser Tyr Ala Lys Ala Gly Ala                 565 570 575 Pro Ala Ala              <210> 50 <211> 1737 <212> DNA <213> Rhizopus oryzae <400> 50 gcctccatcc cgtcctccgc ctccgtgcag ctcgactcct acaactacga cggctccacc 60 ttctccggca aaatctacgt gaagaacatc gcctactcca agaaggtgac cgtgatctac 120 gccgacggct ccgacaactg gaacaacaac ggcaacacca tcgccgcctc ctactccgcc 180 ccgatctccg gctccaacta cgagtactgg accttctccg cctccatcaa cggcatcaag 240 gagttctaca tcaagtacga ggtgtccggc aagacctact acgacaacaa caactccgcc 300 aactaccagg tgtccacctc caagccgacc accaccaccg ccaccgccac caccaccacc 360 gccccgtcca cctccaccac caccccgccg tcccgctccg agccggccac cttcccgacc 420 ggcaactcca ccatctcctc ctggatcaag aagcaggagg gcatctcccg cttcgccatg 480 ctccgcaaca tcaacccgcc gggctccgcc accggcttca tcgccgcctc cctctccacc 540 gccggcccgg actactacta cgcctggacc cgcgacgccg ccctcacctc caacgtgatc 600 gtgtacgagt acaacaccac cctctccggc aacaagacca tcctcaacgt gctcaaggac 660 tacgtgacct tctccgtgaa gacccagtcc acctccaccg tgtgcaactg cctcggcgag 720 ccgaagttca acccggacgc ctccggctac accggcgcct ggggccgccc gcagaacgac 780 ggcccggccg agcgcgccac caccttcatc ctcttcgccg actcctacct cacccagacc 840 aaggacgcct cctacgtgac cggcaccctc aagccggcca tcttcaagga cctcgactac 900 gtggtgaacg tgtggtccaa cggctgcttc gacctctggg aggaggtgaa cggcgtgcac 960 ttctacaccc tcatggtgat gcgcaagggc ctcctcctcg gcgccgactt cgccaagcgc 1020 aacggcgact ccacccgcgc ctccacctac tcctccaccg cctccaccat cgccaacaaa 1080 atctcctcct tctgggtgtc ctccaacaac tggatacagg tgtcccagtc cgtgaccggc 1140 ggcgtgtcca agaagggcct cgacgtgtcc accctcctcg ccgccaacct cggctccgtg 1200 gacgacggct tcttcacccc gggctccgag aagatcctcg ccaccgccgt ggccgtggag 1260 gactccttcg cctccctcta cccgatcaac aagaacctcc cgtcctacct cggcaactcc 1320 atcggccgct acccggagga cacctacaac ggcaacggca actcccaggg caactcctgg 1380 ttcctcgccg tgaccggcta cgccgagctg tactaccgcg ccatcaagga gtggatcggc 1440 aacggcggcg tgaccgtgtc ctccatctcc ctcccgttct tcaagaagtt cgactcctcc 1500 gccacctccg gcaagaagta caccgtgggc acctccgact tcaacaacct cgcccagaac 1560 atcgccctcg ccgccgaccg cttcctctcc accgtgcagc tccacgccca caacaacggc 1620 tccctcgccg aggagttcga ccgcaccacc ggcctctcca ccggcgcccg cgacctcacc 1680 tggtcccacg cctccctcat caccgcctcc tacgccaagg ccggcgcccc ggccgcc 1737 <210> 51 <211> 439 <212> PRT <213> Artificial Sequence   <220> <223> synthetic <400> 51 Met Ala Lys His Leu Ala Ala Met Cys Trp Cys Ser Leu Leu Val Leu  1 5 10 15 Val Leu Leu Cys Leu Gly Ser Gln Leu Ala Gln Ser Gln Val Leu Phe             20 25 30 Gln Gly Phe Asn Trp Glu Ser Trp Lys Lys Gln Gly Gly Trp Tyr Asn         35 40 45 Tyr Leu Leu Gly Arg Val Asp Asp Ile Ala Ala Thr Gly Ala Thr His     50 55 60 Val Trp Leu Pro Gln Pro Ser His Ser Val Ala Pro Gln Gly Tyr Met 65 70 75 80 Pro Gly Arg Leu Tyr Asp Leu Asp Ala Ser Lys Tyr Gly Thr His Ala                 85 90 95 Glu Leu Lys Ser Leu Thr Ala Ala Phe His Ala Lys Gly Val Gln Cys             100 105 110 Val Ala Asp Val Val Ile Asn His Arg Cys Ala Asp Tyr Lys Asp Gly         115 120 125 Arg Gly Ile Tyr Cys Val Phe Glu Gly Gly Thr Pro Asp Ser Arg Leu     130 135 140 Asp Trp Gly Pro Asp Met Ile Cys Ser Asp Asp Thr Gln Tyr Ser Asn 145 150 155 160 Gly Arg Gly His Arg Asp Thr Gly Ala Asp Phe Ala Ala Ala Pro Asp                 165 170 175 Ile Asp His Leu Asn Pro Arg Val Gln Gln Glu Leu Ser Asp Trp Leu             180 185 190 Asn Trp Leu Lys Ser Asp Leu Gly Phe Asp Gly Trp Arg Leu Asp Phe         195 200 205 Ala Lys Gly Tyr Ser Ala Ala Val Ala Lys Val Tyr Val Asp Ser Thr     210 215 220 Ala Pro Thr Phe Val Val Ala Glu Ile Trp Ser Ser Leu His Tyr Asp 225 230 235 240 Gly Asn Gly Glu Pro Ser Ser Asn Gln Asp Ala Asp Arg Gln Glu Leu                 245 250 255 Val Asn Trp Ala Gln Ala Val Gly Gly Pro Ala Ala Ala Phe Asp Phe             260 265 270 Thr Thr Lys Gly Val Leu Gln Ala Ala Val Gln Gly Glu Leu Trp Arg         275 280 285 Met Lys Asp Gly Asn Gly Lys Ala Pro Gly Met Ile Gly Trp Leu Pro     290 295 300 Glu Lys Ala Val Thr Phe Val Asp Asn His Asp Thr Gly Ser Thr Gln 305 310 315 320 Asn Ser Trp Pro Phe Pro Ser Asp Lys Val Met Gln Gly Tyr Ala Tyr                 325 330 335 Ile Leu Thr His Pro Gly Thr Pro Cys Ile Phe Tyr Asp His Val Phe             340 345 350 Asp Trp Asn Leu Lys Gln Glu Ile Ser Ala Leu Ser Ala Val Arg Ser         355 360 365 Arg Asn Gly Ile His Pro Gly Ser Glu Leu Asn Ile Leu Ala Ala Asp     370 375 380 Gly Asp Leu Tyr Val Ala Lys Ile Asp Asp Lys Val Ile Val Lys Ile 385 390 395 400 Gly Ser Arg Tyr Asp Val Gly Asn Leu Ile Pro Ser Asp Phe His Ala                 405 410 415 Val Ala His Gly Asn Asn Tyr Cys Val Trp Glu Lys His Gly Leu Arg             420 425 430  Val Pro Ala Gly Arg His His         435 <210> 52 <211> 1320 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 52 atggcgaagc acttggctgc catgtgctgg tgcagcctcc tagtgcttgt actgctctgc 60 ttgggctccc agctggccca atcccaggtc ctcttccagg ggttcaactg ggagtcgtgg 120 aagaagcaag gtgggtggta caactacctc ctggggcggg tggacgacat cgccgcgacg 180 ggggccacgc acgtctggct cccgcagccg tcgcactcgg tggcgccgca ggggtacatg 240 cccggccggc tctacgacct ggacgcgtcc aagtacggca cccacgcgga gctcaagtcg 300 ctcaccgcgg cgttccacgc caagggcgtc cagtgcgtcg ccgacgtcgt gatcaaccac 360 cgctgcgccg actacaagga cggccgcggc atctactgcg tcttcgaggg cggcacgccc 420 gacagccgcc tcgactgggg ccccgacatg atctgcagcg acgacacgca gtactccaac 480 gggcgcgggc accgcgacac gggggccgac ttcgccgccg cgcccgacat cgaccacctc 540 aacccgcgcg tgcagcagga gctctcggac tggctcaact ggctcaagtc cgacctcggc 600 ttcgacggct ggcgcctcga cttcgccaag ggctactccg ccgccgtcgc caaggtgtac 660 gtcgacagca ccgcccccac cttcgtcgtc gccgagatat ggagctccct ccactacgac 720 ggcaacggcg agccgtccag caaccaggac gccgacaggc aggagctggt caactgggcg 780 caggcggtgg gcggccccgc cgcggcgttc gacttcacca ccaagggcgt gctgcaggcg 840 gccgtccagg gcgagctgtg gcgcatgaag gacggcaacg gcaaggcgcc cgggatgatc 900 ggctggctgc cggagaaggc cgtcacgttc gtcgacaacc acgacaccgg ctccacgcag 960 aactcgtggc cattcccctc cgacaaggtc atgcagggct acgcctatat cctcacgcac 1020 ccaggaactc catgcatctt ctacgaccac gttttcgact ggaacctgaa gcaggagatc 1080 agcgcgctgt ctgcggtgag gtcaagaaac gggatccacc cggggagcga gctgaacatc 1140 ctcgccgccg acggggatct ctacgtcgcc aagattgacg acaaggtcat cgtgaagatc 1200 gggtcacggt acgacgtcgg gaacctgatc ccctcagact tccacgccgt tgcccctggc 1260 aacaactact gcgtttggga gaagcacggt ctgagagttc cagcggggcg gcaccactag 1320 <210> 53 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 53 Ala Thr Gly Gly Thr Thr Thr Thr Ala Thr Thr Thr Gly Ser Gly Gly  1 5 10 15 Val Thr Ser Thr Ser Lys Thr Thr Thr Thr Ala Ser Lys Thr Ser Thr             20 25 30 Thr Thr Ser Ser Thr Ser Cys Thr Thr Pro Thr Ala Val         35 40 45 <210> 54 <211> 137 <212> DNA <213> Artificial Sequence   <220> <223> synthetic <400> 54 gccaccggcg gcaccaccac caccgccacc accaccggct ccggcggcgt gacctccacc 60 tccaagacca ccaccaccgc ctccaagacc tccaccacca cctcctccac ctcctgcacc 120 accccgaccg ccgtgtc 137 <210> 55 <211> 300 <212> PRT <213> Pyrococcus furiosus <400> 55 Ile Tyr Phe Val Glu Lys Tyr His Thr Ser Glu Asp Lys Ser Thr Ser  1 5 10 15 Asn Thr Ser Ser Thr Pro Pro Gln Thr Thr Leu Ser Thr Thr Lys Val             20 25 30 Leu Lys Ile Arg Tyr Pro Asp Asp Gly Glu Trp Pro Gly Ala Pro Ile         35 40 45 Asp Lys Asp Gly Asp Gly Asn Pro Glu Phe Tyr Ile Glu Ile Asn Leu     50 55 60 Trp Asn Ile Leu Asn Ala Thr Gly Phe Ala Glu Met Thr Tyr Asn Leu 65 70 75 80 Thr Ser Gly Val Leu His Tyr Val Gln Gln Leu Asp Asn Ile Val Leu                 85 90 95 Arg Asp Arg Ser Asn Trp Val His Gly Tyr Pro Glu Ile Phe Tyr Gly             100 105 110 Asn Lys Pro Trp Asn Ala Asn Tyr Ala Thr Asp Gly Pro Ile Pro Leu         115 120 125 Pro Ser Lys Val Ser Asn Leu Thr Asp Phe Tyr Leu Thr Ile Ser Tyr     130 135 140 Lys Leu Glu Pro Lys Asn Gly Leu Pro Ile Asn Phe Ala Ile Glu Ser 145 150 155 160 Trp Leu Thr Arg Glu Ala Trp Arg Thr Thr Gly Ile Asn Ser Asp Glu                 165 170 175 Gln Glu Val Met Ile Trp Ile Tyr Tyr Asp Gly Leu Gln Pro Ala Gly             180 185 190 Ser Lys Val Lys Glu Ile Val Val Pro Ile Ile Val Asn Gly Thr Pro         195 200 205 Val Asn Ala Thr Phe Glu Val Trp Lys Ala Asn Ile Gly Trp Glu Tyr     210 215 220 Val Ala Phe Arg Ile Lys Thr Pro Ile Lys Glu Gly Thr Val Thr Ile 225 230 235 240 Pro Tyr Gly Ala Phe Ile Ser Val Ala Ala Asn Ile Ser Ser Leu Pro                 245 250 255 Asn Tyr Thr Glu Leu Tyr Leu Glu Asp Val Glu Ile Gly Thr Glu Phe             260 265 270 Gly Thr Pro Ser Thr Thr Ser Ala His Leu Glu Trp Trp Ile Thr Asn         275 280 285 Ile Thr Leu Thr Pro Leu Asp Arg Pro Leu Ile Ser     290 295 300 <210> 56 <211> 903 <212> DNA <213> Pyrococcus furiosus   <400> 56 atctacttcg tggagaagta ccacacctcc gaggacaagt ccacctccaa cacctcctcc 60 accccgccgc agaccaccct ctccaccacc aaggtgctca agatccgcta cccggacgac 120 ggcgagtggc ccggcgcccc gatcgacaag gacggcgacg gcaacccgga gttctacatc 180 gagatcaacc tctggaacat cctcaacgcc accggcttcg ccgagatgac ctacaacctc 240 actagtggcg tgctccacta cgtgcagcag ctcgacaaca tcgtgctccg cgaccgctcc 300 aactgggtgc acggctaccc ggaaatcttc tacggcaaca agccgtggaa cgccaactac 360 gccaccgacg gcccgatccc gctcccgtcc aaggtgtcca acctcaccga cttctacctc 420 accatctcct acaagctcga gccgaagaac ggtctcccga tcaacttcgc catcgagtcc 480 tggctcaccc gcgaggcctg gcgcaccacc ggcatcaact ccgacgagca ggaggtgatg 540 atctggatct actacgacgg cctccagccc gcgggctcca aggtgaagga gatcgtggtg 600 ccgatcatcg tgaacggcac cccggtgaac gccaccttcg aggtgtggaa ggccaacatc 660 ggctgggagt acgtggcctt ccgcatcaag accccgatca aggagggcac cgtgaccatc 720 ccgtacggcg ccttcatctc cgtggccgcc aacatctcct ccctcccgaa ctacaccgag 780 aagtacctcg aggacgtgga gatcggcacc gagttcggca ccccgtccac cacctccgcc 840 cacctcgagt ggtggatcac caacatcacc ctcaccccgc tcgaccgccc gctcatctcc 900 tag 903 <210> 57 <211> 387 <212> PRT <213> Thermus flavus <400> 57 Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp Thr  1 5 10 15 Val Asp Asn Val Asp Arg Asp Pro Phe Gly Asp Thr Val Arg Glu Arg             20 25 30 Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu Gly Ala Tyr         35 40 45 Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg Gly Thr Pro Pro     50 55 60 Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys Lys Ala Leu Asp Glu 65 70 75 80 Thr Val Leu Lys Val Pro Met Val Thr Ala Asn Leu Phe Ser Glu Pro                 85 90 95 Ala Phe Arg Asp Gly Ala Ser Thr Thr Arg Asp Pro Trp Val Trp Ala             100 105 110 Tyr Ala Leu Arg Lys Ser Leu Glu Thr Met Asp Leu Gly Ala Glu Leu         115 120 125 Gly Ala Glu Ile Tyr Met Phe Trp Met Val Arg Glu Arg Ser Glu Val     130 135 140 Glu Ser Thr Asp Lys Thr Arg Lys Val Trp Asp Trp Val Arg Glu Thr 145 150 155 160 Leu Asn Phe Met Thr Ala Tyr Thr Glu Asp Gln Gly Tyr Gly Tyr Arg                 165 170 175 Phe Ser Val Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe             180 185 190 Thr Thr Val Gly Ser Met Leu Ala Leu Ile His Thr Leu Asp Arg Pro         195 200 205 Glu Arg Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly     210 215 220 Leu Asn Phe Asp His Ala Val Ala Gln Ala Val Asp Ala Gly Lys Leu 225 230 235 240 Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln Asp                 245 250 255 Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Gly Phe Phe Leu Val Asp             260 265 270  Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe Glu Ala His         275 280 285 Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Thr Phe Val Arg Val     290 295 300 Cys Met Arg Thr Tyr Leu Ile Ile Lys Val Arg Ala Glu Thr Phe Arg 305 310 315 320 Glu Asp Pro Glu Val Lys Glu Leu Leu Ala Ala Tyr Tyr Gln Glu Asp                 325 330 335 Pro Ala Thr Leu Ala Leu Leu Asp Pro Tyr Ser Arg Glu Lys Ala Glu             340 345 350 Ala Leu Lys Arg Ala Glu Leu Pro Leu Glu Thr Lys Arg Arg Arg Gly         355 360 365 Tyr Ala Leu Glu Arg Leu Asp Gln Leu Ala Val Glu Tyr Leu Leu Gly     370 375 380 Val Arg Gly 385 <210> 58 <211> 978 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 58 atggggaaga acggcaacct gtgctgcttc tctctgctgc tgcttcttct cgccgggttg 60 gcgtccggcc atcaaatcta cttcgtggag aagtaccaca cctccgagga caagtccacc 120 tccaacacct cctccacccc gccgcagacc accctctcca ccaccaaggt gctcaagatc 180 cgctacccgg acgacggtga gtggcccggc gccccgatcg acaaggacgg cgacggcaac 240 ccggagttct acatcgagat caacctctgg aacatcctca acgccaccgg cttcgccgag 300 atgacctaca acctcactag tggcgtgctc cactacgtgc agcagctcga caacatcgtg 360 ctccgcgacc gctccaactg ggtgcacggc tacccggaaa tcttctacgg caacaagccg 420 tggaacgcca actacgccac cgacggcccg atcccgctcc cgtccaaggt gtccaacctc 480 accgacttct acctcaccat ctcctacaag ctcgagccga agaacggtct cccgatcaac 540 ttcgccatcg agtcctggct cacccgcgag gcctggcgca ccaccggcat caactccgac 600 gagcaggagg tgatgatctg gatctactac gacggcctcc agcccgcggg ctccaaggtg 660 aaggagatcg tggtgccgat catcgtgaac ggcaccccgg tgaacgccac cttcgaggtg 720 tggaaggcca acatcggctg ggagtacgtg gccttccgca tcaagacccc gatcaaggag 780 ggcaccgtga ccatcccgta cggcgccttc atctccgtgg ccgccaacat ctcctccctc 840 ccgaactaca ccgagaagta cctcgaggac gtggagatcg gcaccgagtt cggcaccccg 900 tccaccacct ccgcccacct cgagtggtgg atcaccaaca tcaccctcac cccgctcgac 960 cgcccgctca tctcctag 978 <210> 59 <211> 1920 <212> DNA <213> Aspergillus niger <400> 59 atgtccttcc gctccctcct cgccctctcc ggcctcgtgt gcaccggcct cgccaacgtg 60 atctccaagc gcgccaccct cgactcctgg ctctccaacg aggccaccgt ggcccgcacc 120 gccatcctca acaacatcgg cgccgacggc gcctgggtgt ccggcgccga ctccggcatc 180 gtggtggcct ccccgtccac cgacaacccg gactacttct acacctggac ccgcgactcc 240 ggcctcgtgc tcaagaccct cgtggacctc ttccgcaacg gcgacacctc cctcctctcc 300 accatcgaga actacatctc cgcccaggcc atcgtgcagg gcatctccaa cccgtccggc 360 gacctctcct ccggcgccgg cctcggcgag ccgaagttca acgtggacga gaccgcctac 420  accggctcct ggggccgccc gcagcgcgac ggcccggccc tccgcgccac cgccatgatc 480 ggcttcggcc agtggctcct cgacaacggc tacacctcca ccgccaccga catcgtgtgg 540 ccgctcgtgc gcaacgacct ctcctacgtg gcccagtact ggaaccagac cggctacgac 600 ctctgggagg aggtgaacgg ctcctccttc ttcaccatcg ccgtgcagca ccgcgccctc 660 gtggagggct ccgccttcgc caccgccgtg ggctcctcct gctcctggtg cgactcccag 720 gccccggaga tcctctgcta cctccagtcc ttctggaccg gctccttcat cctcgccaac 780 ttcgactcct cccgctccgg caaggacgcc aacaccctcc tcggctccat ccacaccttc 840 gacccggagg ccgcctgcga cgactccacc ttccagccgt gctccccgcg cgccctcgcc 900 aaccacaagg aggtggtgga ctccttccgc tccatctaca ccctcaacga cggcctctcc 960 gactccgagg ccgtggccgt gggccgctac ccggaggaca cctactacaa cggcaacccg 1020 tggttcctct gcaccctcgc cgccgccgag cagctctacg acgccctcta ccagtgggac 1080 aagcagggct ccctcgaggt gaccgacgtg tccctcgact tcttcaaggc cctctactcc 1140 gacgccgcca ccggcaccta ctcctcctcc tcctccacct actcctccat cgtggacgcc 1200 gtgaagacct tcgccgacgg cttcgtgtcc atcgtggaga cccacgccgc ctccaacggc 1260 tccatgtccg agcagtacga caagtccgac ggcgagcagc tctccgcccg cgacctcacc 1320 tggtcctacg ccgccctcct caccgccaac aaccgccgca actccgtggt gccggcctcc 1380 tggggcgaga cctccgcctc ctccgtgccg ggcacctgcg ccgccacctc cgccatcggc 1440 acctactcct ccgtgaccgt gacctcctgg ccgtccatcg tggccaccgg cggcaccacc 1500 accaccgcca ccccgaccgg ctccggctcc gtgacctcca cctccaagac caccgccacc 1560 gcctccaaga cctccacctc cacctcctcc acctcctgca ccaccccgac cgccgtggcc 1620 gtgaccttcg acctcaccgc caccaccacc tacggcgaga acatctacct cgtgggctcc 1680 atctcccagc tcggcgactg ggagacctcc gacggcatcg ccctctccgc cgacaagtac 1740 acctcctccg acccgctctg gtacgtgacc gtgaccctcc cggccggcga gtccttcgag 1800 tacaagttca tccgcatcga gtccgacgac tccgtggagt gggagtccga cccgaaccgc 1860 gagtacaccg tgccgcaggc ctgcggcacc tccaccgcca ccgtgaccga cacctggcgc 1920 <210> 60 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 60 Ser Glu Lys Asp Glu Leu  1 5 <210> 61 <211> 561 <212> DNA <213> Artificial Sequence <220> <223> Xylanase BD7436 <220> <221> CDS (222) (1) .. (561) <400> 61 atg gct agc acc ttc tac tgg cat ttg tgg acc gac ggc atc ggc acc 48 Met Ala Ser Thr Phe Tyr Trp His Leu Trp Thr Asp Gly Ile Gly Thr 1 5 10 15 gtg aac gct acc aac ggc agc gac ggc aac tac agc gtg agc tgg agc 96 Val Asn Ala Thr Asn Gly Ser Asp Gly Asn Tyr Ser Val Ser Trp Ser             20 25 30 aac tgc ggc aac ttc gtg gtg ggc aag ggc tgg acc acc ggc agc gct 144 Asn Cys Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Ala         35 40 45 acc agg gtg atc aac tac aac gct cat gct ttc agc gtg gtg ggc aac 192 Thr Arg Val Ile Asn Tyr Asn Ala His Ala Phe Ser Val Val Gly Asn     50 55 60 gct tac ttg gct ttg tac ggc tgg acc agg aac agc ttg atc gag tac 240 Ala Tyr Leu Ala Leu Tyr Gly Trp Thr Arg Asn Ser Leu Ile Glu Tyr 65 70 75 80 tac gtg gtg gac agc tgg ggc acc tac agg cca acc ggc acc tac aag 288 Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys                 85 90 95 ggc acc gtg acc agc gac ggc ggc acc tac gac atc tac acc acc acc 336 Gly Thr Val Thr Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr             100 105 110 agg acc aac gct cca agc atc gac ggc aac aac acc acc ttc acc caa 384 Arg Thr Asn Ala Pro Ser Ile Asp Gly Asn Asn Thr Thr Phe Thr Gln         115 120 125 ttc tgg agc gtg agg caa agc aag agg cca atc ggc acc aac aac acc 432 Phe Trp Ser Val Arg Gln Ser Lys Arg Pro Ile Gly Thr Asn Asn Thr     130 135 140 atc acc ttc agc aac cat gtg aac gct tgg aag agc aag ggc atg aac 480 Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser Lys Gly Met Asn 145 150 155 160 ttg ggc agc agc tgg agc tac caa gtg ttg gct acc gag ggc tac caa 528 Leu Gly Ser Ser Trp Ser Tyr Gln Val Leu Ala Thr Glu Gly Tyr Gln                 165 170 175 agc agc ggc tac agc aac gtg acc gtg tgg tag 561 Ser Ser Gly Tyr Ser Asn Val Thr Val Trp             180 185 <210> 62 <211> 186 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 62 Met Ala Ser Thr Phe Tyr Trp His Leu Trp Thr Asp Gly Ile Gly Thr 1 5 10 15 Val Asn Ala Thr Asn Gly Ser Asp Gly Asn Tyr Ser Val Ser Trp Ser             20 25 30 Asn Cys Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Ala         35 40 45 Thr Arg Val Ile Asn Tyr Asn Ala His Ala Phe Ser Val Val Gly Asn     50 55 60 Ala Tyr Leu Ala Leu Tyr Gly Trp Thr Arg Asn Ser Leu Ile Glu Tyr 65 70 75 80 Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys                 85 90 95 Gly Thr Val Thr Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr             100 105 110 Arg Thr Asn Ala Pro Ser Ile Asp Gly Asn Asn Thr Thr Phe Thr Gln         115 120 125 Phe Trp Ser Val Arg Gln Ser Lys Arg Pro Ile Gly Thr Asn Asn Thr     130 135 140 Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser Lys Gly Met Asn 145 150 155 160 Leu Gly Ser Ser Trp Ser Tyr Gln Val Leu Ala Thr Glu Gly Tyr Gln                 165 170 175 Ser Ser Gly Tyr Ser Asn Val Thr Val Trp             180 185 <210> 63 <211> 561 <212> DNA <213> Artificial Sequence <220> <223> Xylanase BD6002A <220> <221> CDS (222) (1) .. (561) <400> 63 atg gct agc acc gac tac tgg caa aac tgg acc gac ggc ggc ggc acc 48 Met Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Thr 1 5 10 15 gtg aac gct acc aac ggc agc gac ggc aac tac agc gtg agc tgg agc 96 Val Asn Ala Thr Asn Gly Ser Asp Gly Asn Tyr Ser Val Ser Trp Ser             20 25 30 aac tgc ggc aac ttc gtg gtg ggc aag ggc tgg acc acc ggc agc gct 144 Asn Cys Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Ala         35 40 45 acc agg gtg atc aac tac aac gct ggc gct ttc agc cca agc ggc aac 192 Thr Arg Val Ile Asn Tyr Asn Ala Gly Ala Phe Ser Pro Ser Gly Asn     50 55 60 ggc tac ttg gct ttg tac ggc tgg acc agg aac agc ttg atc gag tac 240 Gly Tyr Leu Ala Leu Tyr Gly Trp Thr Arg Asn Ser Leu Ile Glu Tyr 65 70 75 80 tac gtg gtg gac agc tgg ggc acc tac agg cca acc ggc acc tac aag 288 Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys                 85 90 95 ggc acc gtg acc agc gac ggc ggc acc tac gac atc tac acc acc acc 336 Gly Thr Val Thr Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr             100 105 110 agg acc aac gct cca agc atc gac ggc aac aac acc acc ttc acc caa 384 Arg Thr Asn Ala Pro Ser Ile Asp Gly Asn Asn Thr Thr Phe Thr Gln         115 120 125 ttc tgg agc gtg agg caa agc aag agg cca atc ggc acc aac aac acc 432 Phe Trp Ser Val Arg Gln Ser Lys Arg Pro Ile Gly Thr Asn Asn Thr     130 135 140 atc acc ttc agc aac cat gtg aac gct tgg aag agc aag ggc atg aac 480 Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser Lys Gly Met Asn 145 150 155 160 ttg ggc agc agc tgg agc tac caa gtg ttg gct acc gag ggc tac caa 528 Leu Gly Ser Ser Trp Ser Tyr Gln Val Leu Ala Thr Glu Gly Tyr Gln                 165 170 175 agc agc ggc tac agc aac gtg acc gtg tgg tag 561 Ser Ser Gly Tyr Ser Asn Val Thr Val Trp             180 185 <210> 64 <211> 186 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 64 Met Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Thr 1 5 10 15 Val Asn Ala Thr Asn Gly Ser Asp Gly Asn Tyr Ser Val Ser Trp Ser             20 25 30 Asn Cys Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Ala         35 40 45 Thr Arg Val Ile Asn Tyr Asn Ala Gly Ala Phe Ser Pro Ser Gly Asn     50 55 60 Gly Tyr Leu Ala Leu Tyr Gly Trp Thr Arg Asn Ser Leu Ile Glu Tyr 65 70 75 80 Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys                 85 90 95 Gly Thr Val Thr Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr             100 105 110 Arg Thr Asn Ala Pro Ser Ile Asp Gly Asn Asn Thr Thr Phe Thr Gln         115 120 125 Phe Trp Ser Val Arg Gln Ser Lys Arg Pro Ile Gly Thr Asn Asn Thr     130 135 140 Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser Lys Gly Met Asn 145 150 155 160 Leu Gly Ser Ser Trp Ser Tyr Gln Val Leu Ala Thr Glu Gly Tyr Gln                 165 170 175 Ser Ser Gly Tyr Ser Asn Val Thr Val Trp             180 185 <210> 65 <211> 561 <212> DNA <213> Artificial Sequence <220> <223> Xylanase BD6002B <220> <221> CDS (222) (1) .. (561) <400> 65 atg gcc tcc acc gac tac tgg cag aac tgg acc gac ggc ggc ggc acc 48 Met Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Thr 1 5 10 15 gtg aac gcc acc aac ggc tcc gac ggc aac tac tcc gtg tcc tgg tcc 96 Val Asn Ala Thr Asn Gly Ser Asp Gly Asn Tyr Ser Val Ser Trp Ser             20 25 30 aac tgc ggc aac ttc gtg gtg ggc aag ggc tgg acc acc ggc tcc gcc 144 Asn Cys Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Ala         35 40 45 acc cgc gtg atc aac tac aac gcc ggc gcc ttc tcc ccg tcc ggc aac 192 Thr Arg Val Ile Asn Tyr Asn Ala Gly Ala Phe Ser Pro Ser Gly Asn     50 55 60 ggc tac ctc gcc ctc tac ggc tgg acc cgc aac tcc ctc atc gag tac 240 Gly Tyr Leu Ala Leu Tyr Gly Trp Thr Arg Asn Ser Leu Ile Glu Tyr 65 70 75 80 tac gtg gtg gac tcc tgg ggc acc tac cgc ccg acc ggc acc tac aag 288 Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys                 85 90 95 ggc acc gtg acc tcc gac ggc ggc acc tac gac atc tac acc acc acc 336 Gly Thr Val Thr Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr             100 105 110 cgc acc aac gcc ccg tcc atc gac ggc aac aac acc acc ttc acc cag 384 Arg Thr Asn Ala Pro Ser Ile Asp Gly Asn Asn Thr Thr Phe Thr Gln         115 120 125 ttc tgg tcc gtg cgc cag tcc aag cgc ccg atc ggc acc aac aac acc 432 Phe Trp Ser Val Arg Gln Ser Lys Arg Pro Ile Gly Thr Asn Asn Thr     130 135 140 atc acc ttc tcc aac cac gtg aac gcc tgg aag tcc aag ggc atg aac 480 Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser Lys Gly Met Asn 145 150 155 160 ctc ggc tcc tcc tgg tcc tac cag gtg ctc gcc acc gag ggc tac cag 528 Leu Gly Ser Ser Trp Ser Tyr Gln Val Leu Ala Thr Glu Gly Tyr Gln                 165 170 175 tcc tcc ggc tac tcc aac gtg acc gtg tgg tga 561 Ser Ser Gly Tyr Ser Asn Val Thr Val Trp             180 185 <210> 66 <211> 186 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 66 Met Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Thr 1 5 10 15 Val Asn Ala Thr Asn Gly Ser Asp Gly Asn Tyr Ser Val Ser Trp Ser             20 25 30 Asn Cys Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Ala         35 40 45 Thr Arg Val Ile Asn Tyr Asn Ala Gly Ala Phe Ser Pro Ser Gly Asn     50 55 60 Gly Tyr Leu Ala Leu Tyr Gly Trp Thr Arg Asn Ser Leu Ile Glu Tyr 65 70 75 80 Tyr Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys                 85 90 95 Gly Thr Val Thr Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr             100 105 110 Arg Thr Asn Ala Pro Ser Ile Asp Gly Asn Asn Thr Thr Phe Thr Gln         115 120 125 Phe Trp Ser Val Arg Gln Ser Lys Arg Pro Ile Gly Thr Asn Asn Thr     130 135 140 Ile Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser Lys Gly Met Asn 145 150 155 160 Leu Gly Ser Ser Trp Ser Tyr Gln Val Leu Ala Thr Glu Gly Tyr Gln                 165 170 175 Ser Ser Gly Tyr Ser Asn Val Thr Val Trp             180 185 <210> 67 <211> 2071 <212> DNA <213> Oryza sativa <220> <221> misc_feature (222) (1) .. (2071) <223> Promoter <400> 67 tccatgctgt cctactactt gcttcatccc cttctacatt ttgttctggt ttttggcctg 60 catttcggat catgatgtat gtgatttcca atctgctgca atatgaatgg agactctgtg 120 ctaaccatca acaacatgaa atgcttatga ggcctttgct gagcagccaa tcttgcctgt 180 gtttatgtct tcacaggccg aattcctctg ttttgttttt caccctcaat atttggaaac 240 atttatctag gttgtttgtg tccaggccta taaatcatac atgatgttgt cgtattggat 300 gtgaatgtgg tggcgtgttc agtgccttgg atttgagttt gatgagagtt gcttctgggt 360 caccactcac cattatcgat gctcctcttc agcataaggt aaaagtcttc cctgtttacg 420 ttattttacc cactatggtt gcttgggttg gttttttcct gattgcttat gccatggaaa 480 gtcatttgat atgttgaact tgaattaact gtagaattgt atacatgttc catttgtgtt 540 gtacttcctt cttttctatt agtagcctca gatgagtgtg aaaaaaacag attatataac 600 ttgccctata aatcatttga aaaaaatatt gtacagtgag aaattgatat atagtgaatt 660 tttaagagca tgttttccta aagaagtata tattttctat gtacaaaggc cattgaagta 720 attgtagata caggataatg tagacttttt ggacttacac tgctaccttt aagtaacaat 780 catgagcaat agtgttgcaa tgatatttag gctgcattcg tttactctct tgatttccat 840 gagcacgctt cccaaactgt taaactctgt gttttttgcc aaaaaaaaat gcataggaaa 900 gttgctttta aaaaatcata tcaatccatt ttttaagtta tagctaatac ttaattaatc 960 atgcgctaat aagtcactct gtttttcgta ctagagagat tgttttgaac cagcactcaa 1020 gaacacagcc ttaacccagc caaataatgc tacaacctac cagtccacac ctcttgtaaa 1080 gcatttgttg catggaaaag ctaagatgac agcaacctgt tcaggaaaac aactgacaag 1140 gtcataggga gagggagctt ttggaaaggt gccgtgcagt tcaaacaatt agttagcagt 1200 agggtgttgg tttttgctca cagcaataag aagttaatca tggtgtaggc aacccaaata 1260 aaacaccaaa atatgcacaa ggcagtttgt tgtattctgt agtacagaca aaactaaaag 1320 taatgaaaga agatgtggtg ttagaaaagg aaacaatatc atgagtaatg tgtgggcatt 1380 atgggaccac gaaataaaaa gaacattttg atgagtcgtg tatcctcgat gagcctcaaa 1440 agttctctca ccccggataa gaaaccctta agcaatgtgc aaagtttgca ttctccactg 1500 acataatgca aaataagata tcatcgatga catagcaact catgcatcat atcatgcctc 1560 tctcaaccta ttcattccta ctcatctaca taagtatctt cagctaaatg ttagaacata 1620 aacccataag tcacgtttga tgagtattag gcgtgacaca tgacaaatca cagactcaag 1680 caagataaag caaaatgatg tgtacataaa actccagagc tatatgtcat attgcaaaaa 1740 gaggagagct tataagacaa ggcatgactc acaaaaattc atttgccttt cgtgtcaaaa 1800 agaggagggc tttacattat ccatgtcata ttgcaaaaga aagagagaaa gaacaacaca 1860 atgctgcgtc aattatacat atctgtatgt ccatcattat tcatccacct ttcgtgtacc 1920 acacttcata tatcatgagt cacttcatgt ctggacatta acaaactcta tcttaacatt 1980 tagatgcaag agcctttatc tcactataaa tgcacgatga tttctcattg tttctcacaa 2040 aaagcattca gttcattagt cctacaacaa c 2071 <210> 68 <211> 79 <212> PRT <213> Zea mays <220> <221> SIGNAL (222) (1) .. (79) <223> Maize waxy signal sequence. <400> 68 Met Leu Ala Ala Leu Ala Thr Ser Gln Leu Val Ala Thr Arg Ala Gly 1 5 10 15 Leu Gly Val Pro Asp Ala Ser Thr Phe Arg Arg Gly Ala Ala Gln Gly             20 25 30 Leu Arg Gly Ala Arg Ala Ser Ala Ala Ala Asp Thr Leu Ser Met Arg         35 40 45 Thr Ser Ala Arg Ala Ala Pro Arg His Gln His Gln Gln Ala Arg Arg     50 55 60 Gly Ala Arg Phe Pro Ser Leu Val Val Cys Ala Ser Ala Gly Ala 65 70 75 <210> 69 <211> 1005 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Bromelain Sequence <220> <221> CDS (222) (1) .. (1005) <223> Synthetic Bromelain <400> 69 atg gcc tgg aag gtg cag gtg gtg ttc ctc ttc ctc ttc ctc tgc gtg 48 Met Ala Trp Lys Val Gln Val Val Phe Leu Phe Leu Phe Leu Cys Val 1 5 10 15 atg tgg gcc tcc ccg tcc gcc gcc tcc gcg gac gag ccg tcc gac ccg 96 Met Trp Ala Ser Pro Ser Ala Ala Ser Ala Asp Glu Pro Ser Asp Pro             20 25 30 atg atg aag cgc ttc gag gag tgg atg gtg gag tac ggc cgc gtg tac 144 Met Met Lys Arg Phe Glu Glu Trp Met Val Glu Tyr Gly Arg Val Tyr         35 40 45 aag gac aac gac gag aag atg cgc cgc ttc cag atc ttc aag aac aac 192 Lys Asp Asn Asp Glu Lys Met Arg Arg Phe Gln Ile Phe Lys Asn Asn     50 55 60 gtg aac cac atc gag acc ttc aac tcc cgc aac gag aac tcc tac acc 240 Val Asn His Ile Glu Thr Phe Asn Ser Arg Asn Glu Asn Ser Tyr Thr 65 70 75 80 ctc ggc atc aac cag ttc acc gac atg acc aac aac gag ttc atc gcc 288 Leu Gly Ile Asn Gln Phe Thr Asp Met Thr Asn Asn Glu Phe Ile Ala                 85 90 95 cag tac acc ggc ggc atc tcc cgc ccg ctc aac atc gag cgc gag ccg 336 Gln Tyr Thr Gly Gly Ile Ser Arg Pro Leu Asn Ile Glu Arg Glu Pro             100 105 110 gtg gtg tcc ttc gac gac gtg gac atc tcc gcc gtg ccg cag tcc atc 384 Val Val Ser Phe Asp Asp Val Asp Ile Ser Ala Val Pro Gln Ser Ile         115 120 125 gac tgg cgc gac tac ggc gcc gtg acc tcc gtg aag aac cag aac ccg 432 Asp Trp Arg Asp Tyr Gly Ala Val Thr Ser Ser Val Lys Asn Gln Asn Pro     130 135 140 tgc ggc gcc tgc tgg gcc ttc gcc gcc atc gcc acc gtg gag tcc atc 480 Cys Gly Ala Cys Trp Ala Phe Ala Ala Ile Ala Thr Val Glu Ser Ile 145 150 155 160 tac aag atc aag aag ggc atc ctc gag ccg ctc tcc gag cag cag gtg 528 Tyr Lys Ile Lys Lys Gly Ile Leu Glu Pro Leu Ser Glu Gln Gln Val                 165 170 175 ctc gac tgc gcc aag ggc tac ggc tgc aag ggc ggc tgg gag ttc cgc 576 Leu Asp Cys Ala Lys Gly Tyr Gly Cys Lys Gly Gly Trp Glu Phe Arg             180 185 190 gcc ttc gag ttc atc atc tcc aac aag ggc gtg gcc tcc ggc gcc atc 624 Ala Phe Glu Phe Ile Ile Ser Asn Lys Gly Val Ala Ser Gly Ala Ile         195 200 205 tac ccg tac aag gcc gcc aag ggc acc tgc aag acc gac ggc gtg ccg 672 Tyr Pro Tyr Lys Ala Ala Lys Gly Thr Cys Lys Thr Asp Gly Val Pro     210 215 220 aac tcc gcc tac atc acc ggc tac gcc cgc gtg ccg cgc aac aac gag 720 Asn Ser Ala Tyr Ile Thr Gly Tyr Ala Arg Val Pro Arg Asn Asn Glu 225 230 235 240 tcc tcc atg atg tac gcc gtg tcc aag cag ccg atc acc gtg gcc gtg 768 Ser Ser Met Met Tyr Ala Val Ser Lys Gln Pro Ile Thr Val Ala Val                 245 250 255 gac gcc aac gcc aac ttc cag tac tac aag tcc ggc gtg ttc aac ggc 816 Asp Ala Asn Ala Asn Phe Gln Tyr Tyr Lys Ser Gly Val Phe Asn Gly             260 265 270 ccg tgc ggc acc tcc ctc aac cac gcc gtg acc gcc atc ggc tac ggc 864 Pro Cys Gly Thr Ser Leu Asn His Ala Val Thr Ala Ile Gly Tyr Gly         275 280 285 cag gac tcc atc atc tac ccg aag aag tgg ggc gcc aag tgg ggc gag 912 Gln Asp Ser Ile Ile Tyr Pro Lys Lys Trp Gly Ala Lys Trp Gly Glu     290 295 300 gcc ggc tac atc cgc atg gcc cgc gac gtg tcc tcc tcc tcc ggc atc 960 Ala Gly Tyr Ile Arg Met Ala Arg Asp Val Ser Ser Ser Ser Gly Ile 305 310 315 320 tgc ggc atc gcc atc gac ccg ctc tac ccg acc ctc gag gag tag 1005 Cys Gly Ile Ala Ile Asp Pro Leu Tyr Pro Thr Leu Glu Glu                 325 330 <210> 70 <211> 334 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 70 Met Ala Trp Lys Val Gln Val Val Phe Leu Phe Leu Phe Leu Cys Val 1 5 10 15 Met Trp Ala Ser Pro Ser Ala Ala Ser Ala Asp Glu Pro Ser Asp Pro             20 25 30 Met Met Lys Arg Phe Glu Glu Trp Met Val Glu Tyr Gly Arg Val Tyr         35 40 45 Lys Asp Asn Asp Glu Lys Met Arg Arg Phe Gln Ile Phe Lys Asn Asn     50 55 60 Val Asn His Ile Glu Thr Phe Asn Ser Arg Asn Glu Asn Ser Tyr Thr 65 70 75 80 Leu Gly Ile Asn Gln Phe Thr Asp Met Thr Asn Asn Glu Phe Ile Ala                 85 90 95 Gln Tyr Thr Gly Gly Ile Ser Arg Pro Leu Asn Ile Glu Arg Glu Pro             100 105 110 Val Val Ser Phe Asp Asp Val Asp Ile Ser Ala Val Pro Gln Ser Ile         115 120 125 Asp Trp Arg Asp Tyr Gly Ala Val Thr Ser Ser Val Lys Asn Gln Asn Pro     130 135 140 Cys Gly Ala Cys Trp Ala Phe Ala Ala Ile Ala Thr Val Glu Ser Ile 145 150 155 160 Tyr Lys Ile Lys Lys Gly Ile Leu Glu Pro Leu Ser Glu Gln Gln Val                 165 170 175 Leu Asp Cys Ala Lys Gly Tyr Gly Cys Lys Gly Gly Trp Glu Phe Arg             180 185 190 Ala Phe Glu Phe Ile Ile Ser Asn Lys Gly Val Ala Ser Gly Ala Ile         195 200 205 Tyr Pro Tyr Lys Ala Ala Lys Gly Thr Cys Lys Thr Asp Gly Val Pro     210 215 220 Asn Ser Ala Tyr Ile Thr Gly Tyr Ala Arg Val Pro Arg Asn Asn Glu 225 230 235 240 Ser Ser Met Met Tyr Ala Val Ser Lys Gln Pro Ile Thr Val Ala Val                 245 250 255 Asp Ala Asn Ala Asn Phe Gln Tyr Tyr Lys Ser Gly Val Phe Asn Gly             260 265 270 Pro Cys Gly Thr Ser Leu Asn His Ala Val Thr Ala Ile Gly Tyr Gly         275 280 285 Gln Asp Ser Ile Ile Tyr Pro Lys Lys Trp Gly Ala Lys Trp Gly Glu     290 295 300 Ala Gly Tyr Ile Arg Met Ala Arg Asp Val Ser Ser Ser Ser Gly Ile 305 310 315 320 Cys Gly Ile Ala Ile Asp Pro Leu Tyr Pro Thr Leu Glu Glu                 325 330 <210> 71 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Bromealin signal sequence <400> 71 atggcctgga aggtgcaggt ggtgttcctc ttcctcttcc tctgcgtgat gtgggcctcc 60 ccgtccgccg cctccgcc 78 <210> 72 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Bromealin signal peptide <400> 72 Met Ala Trp Lys Val Gln Val Val Phe Leu Phe Leu Phe Leu Cys Val 1 5 10 15 Met Trp Ala Ser Pro Ser Ala Ala Ser Ala             20 25 <210> 73 <211> 1050 <212> DNA <213> Artificial Sequence <220> <223> pSYN11000 <400> 73 atggcctgga aggtgcaggt ggtgttcctc ttcctcttcc tctgcgtgat gtgggcctcc 60 ccgtccgccg cctccgcgga cgagccgtcc gacccgatga tgaagcgctt cgaggagtgg 120 atggtggagt acggccgcgt gtacaaggac aacgacgaga agatgcgccg cttccagatc 180 ttcaagaaca acgtgaacca catcgagacc ttcaactccc gcaacgagaa ctcctacacc 240 ctcggcatca accagttcac cgacatgacc aacaacgagt tcatcgccca gtacaccggc 300 ggcatctccc gcccgctcaa catcgagcgc gagccggtgg tgtccttcga cgacgtggac 360 atctccgccg tgccgcagtc catcgactgg cgcgactacg gcgccgtgac ctccgtgaag 420 aaccagaacc cgtgcggcgc ctgctgggcc ttcgccgcca tcgccaccgt ggagtccatc 480 tacaagatca agaagggcat cctcgagccg ctctccgagc agcaggtgct cgactgcgcc 540 aagggctacg gctgcaaggg cggctgggag ttccgcgcct tcgagttcat catctccaac 600 aagggcgtgg cctccggcgc catctacccg tacaaggccg ccaagggcac ctgcaagacc 660 gacggcgtgc cgaactccgc ctacatcacc ggctacgccc gcgtgccgcg caacaacgag 720 tcctccatga tgtacgccgt gtccaagcag ccgatcaccg tggccgtgga cgccaacgcc 780 aacttccagt actacaagtc cggcgtgttc aacggcccgt gcggcacctc cctcaaccac 840 gccgtgaccg ccatcggcta cggccaggac tccatcatct acccgaagaa gtggggcgcc 900 aagtggggcg aggccggcta catccgcatg gcccgcgacg tgtcctcctc ctccggcatc 960 tgcggcatcg ccatcgaccc gctctacccg accctcgagg aggtgttcgc cgaggccatc 1020 gccgccaact ccaccctcgt ggccgagtag 1050 <210> 74 <211> 1067 <212> DNA <213> Artificial Sequence <220> <223> pSYN11589 <400> 74 tggcctggaa ggtgcaggtg gtgttcctct tcctcttcct ctgcgtgatg tgggcctccc 60 cgtccgccgc ctccgcctcc tcctcctcct tcgccgactc caacccgatc cgcccggtga 120 ccgaccgcgc cgcctccacc gacgagccgt ccgacccgat gatgaagcgc ttcgaggagt 180 ggatggtgga gtacggccgc gtgtacaagg acaacgacga gaagatgcgc cgcttccaga 240 tcttcaagaa caacgtgaac cacatcgaga ccttcaactc ccgcaacgag aactcctaca 300 ccctcggcat caaccagttc accgacatga ccaacaacga gttcatcgcc cagtacaccg 360 gcggcatctc ccgcccgctc aacatcgagc gcgagccggt ggtgtccttc gacgacgtgg 420 acatctccgc cgtgccgcag tccatcgact ggcgcgacta cggcgccgtg acctccgtga 480 agaaccagaa cccgtgcggc gcctgctggg ccttcgccgc catcgccacc gtggagtcca 540 tctacaagat caagaagggc atcctcgagc cgctctccga gcagcaggtg ctcgactgcg 600 ccaagggcta cggctgcaag ggcggctggg agttccgcgc cttcgagttc atcatctcca 660 acaagggcgt ggcctccggc gccatctacc cgtacaaggc cgccaagggc acctgcaaga 720 ccgacggcgt gccgaactcc gcctacatca ccggctacgc ccgcgtgccg cgcaacaacg 780 agtcctccat gatgtacgcc gtgtccaagc agccgatcac cgtggccgtg gacgccaacg 840 ccaacttcca gtactacaag tccggcgtgt tcaacggccc gtgcggcacc tccctcaacc 900 acgccgtgac cgccatcggc tacggccagg actccatcat ctacccgaag aagtggggcg 960 ccaagtgggg cgaggccggc tacatccgca tggcccgcga cgtgtcctcc tcctccggca 1020 tctgcggcat cgccatcgac ccgctctacc cgaccctcga ggagtag 1067 <210> 75 <211> 1023 <212> DNA <213> Artificial Sequence <220> <223> pSYN11587 Sequence <400> 75 atggcctgga aggtgcaggt ggtgttcctc ttcctcttcc tctgcgtgat gtgggcctcc 60 ccgtccgccg cctccgcgga cgagccgtcc gacccgatga tgaagcgctt cgaggagtgg 120 atggtggagt acggccgcgt gtacaaggac aacgacgaga agatgcgccg cttccagatc 180 ttcaagaaca acgtgaacca catcgagacc ttcaactccc gcaacgagaa ctcctacacc 240 ctcggcatca accagttcac cgacatgacc aacaacgagt tcatcgccca gtacaccggc 300 ggcatctccc gcccgctcaa catcgagcgc gagccggtgg tgtccttcga cgacgtggac 360 atctccgccg tgccgcagtc catcgactgg cgcgactacg gcgccgtgac ctccgtgaag 420 aaccagaacc cgtgcggcgc ctgctgggcc ttcgccgcca tcgccaccgt ggagtccatc 480 tacaagatca agaagggcat cctcgagccg ctctccgagc agcaggtgct cgactgcgcc 540 aagggctacg gctgcaaggg cggctgggag ttccgcgcct tcgagttcat catctccaac 600 aagggcgtgg cctccggcgc catctacccg tacaaggccg ccaagggcac ctgcaagacc 660 gacggcgtgc cgaactccgc ctacatcacc ggctacgccc gcgtgccgcg caacaacgag 720 tcctccatga tgtacgccgt gtccaagcag ccgatcaccg tggccgtgga cgccaacgcc 780 aacttccagt actacaagtc cggcgtgttc aacggcccgt gcggcacctc cctcaaccac 840 gccgtgaccg ccatcggcta cggccaggac tccatcatct acccgaagaa gtggggcgcc 900 aagtggggcg aggccggcta catccgcatg gcccgcgacg tgtcctcctc ctccggcatc 960 tgcggcatcg ccatcgaccc gctctacccg accctcgagg agtccgagaa ggacgagctg 1020 tag 1023 <210> 76 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> pSYN12169 Sequence <400> 76 atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccatg 60 gcggacgagc cgtccgaccc gatgatgaag cgcttcgagg agtggatggt ggagtacggc 120 cgcgtgtaca aggacaacga cgagaagatg cgccgcttcc agatcttcaa gaacaacgtg 180 aaccacatcg agaccttcaa ctcccgcaac gagaactcct acaccctcgg catcaaccag 240 ttcaccgaca tgaccaacaa cgagttcatc gcccagtaca ccggcggcat ctcccgcccg 300 ctcaacatcg agcgcgagcc ggtggtgtcc ttcgacgacg tggacatctc cgccgtgccg 360 cagtccatcg actggcgcga ctacggcgcc gtgacctccg tgaagaacca gaacccgtgc 420 ggcgcctgct gggccttcgc cgccatcgcc accgtggagt ccatctacaa gatcaagaag 480 ggcatcctcg agccgctctc cgagcagcag gtgctcgact gcgccaaggg ctacggctgc 540 aagggcggct gggagttccg cgccttcgag ttcatcatct ccaacaaggg cgtggcctcc 600 ggcgccatct acccgtacaa ggccgccaag ggcacctgca agaccgacgg cgtgccgaac 660 tccgcctaca tcaccggcta cgcccgcgtg ccgcgcaaca acgagtcctc catgatgtac 720 gccgtgtcca agcagccgat caccgtggcc gtggacgcca acgccaactt ccagtactac 780 aagtccggcg tgttcaacgg cccgtgcggc acctccctca accacgccgt gaccgccatc 840 ggctacggcc aggactccat catctacccg aagaagtggg gcgccaagtg gggcgaggcc 900 ggctacatcc gcatggcccg cgacgtgtcc tcctcctccg gcatctgcgg catcgccatc 960 gacccgctct acccgaccct cgaggagtag 990 <210> 77 <211> 1170 <212> DNA <213> Artificial Sequence <220> <223> pSYN12575 Sequence <400> 77 atgctggcgg ctctggccac gtcgcagctc gtcgcaacgc gcgccggcct gggcgtcccg 60 gacgcgtcca cgttccgccg cggcgccgcg cagggcctga ggggggcccg ggcgtcggcg 120 gcggcggaca cgctcagcat gcggaccagc gcgcgcgcgg cgcccaggca ccagcaccag 180 caggcgcgcc gcggggccag gttcccgtcg ctcgtcgtgt gcgccagcgc cggcgccatg 240 gcggacgagc cgtccgaccc gatgatgaag cgcttcgagg agtggatggt ggagtacggc 300 cgcgtgtaca aggacaacga cgagaagatg cgccgcttcc agatcttcaa gaacaacgtg 360 aaccacatcg agaccttcaa ctcccgcaac gagaactcct acaccctcgg catcaaccag 420 ttcaccgaca tgaccaacaa cgagttcatc gcccagtaca ccggcggcat ctcccgcccg 480 ctcaacatcg agcgcgagcc ggtggtgtcc ttcgacgacg tggacatctc cgccgtgccg 540 cagtccatcg actggcgcga ctacggcgcc gtgacctccg tgaagaacca gaacccgtgc 600 ggcgcctgct gggccttcgc cgccatcgcc accgtggagt ccatctacaa gatcaagaag 660 ggcatcctcg agccgctctc cgagcagcag gtgctcgact gcgccaaggg ctacggctgc 720 aagggcggct gggagttccg cgccttcgag ttcatcatct ccaacaaggg cgtggcctcc 780 ggcgccatct acccgtacaa ggccgccaag ggcacctgca agaccgacgg cgtgccgaac 840 tccgcctaca tcaccggcta cgcccgcgtg ccgcgcaaca acgagtcctc catgatgtac 900 gccgtgtcca agcagccgat caccgtggcc gtggacgcca acgccaactt ccagtactac 960 aagtccggcg tgttcaacgg cccgtgcggc acctccctca accacgccgt gaccgccatc 1020 ggctacggcc aggactccat catctacccg aagaagtggg gcgccaagtg gggcgaggcc 1080 ggctacatcc gcatggcccg cgacgtgtcc tcctcctccg gcatctgcgg catcgccatc 1140 gacccgctct acccgaccct cgaggagtag 1170 <210> 78 <211> 1068 <212> DNA <213> Artificial Sequence <220> <223> pSM270 Sequence <400> 78 atggcctgga aggtgcaggt ggtgttcctc ttcctcttcc tctgcgtgat gtgggcctcc 60 ccgtccgccg cctccgcctc ctcctcctcc ttcgccgact ccaacccgat ccgcccggtg 120 accgaccgcg ccgcctccac cgacgagccg tccgacccga tgatgaagcg cttcgaggag 180 tggatggtgg agtacggccg cgtgtacaag gacaacgacg agaagatgcg ccgcttccag 240 atcttcaaga acaacgtgaa ccacatcgag accttcaact cccgcaacga gaactcctac 300 accctcggca tcaaccagtt caccgacatg accaacaacg agttcatcgc ccagtacacc 360 ggcggcatct cccgcccgct caacatcgag cgcgagccgg tggtgtcctt cgacgacgtg 420 gacatctccg ccgtgccgca gtccatcgac tggcgcgact acggcgccgt gacctccgtg 480 aagaaccaga acccgtgcgg cgcctgctgg gccttcgccg ccatcgccac cgtggagtcc 540 atctacaaga tcaagaaggg catcctcgag ccgctctccg agcagcaggt gctcgactgc 600 gccaagggct acggctgcaa gggcggctgg gagttccgcg ccttcgagtt catcatctcc 660 aacaagggcg tggcctccgg cgccatctac ccgtacaagg ccgccaaggg cacctgcaag 720 accgacggcg tgccgaactc cgcctacatc accggctacg cccgcgtgcc gcgcaacaac 780 gagtcctcca tgatgtacgc cgtgtccaag cagccgatca ccgtggccgt ggacgccaac 840 gccaacttcc agtactacaa gtccggcgtg ttcaacggcc cgtgcggcac ctccctcaac 900 cacgccgtga ccgccatcgg ctacggccag gactccatca tctacccgaa gaagtggggc 960 gccaagtggg gcgaggccgg ctacatccgc atggcccgcg acgtgtcctc ctcctccggc 1020 atctgcggca tcgccatcga cccgctctac ccgaccctcg aggagtag 1068 <210> 79 <211> 1497 <212> DNA <213> Trichoderma reesei <220> <221> CDS (222) (1) .. (1497) <223> Trichoderma reesei cellobiohyrodlase I <400> 79 atg cag tcg gcg tgt act ctc caa tcg gag act cac ccg cct ctg aca 48 Met Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr 1 5 10 15 tgg cag aaa tgc tcg tct ggt ggc acg tgc act caa cag aca ggc tcc 96 Trp Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser             20 25 30 gtg gtc atc gac gcc aac tgg cgc tgg act cac gct acg aac agc agc 144 Val Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser         35 40 45 acg aac tgc tac gat ggc aac act tgg agc tcg acc cta tgt cct gac 192 Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp     50 55 60 aac gag acc tgc gcg aag aac tgc tgt ctg gac ggt gcc gcc tac gcg 240 Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala 65 70 75 80 tcc acg tac gga gtt acc acg agc ggt aac agc ctc tcc att ggc ttt 288 Ser Thr Tyr Gly Val Thr Thr Ser Ser Gly Asn Ser Leu Ser Ile Gly Phe                 85 90 95 gtc acc cag tct gcg cag aag aac gtt ggc gct cgc ctt tac ctt atg 336 Val Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met             100 105 110 gcg agc gac acg acc tac cag gaa ttc acc ctg ctt ggc aac gag ttc 384 Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe         115 120 125 tct ttc gat gtt gat gtt tcg cag ctg ccg tgc ggc ttg aac gga gct 432 Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala     130 135 140 ctc tac ttc gtg tcc atg gac gcg gat ggt ggc gtg agc aag tat ccc 480 Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro 145 150 155 160 acc aac acc gct ggc gcc aag tac ggc acg ggg tac tgt gac agc cag 528 Thr Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln                 165 170 175 tgt ccc cgc gat ctg aag ttc atc aat ggc cag gcc aac gtt gag ggc 576 Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly             180 185 190 tgg gag ccg tca tcc aac aac gcg aac acg ggc att gga gga cac gga 624 Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly         195 200 205 agc tgc tgc tct gag atg gat atc tgg gag gcc aac tcc atc tcc gag 672 Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu     210 215 220 gct ctt acc ccc cac cct tgc acg act gtc ggc cag gag atc tgc gag 720 Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu 225 230 235 240 ggt gat ggg tgc ggc gga act tac tcc gat aac aga tat ggc ggc act 768 Gly Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr                 245 250 255 tgc gat ccc gat ggc tgc gac tgg aac cca tac cgc ctg ggc aac acc 816 Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr             260 265 270 agc ttc tac ggc cct ggc tct agc ttt acc ctc gat acc acc aag aaa 864 Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys         275 280 285 ttg acc gtt gtc acc cag ttc gag acg tcg ggt gcc atc aac cga tac 912 Leu Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr     290 295 300 tat gtc cag aat ggc gtc act ttc cag cag ccc aac gcc gag ctt ggt 960 Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly 305 310 315 320 agt tac tct ggc aac gag ctc aac gat gat tac tgc aca gct gag gag 1008 Ser Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu                 325 330 335 gca gaa ttc ggc gga tcc tct ttc tca gac aag ggc ggc ctg act cag 1056 Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln             340 345 350 ttc aag aag gct acc tct ggc ggc atg gtt ctg gtc atg agt ctg tgg 1104 Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp         355 360 365 gat gat tac tac gcc aac atg ctg tgg ctg gac tcc acc tac ccg aca 1152 Asp Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr     370 375 380 aac gag acc tcc tcc aca ccc ggt gcc gtg cgc gga agc tgc tcc acc 1200 Asn Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr 385 390 395 400 agc tcc ggt gtc cct gct cag gtc gaa tct cag tct ccc aac gcc aag 1248 Ser Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys                 405 410 415 gtc acc ttc tcc aac atc aag ttc gga ccc att ggc agc acc ggc aac 1296 Val Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn             420 425 430 cct agc ggc ggc aac cct ccc ggc gga aac ccg cct ggc acc acc acc 1344 Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr         435 440 445 acc cgc cgc cca gcc act acc act gga agc tct ccc gga cct acc cag 1392 Thr Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln     450 455 460 tct cac tac ggc cag tgc ggc ggt att ggc tac agc ggc ccc acg gtc 1440 Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val 465 470 475 480 tgc gcc agc ggc aca act tgc cag gtc ctg aac cct tac tac tct cag 1488 Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln                 485 490 495 tgc ctg taa 1497 Cys leu                                                                          <210> 80 <211> 498 <212> PRT <213> Trichoderma reesei <400> 80 Met Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr 1 5 10 15 Trp Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser             20 25 30 Val Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser         35 40 45 Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp     50 55 60 Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala 65 70 75 80 Ser Thr Tyr Gly Val Thr Thr Ser Ser Gly Asn Ser Leu Ser Ile Gly Phe                 85 90 95 Val Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met             100 105 110 Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe         115 120 125 Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala     130 135 140 Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro 145 150 155 160 Thr Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln                 165 170 175 Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly             180 185 190 Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly         195 200 205 Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu     210 215 220 Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu 225 230 235 240 Gly Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr                 245 250 255 Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr             260 265 270 Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys         275 280 285 Leu Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr     290 295 300 Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly 305 310 315 320 Ser Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu                 325 330 335 Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln             340 345 350 Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp         355 360 365 Asp Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr     370 375 380 Asn Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr 385 390 395 400 Ser Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys                 405 410 415 Val Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn             420 425 430 Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr         435 440 445 Thr Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln     450 455 460 Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val 465 470 475 480 Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln                 485 490 495 Cys leu          <210> 81 <211> 1365 <212> DNA <213> Trichoderma reesei <220> <221> CDS (222) (1) .. (1365) Trichoderma reesei cellobiohydrolase II <400> 81 atg gtg cct cta gag gag cgg caa gct tgc tca agc gtc tgg ggc caa 48 Met Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly Gln 1 5 10 15 tgt ggt ggc cag aat tgg tcg ggt ccg act tgc tgt gct tcc gga agc 96 Cys Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala Ser Gly Ser             20 25 30 aca tgc gtc tac tcc aac gac tat tac tcc cag tgt ctt ccc ggc gct 144 Thr Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly Ala         35 40 45 gca agc tca agc tcg tcc acg cgc gcc gcg tcg acg act tca cga gta 192 Ala Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg Val     50 55 60 tcc ccc aca aca tcc cgg tcg agc tcc gcg acg cct cca cct ggt tct 240 Ser Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly Ser 65 70 75 80 acc act acc aga gta cct cca gtc gga tcg gga acc gct acg tat tca 288 Thr Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr Ser                 85 90 95 ggc aac cct ttt gtt ggg gtc act cct tgg gcc aat gca tat tac gcc 336 Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala             100 105 110 tct gaa gtt agc agc ctc gct att cct agc ttg act gga gcc atg gcc 384 Ser Glu Val Ser Ser Le Le Ala Ile Pro Ser Leu Thr Gly Ala Met Ala         115 120 125 act gct gca gca gct gtc gca aag gtt ccc tct ttt atg tgg cta gat 432 Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp     130 135 140 act ctt gac aag acc cct ctc atg gag caa acc ttg gcc gac atc cgc 480 Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg 145 150 155 160 acc gcc aac aag aat ggc ggt aac tat gcc gga cag ttt gtg gtg tat 528 Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr                 165 170 175 gac ttg ccg gat cgc gat tgc gct gcc ctt gcc tcg aat ggc gaa tac 576 Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu Tyr             180 185 190 tct att gcc gat ggt ggc gtc gcc aaa tat aag aac tat atc gac acc 624 Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp Thr         195 200 205 att cgt caa att gtc gtg gaa tat tcc gat atc cgg acc ctc ctg gtt 672 Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu Val     210 215 220 att gag cct gac tct ctt gcc aac ctg gtg acc aac ctc ggt act cca 720 Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr Pro 225 230 235 240 aag tgt gcc aat gct cag tca gcc tac ctt gag tgc atc aac tac gcc 768 Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr Ala                 245 250 255 gtc aca cag ctg aac ctt cca aat gtt gcg atg tat ttg gac gct ggc 816 Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly             260 265 270 cat gca gga tgg ctt ggc tgg ccg gca aac caa gac ccg gcc gct cag 864 His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala Gln         275 280 285 cta ttt gca aat gtt tac aag aat gca tcg tct ccg aga gct ctt cgc 912 Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu Arg     290 295 300 gga ttg gca acc aat gtc gcc aac tac aac ggg tgg aac att acc agc 960 Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr Ser 305 310 315 320 ccc cca tcg tac acg caa ggc aac gct gtc tac aac gag aag ctg tac 1008 Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu Tyr                 325 330 335 atc cac gct att gga cct ctt ctt gcc aat cac ggc tgg tcc aac gcc 1056 Ile His Ala Ile Gly Pro Leu Leu Ala Asn His Gly Trp Ser Asn Ala             340 345 350 ttc ttc atc act gat caa ggt cga tcg gga aag cag cct acc gga cag 1104 Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln         355 360 365 caa cag tgg gga gac tgg tgc aat gtg atc ggc acc gga ttt ggt att 1152 Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly Ile     370 375 380 cgc cca tcc gca aac act ggg gac tcg ttg ctg gat tcg ttt gtc tgg 1200 Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val Trp 385 390 395 400 gtc aag cca ggc ggc gag tgt gac ggc acc agc gac agc agt gcg cca 1248 Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser Ala Pro                 405 410 415 cga ttt gac tcc cac tgt gcg ctc cca gat gcc ttg caa ccg gcg cct 1296 Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala Pro             420 425 430 caa gct ggt gct tgg ttc caa gcc tac ttt gtg cag ctt ctc aca aac 1344 Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr Asn         435 440 445 gca aac cca tcg ttc ctg tag 1365 Ala Asn Pro Ser Phe Leu     450 <210> 82 <211> 454 <212> PRT <213> Trichoderma reesei <400> 82 Met Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly Gln 1 5 10 15 Cys Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala Ser Gly Ser             20 25 30 Thr Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly Ala         35 40 45 Ala Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg Val     50 55 60 Ser Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly Ser 65 70 75 80 Thr Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr Ser                 85 90 95 Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr Ala             100 105 110 Ser Glu Val Ser Ser Le Le Ala Ile Pro Ser Leu Thr Gly Ala Met Ala         115 120 125 Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu Asp     130 135 140 Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile Arg 145 150 155 160 Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val Tyr                 165 170 175 Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu Tyr             180 185 190 Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp Thr         195 200 205 Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu Val     210 215 220 Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr Pro 225 230 235 240 Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr Ala                 245 250 255 Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly             260 265 270 His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala Gln         275 280 285 Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu Arg     290 295 300 Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr Ser 305 310 315 320 Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu Tyr                 325 330 335 Ile His Ala Ile Gly Pro Leu Leu Ala Asn His Gly Trp Ser Asn Ala             340 345 350 Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln         355 360 365 Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly Ile     370 375 380 Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val Trp 385 390 395 400 Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser Ala Pro                 405 410 415 Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala Pro             420 425 430 Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr Asn         435 440 445 Ala Asn Pro Ser Phe Leu     450 <210> 83 <211> 1317 <212> DNA <213> Trichoderma reesei <220> <221> CDS (222) (1) .. (1317) <223> Trichoderma reesei endoglucanase I <400> 83 atg cag caa ccg gga acc agc acc ccc gag gtc cat ccc aag ttg aca 48 Met Gln Gln Pro Gly Thr Ser Thr Pro Glu Val His Pro Lys Leu Thr 1 5 10 15 acc tac aag tgc aca aag tcc ggg ggg tgc gtg gcc cag gac acc tcg 96 Thr Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val Ala Gln Asp Thr Ser             20 25 30 gtg gtc ctt gac tgg aac tac cgc tgg atg cac gac gca aac tac aac 144 Val Val Leu Asp Trp Asn Tyr Arg Trp Met His Asp Ala Asn Tyr Asn         35 40 45 tcg tgc acc gtc aac ggc ggc gtc aac acc acg ctc tgc cct gac gag 192 Ser Cys Thr Val Asn Gly Gly Val Asn Thr Thr Leu Cys Pro Asp Glu     50 55 60 gcg acc tgt ggc aag aac tgc ttc atc gag ggc gtc gac tac gcc gcc 240 Ala Thr Cys Gly Lys Asn Cys Phe Ile Glu Gly Val Asp Tyr Ala Ala 65 70 75 80 tcg ggc gtc acg acc tcg ggc agc agc ctc acc atg aac cag tac atg 288 Ser Gly Val Thr Thr Ser Gly Ser Ser Leu Thr Met Asn Gln Tyr Met                 85 90 95 ccc agc agc tct ggc ggc tac agc agc gtc tct cct cgg ctg tat ctc 336 Pro Ser Ser Ser Gly Gly Tyr Ser Ser Val Ser Pro Arg Leu Tyr Leu             100 105 110 ctg gac tct gac ggt gag tac gtg atg ctg aag ctc aac ggc cag gag 384 Leu Asp Ser Asp Gly Glu Tyr Val Met Leu Lys Leu Asn Gly Gln Glu         115 120 125 ctg agc ttc gac gtc gac ctc tct gct ctg ccg tgt gga gag aac ggc 432 Leu Ser Phe Asp Val Asp Leu Ser Ala Leu Pro Cys Gly Glu Asn Gly     130 135 140 tcg ctc tac ctg tct cag atg gac gag aac ggg ggc gcc aac cag tat 480 Ser Leu Tyr Leu Ser Gln Met Asp Glu Asn Gly Gly Ala Asn Gln Tyr 145 150 155 160 aac acg gcc ggt gcc aac tac ggg agc ggc tac tgc gat gct cag tgc 528 Asn Thr Ala Gly Ala Asn Tyr Gly Ser Gly Tyr Cys Asp Ala Gln Cys                 165 170 175 ccc gtc cag aca tgg agg aac ggc acc ctc aac act agc cac cag ggc 576 Pro Val Gln Thr Trp Arg Asn Gly Thr Leu Asn Thr Ser His Gln Gly             180 185 190 ttc tgc tgc aac gag atg gat atc ctg gag ggc aac tcg agg gcg aat 624 Phe Cys Cys Asn Glu Met Asp Ile Leu Glu Gly Asn Ser Arg Ala Asn         195 200 205 gcc ttg acc cct cac tct tgc acg gcc acg gcc tgc gac tct gcc ggt 672 Ala Leu Thr Pro His Ser Cys Thr Ala Thr Ala Cys Asp Ser Ala Gly     210 215 220 tgc ggc ttc aac ccc tat ggc agc ggc tac aaa agc tac tac ggc ccc 720 Cys Gly Phe Asn Pro Tyr Gly Ser Gly Tyr Lys Ser Tyr Tyr Gly Pro 225 230 235 240 gga gat acc gtt gac acc tcc aag acc ttc acc atc atc acc cag ttc 768 Gly Asp Thr Val Asp Thr Ser Lys Thr Phe Thr Ile Ile Thr Gln Phe                 245 250 255 aac acg gac aac ggc tcg ccc tcg ggc aac ctt gtg agc atc acc cgc 816 Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu Val Ser Ile Thr Arg             260 265 270 aag tac cag caa aac ggc gtc gac atc ccc agc gcc cag ccc ggc ggc 864 Lys Tyr Gln Gln Asn Gly Val Asp Ile Pro Ser Ala Gln Pro Gly Gly         275 280 285 gac acc atc tcg tcc tgc ccg tcc gcc tca gcc tac ggc ggc ctc gcc 912 Asp Thr Ile Ser Ser Cys Pro Ser Ala Ser Ala Tyr Gly Gly Leu Ala     290 295 300 acc atg ggc aag gcc ctg agc agc ggc atg gtg ctc gtg ttc agc att 960 Thr Met Gly Lys Ala Leu Ser Ser Gly Met Val Leu Val Phe Ser Ile 305 310 315 320 tgg aac gac aac agc cag tac atg aac tgg ctc gac agc ggc aac gcc 1008 Trp Asn Asp Asn Ser Gln Tyr Met Asn Trp Leu Asp Ser Gly Asn Ala                 325 330 335 ggc ccc tgc agc agc acc gag ggc aac cca tcc aac acc ctg gcc aac 1056 Gly Pro Cys Ser Ser Thr Glu Gly Asn Pro Ser Asn Thr Leu Ala Asn             340 345 350 aac ccc aac acg cac gtc gtc ttc tcc aac atc cgc tgg gga gac att 1104 Asn Pro Asn Thr His Val Val Phe Ser Asn Ile Arg Trp Gly Asp Ile         355 360 365 ggg tct act acg aac tcg act gcg ccc ccg ccc ccg cct gcg tcc agc 1152 Gly Ser Thr Thr Asn Ser Thr Ala Pro Pro Pro Pro Ala Ser Ser     370 375 380 acg acg ttt tcg act aca cgg agg agc tcg acg act tcg agc agc ccg 1200 Thr Thr Phe Ser Thr Thr Arg Arg Ser Ser Thr Thr Ser Ser Ser Pro 385 390 395 400 agc tgc acg cag act cac tgg ggg cag tgc ggt ggc att ggg tac agc 1248 Ser Cys Thr Gln Thr His Trp Gly Gln Cys Gly Gly Ile Gly Tyr Ser                 405 410 415 ggg tgc aag acg tgc acg tcg ggc act acg tgc cag tat agc aac gac 1296 Gly Cys Lys Thr Cys Thr Ser Gly Thr Thr Cys Gln Tyr Ser Asn Asp             420 425 430 tac tac tcg caa tgc ctt tag 1317 Tyr Tyr Ser Gln Cys Leu         435 <210> 84 <211> 438 <212> PRT <213> Trichoderma reesei <400> 84 Met Gln Gln Pro Gly Thr Ser Thr Pro Glu Val His Pro Lys Leu Thr 1 5 10 15 Thr Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val Ala Gln Asp Thr Ser             20 25 30 Val Val Leu Asp Trp Asn Tyr Arg Trp Met His Asp Ala Asn Tyr Asn         35 40 45 Ser Cys Thr Val Asn Gly Gly Val Asn Thr Thr Leu Cys Pro Asp Glu     50 55 60 Ala Thr Cys Gly Lys Asn Cys Phe Ile Glu Gly Val Asp Tyr Ala Ala 65 70 75 80 Ser Gly Val Thr Thr Ser Gly Ser Ser Leu Thr Met Asn Gln Tyr Met                 85 90 95 Pro Ser Ser Ser Gly Gly Tyr Ser Ser Val Ser Pro Arg Leu Tyr Leu             100 105 110 Leu Asp Ser Asp Gly Glu Tyr Val Met Leu Lys Leu Asn Gly Gln Glu         115 120 125 Leu Ser Phe Asp Val Asp Leu Ser Ala Leu Pro Cys Gly Glu Asn Gly     130 135 140 Ser Leu Tyr Leu Ser Gln Met Asp Glu Asn Gly Gly Ala Asn Gln Tyr 145 150 155 160 Asn Thr Ala Gly Ala Asn Tyr Gly Ser Gly Tyr Cys Asp Ala Gln Cys                 165 170 175 Pro Val Gln Thr Trp Arg Asn Gly Thr Leu Asn Thr Ser His Gln Gly             180 185 190 Phe Cys Cys Asn Glu Met Asp Ile Leu Glu Gly Asn Ser Arg Ala Asn         195 200 205 Ala Leu Thr Pro His Ser Cys Thr Ala Thr Ala Cys Asp Ser Ala Gly     210 215 220 Cys Gly Phe Asn Pro Tyr Gly Ser Gly Tyr Lys Ser Tyr Tyr Gly Pro 225 230 235 240 Gly Asp Thr Val Asp Thr Ser Lys Thr Phe Thr Ile Ile Thr Gln Phe                 245 250 255 Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu Val Ser Ile Thr Arg             260 265 270 Lys Tyr Gln Gln Asn Gly Val Asp Ile Pro Ser Ala Gln Pro Gly Gly         275 280 285 Asp Thr Ile Ser Ser Cys Pro Ser Ala Ser Ala Tyr Gly Gly Leu Ala     290 295 300 Thr Met Gly Lys Ala Leu Ser Ser Gly Met Val Leu Val Phe Ser Ile 305 310 315 320 Trp Asn Asp Asn Ser Gln Tyr Met Asn Trp Leu Asp Ser Gly Asn Ala                 325 330 335 Gly Pro Cys Ser Ser Thr Glu Gly Asn Pro Ser Asn Thr Leu Ala Asn             340 345 350 Asn Pro Asn Thr His Val Val Phe Ser Asn Ile Arg Trp Gly Asp Ile         355 360 365 Gly Ser Thr Thr Asn Ser Thr Ala Pro Pro Pro Pro Ala Ser Ser     370 375 380 Thr Thr Phe Ser Thr Thr Arg Arg Ser Ser Thr Thr Ser Ser Ser Pro 385 390 395 400 Ser Cys Thr Gln Thr His Trp Gly Gln Cys Gly Gly Ile Gly Tyr Ser                 405 410 415 Gly Cys Lys Thr Cys Thr Ser Gly Thr Thr Cys Gln Tyr Ser Asn Asp             420 425 430 Tyr Tyr Ser Gln Cys Leu         435 <210> 85 <211> 954 <212> DNA <213> Artificial Sequence <220> <223> 6GP1 <220> <221> CDS (222) (1) .. (954) <223> 6GP1 <400> 85 atg ggc gtg gac ccg ttc gag cgc aac aag atc ctc ggc cgc ggc atc 48 Met Gly Val Asp Pro Phe Glu Arg Asn Lys Ile Leu Gly Arg Gly Ile 1 5 10 15 aac atc ggc aac gcc ctg gag gcc ccg aac gag ggc gac tgg ggc gtg 96 Asn Ile Gly Asn Ala Leu Glu Ala Pro Asn Glu Gly Asp Trp Gly Val             20 25 30 gtg atc aag gac gag ttc ttc gac atc atc aag gag gcc ggc ttc tcc 144 Val Ile Lys Asp Glu Phe Phe Asp Ile Ile Lys Glu Ala Gly Phe Ser         35 40 45 cac gtg cgc atc ccg atc cgc tgg tcc acc cac gcc tac gcc ttc ccg 192 His Val Arg Ile Pro Ile Arg Trp Ser Thr His Ala Tyr Ala Phe Pro     50 55 60 ccg tac aag atc atg gac cgc ttc ttc aag cgc gtg gac gag gtg atc 240 Pro Tyr Lys Ile Met Asp Arg Phe Phe Lys Arg Val Asp Glu Val Ile 65 70 75 80 aac ggc gcc ctc aag cgc ggc ctc gcc gtg gcc atc aac atc cac cac 288 Asn Gly Ala Leu Lys Arg Gly Leu Ala Val Ala Ile Asn Ile His His                 85 90 95 tac gag gag ctc atg aac gac ccg gag gag cac aag gag cgc ttc ctc 336 Tyr Glu Glu Leu Met Asn Asp Pro Glu Glu His Lys Glu Arg Phe Leu             100 105 110 gcc ctc tgg aag cag atc gcc gac cgc tac aag gac tac ccg gag acc 384 Ala Leu Trp Lys Gln Ile Ala Asp Arg Tyr Lys Asp Tyr Pro Glu Thr         115 120 125 ctc ttc ttc gag atc ctc aac gag ccg cac ggc aac ctc acc ccg gag 432 Leu Phe Phe Glu Ile Leu Asn Glu Pro His Gly Asn Leu Thr Pro Glu     130 135 140 aag tgg aac gag ctg ctc gag gag gcc ctc aag gtg atc cgc tcc atc 480 Lys Trp Asn Glu Leu Leu Glu Glu Ala Leu Lys Val Ile Arg Ser Ile 145 150 155 160 gac aag aag cac acc atc atc att ggc acc gca gag tgg gga ggc atc 528 Asp Lys Lys His Thr Ile Ile Ile Gly Thr Ala Glu Trp Gly Gly Ile                 165 170 175 tcc gcc ctc gag aag ctc tcc gtg ccg aag tgg gag aag aat tcc atc 576 Ser Ala Leu Glu Lys Leu Ser Val Pro Lys Trp Glu Lys Asn Ser Ile             180 185 190 gtg acc atc cac tac tac aac ccg ttc gag ttc acg cac cag ggc gcc 624 Val Thr Ile His Tyr Tyr Asn Pro Phe Glu Phe Thr His Gln Gly Ala         195 200 205 gag tgg gtg gag ggc tcc gag aag tgg ctt ggc cgc aag tgg ggc tcc 672 Glu Trp Val Glu Gly Ser Glu Lys Trp Leu Gly Arg Lys Trp Gly Ser     210 215 220 ccg gac gac cag aag cac ctc atc gag gag ttc aac ttc atc gag gag 720 Pro Asp Asp Gln Lys His Leu Ile Glu Glu Phe Asn Phe Ile Glu Glu 225 230 235 240 tgg tcc aag aag aac aag cgc ccg atc tac atc ggc gag ttt ggc gcc 768 Trp Ser Lys Lys Asn Lys Arg Pro Ile Tyr Ile Gly Glu Phe Gly Ala                 245 250 255 tac cgc aag gcc gac ctc gag tcc cgc atc aag tgg acc tcc ttc gtg 816 Tyr Arg Lys Ala Asp Leu Glu Ser Arg Ile Lys Trp Thr Ser Phe Val             260 265 270 gtg cgt gag atg gag aag cgc cgc tgg tcc tgg gcc tac tgg gag ttc 864 Val Arg Glu Met Glu Lys Arg Arg Trp Ser Trp Ala Tyr Trp Glu Phe         275 280 285 tgc tcc ggc ttc ggc gtg tac gac acc ctc cgc aag acc tgg aac aag 912 Cys Ser Gly Phe Gly Val Tyr Asp Thr Leu Arg Lys Thr Trp Asn Lys     290 295 300 gac ctc ctc gag gcc ctc atc ggc ggc gac tcc atc gag tag 954 Asp Leu Leu Glu Ala Leu Ile Gly Gly Asp Ser Ile Glu 305 310 315 <210> 86 <211> 317 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 86 Met Gly Val Asp Pro Phe Glu Arg Asn Lys Ile Leu Gly Arg Gly Ile 1 5 10 15 Asn Ile Gly Asn Ala Leu Glu Ala Pro Asn Glu Gly Asp Trp Gly Val             20 25 30 Val Ile Lys Asp Glu Phe Phe Asp Ile Ile Lys Glu Ala Gly Phe Ser         35 40 45 His Val Arg Ile Pro Ile Arg Trp Ser Thr His Ala Tyr Ala Phe Pro     50 55 60 Pro Tyr Lys Ile Met Asp Arg Phe Phe Lys Arg Val Asp Glu Val Ile 65 70 75 80 Asn Gly Ala Leu Lys Arg Gly Leu Ala Val Ala Ile Asn Ile His His                 85 90 95 Tyr Glu Glu Leu Met Asn Asp Pro Glu Glu His Lys Glu Arg Phe Leu             100 105 110 Ala Leu Trp Lys Gln Ile Ala Asp Arg Tyr Lys Asp Tyr Pro Glu Thr         115 120 125 Leu Phe Phe Glu Ile Leu Asn Glu Pro His Gly Asn Leu Thr Pro Glu     130 135 140 Lys Trp Asn Glu Leu Leu Glu Glu Ala Leu Lys Val Ile Arg Ser Ile 145 150 155 160 Asp Lys Lys His Thr Ile Ile Ile Gly Thr Ala Glu Trp Gly Gly Ile                 165 170 175 Ser Ala Leu Glu Lys Leu Ser Val Pro Lys Trp Glu Lys Asn Ser Ile             180 185 190 Val Thr Ile His Tyr Tyr Asn Pro Phe Glu Phe Thr His Gln Gly Ala         195 200 205 Glu Trp Val Glu Gly Ser Glu Lys Trp Leu Gly Arg Lys Trp Gly Ser     210 215 220 Pro Asp Asp Gln Lys His Leu Ile Glu Glu Phe Asn Phe Ile Glu Glu 225 230 235 240 Trp Ser Lys Lys Asn Lys Arg Pro Ile Tyr Ile Gly Glu Phe Gly Ala                 245 250 255 Tyr Arg Lys Ala Asp Leu Glu Ser Arg Ile Lys Trp Thr Ser Phe Val             260 265 270 Val Arg Glu Met Glu Lys Arg Arg Trp Ser Trp Ala Tyr Trp Glu Phe         275 280 285 Cys Ser Gly Phe Gly Val Tyr Asp Thr Leu Arg Lys Thr Trp Asn Lys     290 295 300 Asp Leu Leu Glu Ala Leu Ile Gly Gly Asp Ser Ile Glu 305 310 315 <210> 87 <211> 1248 <212> DNA Hordeum vulgare <220> <221> CDS (222) (1) .. (1248) Barley Amy I amylase <400> 87 atg gca cac caa gtc ctc ttt cag ggg ttc aac tgg gag tcg tgg aag 48 Met Ala His Gln Val Leu Phe Gln Gly Phe Asn Trp Glu Ser Trp Lys 1 5 10 15 cag agc ggc ggg tgg tac aac atg atg atg ggc aag gtc gac gac atc 96 Gln Ser Gly Gly Trp Tyr Asn Met Met Met Gly Lys Val Asp Asp Ile             20 25 30 gcc gct gcc gga gtc acc cac gtc tgg ctg cca ccg ccg tcg cac tcc 144 Ala Ala Ala Gly Val Thr His Val Trp Leu Pro Pro Pro Ser His Ser         35 40 45 gtc tcc aac gaa ggt tac atg cct ggt cgg ctg tac gac atc gac gcg 192 Val Ser Asn Glu Gly Tyr Met Pro Gly Arg Leu Tyr Asp Ile Asp Ala     50 55 60 tcc aag tac ggc aac gcg gcg gag ctc aag tcg ctc atc ggc gcg ctc 240 Ser Lys Tyr Gly Asn Ala Ala Glu Leu Lys Ser Leu Ile Gly Ala Leu 65 70 75 80 cac ggc aag ggc gtg cag gcc atc gcc gac atc gtc atc aac cac cgc 288 His Gly Lys Gly Val Gln Ala Ile Ala Asp Ile Val Ile Asn His Arg                 85 90 95 tgc gcc gac tac aag gat agc cgc ggc atc tac tgc atc ttc gag ggc 336 Cys Ala Asp Tyr Lys Asp Ser Arg Gly Ile Tyr Cys Ile Phe Glu Gly             100 105 110 ggc acc tcc gac ggc cgc ctc gac tgg ggc ccc cac atg atc tgt cgc 384 Gly Thr Ser Asp Gly Arg Leu Asp Trp Gly Pro His Met Ile Cys Arg         115 120 125 gac gac acc aaa tac tcc gat ggc acc gca aac ctc gac acc gga gcc 432 Asp Asp Thr Lys Tyr Ser Asp Gly Thr Ala Asn Leu Asp Thr Gly Ala     130 135 140 gac ttc gcc gcc gcg ccc gac atc gac cac ctc aac gac cgg gtc cag 480 Asp Phe Ala Ala Ala Pro Asp Ile Asp His Leu Asn Asp Arg Val Gln 145 150 155 160 cgc gag ctc aag gag tgg ctc ctc tgg ctc aag ag agc gac ctc ggc ttc 528 Arg Glu Leu Lys Glu Trp Leu Leu Trp Leu Lys Ser Asp Leu Gly Phe                 165 170 175 gac gcg tgg cgc ctt gac ttc gcc agg ggc tac tcg ccg gag atg gcc 576 Asp Ala Trp Arg Leu Asp Phe Ala Arg Gly Tyr Ser Pro Glu Met Ala             180 185 190 aag gtg tac atc gac ggc aca tcc ccg agc ctc gcc gtg gcc gag gtg 624 Lys Val Tyr Ile Asp Gly Thr Ser Pro Ser Leu Ala Val Ala Glu Val         195 200 205 tgg gac aat atg gcc acc ggc ggc gac ggc aag ccc aac tac gac cag 672 Trp Asp Asn Met Ala Thr Gly Gly Asp Gly Lys Pro Asn Tyr Asp Gln     210 215 220 gac gcg cac cgg cag aat ctg gtg aac tgg gtg gac aag gtg ggc ggc 720 Asp Ala His Arg Gln Asn Leu Val Asn Trp Val Asp Lys Val Gly Gly 225 230 235 240 gcg gcc tcg gca ggc atg gtg ttc gac ttc acg acc aaa ggg ata ctg 768 Ala Ala Ser Ala Gly Met Val Phe Asp Phe Thr Thr Lys Gly Ile Leu                 245 250 255 aac gct gcc gtg gag ggc gag ctg tgg agg ctg atc gac ccg cag ggg 816 Asn Ala Ala Val Glu Gly Glu Leu Trp Arg Leu Ile Asp Pro Gln Gly             260 265 270 aag gcc ccc ggc gtg atg gga tgg tgg ccg gcc aag gcc gtc acc ttc 864 Lys Ala Pro Gly Val Met Gly Trp Trp Pro Ala Lys Ala Val Thr Phe         275 280 285 gtc gac aac cac gat aca ggc tcc acg cag gcc atg tgg cca ttc ccc 912 Val Asp Asn His Asp Thr Gly Ser Thr Gln Ala Met Trp Pro Phe Pro     290 295 300 tcc gac aag gtc atg cag ggc tac gcg tac atc ctc acc cac ccc ggc 960 Ser Asp Lys Val Met Gln Gly Tyr Ala Tyr Ile Leu Thr His Pro Gly 305 310 315 320 atc cca tgc atc ttc tac gac cat ttc ttc aac tgg ggg ttt aag gac 1008 Ile Pro Cys Ile Phe Tyr Asp His Phe Phe Asn Trp Gly Phe Lys Asp                 325 330 335 cag atc gcg gcg ctg gtg gcg atc agg aag cgc aac ggc atc acg gcg 1056 Gln Ile Ala Ala Leu Val Ala Ile Arg Lys Arg Asn Gly Ile Thr Ala             340 345 350 acg agc gct ctg aag atc ctc atg cac gaa gga gat gcc tac gtc gcc 1104 Thr Ser Ala Leu Lys Ile Leu Met His Glu Gly Asp Ala Tyr Val Ala         355 360 365 gag ata gac ggc aag gtg gtg gtg aag atc ggg tcc agg tac gac gtc 1152 Glu Ile Asp Gly Lys Val Val Val Lys Ile Gly Ser Arg Tyr Asp Val     370 375 380 ggg gcg gtg atc ccg gcc ggg ttc gtg acc tcg gca cac ggc aac gac 1200 Gly Ala Val Ile Pro Ala Gly Phe Val Thr Ser Ala His Gly Asn Asp 385 390 395 400 tac gcc gtc tgg gag aag aac ggt gcc gcg gca aca cta caa cgg agc 1248 Tyr Ala Val Trp Glu Lys Asn Gly Ala Ala Ala Thr Leu Gln Arg Ser                 405 410 415 <210> 88 <211> 416 <212> PRT Hordeum vulgare <400> 88 Met Ala His Gln Val Leu Phe Gln Gly Phe Asn Trp Glu Ser Trp Lys 1 5 10 15 Gln Ser Gly Gly Trp Tyr Asn Met Met Met Gly Lys Val Asp Asp Ile             20 25 30 Ala Ala Ala Gly Val Thr His Val Trp Leu Pro Pro Pro Ser His Ser         35 40 45 Val Ser Asn Glu Gly Tyr Met Pro Gly Arg Leu Tyr Asp Ile Asp Ala     50 55 60 Ser Lys Tyr Gly Asn Ala Ala Glu Leu Lys Ser Leu Ile Gly Ala Leu 65 70 75 80 His Gly Lys Gly Val Gln Ala Ile Ala Asp Ile Val Ile Asn His Arg                 85 90 95 Cys Ala Asp Tyr Lys Asp Ser Arg Gly Ile Tyr Cys Ile Phe Glu Gly             100 105 110 Gly Thr Ser Asp Gly Arg Leu Asp Trp Gly Pro His Met Ile Cys Arg         115 120 125 Asp Asp Thr Lys Tyr Ser Asp Gly Thr Ala Asn Leu Asp Thr Gly Ala     130 135 140 Asp Phe Ala Ala Ala Pro Asp Ile Asp His Leu Asn Asp Arg Val Gln 145 150 155 160 Arg Glu Leu Lys Glu Trp Leu Leu Trp Leu Lys Ser Asp Leu Gly Phe                 165 170 175 Asp Ala Trp Arg Leu Asp Phe Ala Arg Gly Tyr Ser Pro Glu Met Ala             180 185 190 Lys Val Tyr Ile Asp Gly Thr Ser Pro Ser Leu Ala Val Ala Glu Val         195 200 205 Trp Asp Asn Met Ala Thr Gly Gly Asp Gly Lys Pro Asn Tyr Asp Gln     210 215 220 Asp Ala His Arg Gln Asn Leu Val Asn Trp Val Asp Lys Val Gly Gly 225 230 235 240 Ala Ala Ser Ala Gly Met Val Phe Asp Phe Thr Thr Lys Gly Ile Leu                 245 250 255 Asn Ala Ala Val Glu Gly Glu Leu Trp Arg Leu Ile Asp Pro Gln Gly             260 265 270 Lys Ala Pro Gly Val Met Gly Trp Trp Pro Ala Lys Ala Val Thr Phe         275 280 285 Val Asp Asn His Asp Thr Gly Ser Thr Gln Ala Met Trp Pro Phe Pro     290 295 300 Ser Asp Lys Val Met Gln Gly Tyr Ala Tyr Ile Leu Thr His Pro Gly 305 310 315 320 Ile Pro Cys Ile Phe Tyr Asp His Phe Phe Asn Trp Gly Phe Lys Asp                 325 330 335 Gln Ile Ala Ala Leu Val Ala Ile Arg Lys Arg Asn Gly Ile Thr Ala             340 345 350 Thr Ser Ala Leu Lys Ile Leu Met His Glu Gly Asp Ala Tyr Val Ala         355 360 365 Glu Ile Asp Gly Lys Val Val Val Lys Ile Gly Ser Arg Tyr Asp Val     370 375 380 Gly Ala Val Ile Pro Ala Gly Phe Val Thr Ser Ala His Gly Asn Asp 385 390 395 400 Tyr Ala Val Trp Glu Lys Asn Gly Ala Ala Ala Thr Leu Gln Arg Ser                 405 410 415 <210> 89 <211> 1401 <212> DNA <213> Artificial Sequence <220> <223> Trichoderma reesei ß-Glucosidase 2 <220> <221> CDS (222) (1) .. (1401) <223> Trichoderma reesei ß-Glucosidase 2 <400> 89 atg ttg ccc aag gac ttt cag tgg ggg ttc gcc acg gct gcc tac cag 48 Met Leu Pro Lys Asp Phe Gln Trp Gly Phe Ala Thr Ala Ala Tyr Gln 1 5 10 15 atc gag ggc gcc gtc gac cag gac ggc cgc ggc ccc agc atc tgg gac 96 Ile Glu Gly Ala Val Asp Gln Asp Gly Arg Gly Pro Ser Ile Trp Asp             20 25 30 acg ttc tgc gcg cag ccc ggc aag atc gcc gac ggc tcg tcg ggc gtg 144 Thr Phe Cys Ala Gln Pro Gly Lys Ile Ala Asp Gly Ser Ser Gly Val         35 40 45 acg gcg tgc gac tcg tac aac cgc acg gcc gag gac att gcg ctg ctg 192 Thr Ala Cys Asp Ser Tyr Asn Arg Thr Ala Glu Asp Ile Ala Leu Leu     50 55 60 aag tcg ctc ggg gcc aag agc tac cgc ttc tcc atc tcg tgg tcg cgc 240 Lys Ser Leu Gly Ala Lys Ser Tyr Arg Phe Ser Ile Ser Trp Ser Arg 65 70 75 80 atc atc ccc gag ggc ggc cgc ggc gat gcc gtc aac cag gcg ggc atc 288 Ile Ile Pro Glu Gly Gly Arg Gly Asp Ala Val Asn Gln Ala Gly Ile                 85 90 95 gac cac tac gtc aag ttc gtc gac gac ctg ctc gac gcc ggc atc acg 336 Asp His Tyr Val Lys Phe Val Asp Asp Leu Leu Asp Ala Gly Ile Thr             100 105 110 ccc ttc atc acc ctc ttc cac tgg gac ctg ccc gag ggc ctg cat cag 384 Pro Phe Ile Thr Leu Phe His Trp Asp Leu Pro Glu Gly Leu His Gln         115 120 125 cgg tac ggg ggg ctg ctg aac cgc acc gag ttc ccg ctc gac ttt gaa 432 Arg Tyr Gly Gly Leu Leu Asn Arg Thr Glu Phe Pro Leu Asp Phe Glu     130 135 140 aac tac gcc cgc gtc atg ttc agg gcg ctg ccc aag gtg cgc aac tgg 480 Asn Tyr Ala Arg Val Met Phe Arg Ala Leu Pro Lys Val Arg Asn Trp 145 150 155 160 atc acc ttc aac gag ccg ctg tgc tcg gcc atc ccg ggc tac ggc tcc 528 Ile Thr Phe Asn Glu Pro Leu Cys Ser Ala Ile Pro Gly Tyr Gly Ser                 165 170 175 ggc acc ttc gcc ccc ggc cgg cag agc acc tcg gag ccg tgg acc gtc 576 Gly Thr Phe Ala Pro Gly Arg Gln Ser Thr Ser Glu Pro Trp Thr Val             180 185 190 ggc cac aac atc ctc gtc gcc cac ggc cgc gcc gtc aag gcg tac cgc 624 Gly His Asn Ile Leu Val Ala His Gly Arg Ala Val Lys Ala Tyr Arg         195 200 205 gac gac ttc aag ccc gcc agc ggc gac ggc cag atc ggc atc gtc ctc 672 Asp Asp Phe Lys Pro Ala Ser Gly Asp Gly Gln Ile Gly Ile Val Leu     210 215 220 aac ggc gac ttc acc tac ccc tgg gac gcc gcc gac ccg gcc gac aag 720 Asn Gly Asp Phe Thr Tyr Pro Trp Asp Ala Ala Asp Pro Ala Asp Lys 225 230 235 240 gag gcg gcc gag cgg cgc ctc gag ttc ttc acg gcc tgg ttc gcg gac 768 Glu Ala Ala Glu Arg Arg Leu Glu Phe Phe Thr Ala Trp Phe Ala Asp                 245 250 255 ccc atc tac ttg ggc gac tac ccg gcg tcg atg cgc aag cag ctg ggc 816 Pro Ile Tyr Leu Gly Asp Tyr Pro Ala Ser Met Arg Lys Gln Leu Gly             260 265 270 gac cgg ctg ccg acc ttt acg ccc gag gag cgc gcc ctc gtc cac ggc 864 Asp Arg Leu Pro Thr Phe Thr Pro Glu Glu Arg Ala Leu Val His Gly         275 280 285 tcc aac gac ttt tac ggc atg aac cac tac acg tcc aac tac atc cgc 912 Ser Asn Asp Phe Tyr Gly Met Asn His Tyr Thr Ser Asn Tyr Ile Arg     290 295 300 cac cgc agc tcg ccc gcc tcc gcc gac gac acc gtc ggc aac gtc gac 960 His Arg Ser Ser Pro Ala Ser Ala Asp Asp Thr Val Gly Asn Val Asp 305 310 315 320 gtg ctc ttc acc aac aag cag ggc aac tgc atc ggc ccc gag acg cag 1008 Val Leu Phe Thr Asn Lys Gln Gly Asn Cys Ile Gly Pro Glu Thr Gln                 325 330 335 tcc ccc tgg ctg cgc ccc tgt gcc gcc ggc ttc cgc gac ttc ctg gtg 1056 Ser Pro Trp Leu Arg Pro Cys Ala Ala Gly Phe Arg Asp Phe Leu Val             340 345 350 tgg atc agc aag agg tac ggc tac ccg ccc atc tac gtg acg gag aac 1104 Trp Ile Ser Lys Arg Tyr Gly Tyr Pro Pro Ile Tyr Val Thr Glu Asn         355 360 365 ggc acg agc atc aag ggc gag agc gac ttg ccc aag gag aag att ctc 1152 Gly Thr Ser Ile Lys Gly Glu Ser Asp Leu Pro Lys Glu Lys Ile Leu     370 375 380 gaa gat gac ttc agg gtc aag tac tat aac gag tac atc cgt gcc atg 1200 Glu Asp Asp Phe Arg Val Lys Tyr Tyr Asn Glu Tyr Ile Arg Ala Met 385 390 395 400 gtt acc gcc gtg gag ctg gac ggg gtc aac gtc aag ggg tac ttt gcc 1248 Val Thr Ala Val Glu Leu Asp Gly Val Asn Val Lys Gly Tyr Phe Ala                 405 410 415 tgg tcg ctc atg gac aac ttt gag tgg gcg gac ggc tac gtg acg agg 1296 Trp Ser Leu Met Asp Asn Phe Glu Trp Ala Asp Gly Tyr Val Thr Arg             420 425 430 ttt ggg gtt acg tat gtg gat tat gag aat ggg cag aag cgg ttc ccc 1344 Phe Gly Val Thr Tyr Val Asp Tyr Glu Asn Gly Gln Lys Arg Phe Pro         435 440 445 aag aag agc gca aag agc ttg aag ccg ctg ttt gac gag ctg att gcg 1392 Lys Lys Ser Ala Lys Ser Leu Lys Pro Leu Phe Asp Glu Leu Ile Ala     450 455 460 gcg gcg tga 1401 Ala Ala 465 <210> 90 <211> 466 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 90 Met Leu Pro Lys Asp Phe Gln Trp Gly Phe Ala Thr Ala Ala Tyr Gln 1 5 10 15 Ile Glu Gly Ala Val Asp Gln Asp Gly Arg Gly Pro Ser Ile Trp Asp             20 25 30 Thr Phe Cys Ala Gln Pro Gly Lys Ile Ala Asp Gly Ser Ser Gly Val         35 40 45 Thr Ala Cys Asp Ser Tyr Asn Arg Thr Ala Glu Asp Ile Ala Leu Leu     50 55 60 Lys Ser Leu Gly Ala Lys Ser Tyr Arg Phe Ser Ile Ser Trp Ser Arg 65 70 75 80 Ile Ile Pro Glu Gly Gly Arg Gly Asp Ala Val Asn Gln Ala Gly Ile                 85 90 95 Asp His Tyr Val Lys Phe Val Asp Asp Leu Leu Asp Ala Gly Ile Thr             100 105 110 Pro Phe Ile Thr Leu Phe His Trp Asp Leu Pro Glu Gly Leu His Gln         115 120 125 Arg Tyr Gly Gly Leu Leu Asn Arg Thr Glu Phe Pro Leu Asp Phe Glu     130 135 140 Asn Tyr Ala Arg Val Met Phe Arg Ala Leu Pro Lys Val Arg Asn Trp 145 150 155 160 Ile Thr Phe Asn Glu Pro Leu Cys Ser Ala Ile Pro Gly Tyr Gly Ser                 165 170 175 Gly Thr Phe Ala Pro Gly Arg Gln Ser Thr Ser Glu Pro Trp Thr Val             180 185 190 Gly His Asn Ile Leu Val Ala His Gly Arg Ala Val Lys Ala Tyr Arg         195 200 205 Asp Asp Phe Lys Pro Ala Ser Gly Asp Gly Gln Ile Gly Ile Val Leu     210 215 220 Asn Gly Asp Phe Thr Tyr Pro Trp Asp Ala Ala Asp Pro Ala Asp Lys 225 230 235 240 Glu Ala Ala Glu Arg Arg Leu Glu Phe Phe Thr Ala Trp Phe Ala Asp                 245 250 255 Pro Ile Tyr Leu Gly Asp Tyr Pro Ala Ser Met Arg Lys Gln Leu Gly             260 265 270 Asp Arg Leu Pro Thr Phe Thr Pro Glu Glu Arg Ala Leu Val His Gly         275 280 285 Ser Asn Asp Phe Tyr Gly Met Asn His Tyr Thr Ser Asn Tyr Ile Arg     290 295 300 His Arg Ser Ser Pro Ala Ser Ala Asp Asp Thr Val Gly Asn Val Asp 305 310 315 320 Val Leu Phe Thr Asn Lys Gln Gly Asn Cys Ile Gly Pro Glu Thr Gln                 325 330 335 Ser Pro Trp Leu Arg Pro Cys Ala Ala Gly Phe Arg Asp Phe Leu Val             340 345 350 Trp Ile Ser Lys Arg Tyr Gly Tyr Pro Pro Ile Tyr Val Thr Glu Asn         355 360 365 Gly Thr Ser Ile Lys Gly Glu Ser Asp Leu Pro Lys Glu Lys Ile Leu     370 375 380 Glu Asp Asp Phe Arg Val Lys Tyr Tyr Asn Glu Tyr Ile Arg Ala Met 385 390 395 400 Val Thr Ala Val Glu Leu Asp Gly Val Asn Val Lys Gly Tyr Phe Ala                 405 410 415 Trp Ser Leu Met Asp Asn Phe Glu Trp Ala Asp Gly Tyr Val Thr Arg             420 425 430 Phe Gly Val Thr Tyr Val Asp Tyr Glu Asn Gly Gln Lys Arg Phe Pro         435 440 445 Lys Lys Ser Ala Lys Ser Leu Lys Pro Leu Phe Asp Glu Leu Ile Ala     450 455 460 Ala Ala 465 <210> 91 <211> 2103 <212> DNA <213> Artificial Sequence <220> <223> Trichoderma reesei ß-Glucosidase D <220> <221> CDS (222) (1) .. (2103) <223> Trichoderma reesei ß-Glucosidase D <400> 91 atg att ctc ggc tgt gaa agc aca ggt gtc atc tct gcc gtc aaa cac 48 Met Ile Leu Gly Cys Glu Ser Thr Gly Val Ile Ser Ala Val Lys His 1 5 10 15 ttt gtc gcc aac gac cag gag cac gag cgg cga gcg gtc gac tgt ctc 96 Phe Val Ala Asn Asp Gln Glu His Glu Arg Arg Ala Val Asp Cys Leu             20 25 30 atc acc cag cgg gct ctc cgg gag gtc tat ctg cga ccc ttc cag atc 144 Ile Thr Gln Arg Ala Leu Arg Glu Val Tyr Leu Arg Pro Phe Gln Ile         35 40 45 gta gcc cga gat gca agg ccc ggc gca ttg atg aca tcc tac aac aag 192 Val Ala Arg Asp Ala Arg Pro Gly Ala Leu Met Thr Ser Tyr Asn Lys     50 55 60 gtc aat ggc aag cac gtc gct gac agc gcc gag ttc ctt cag ggc att 240 Val Asn Gly Lys His Val Ala Asp Ser Ala Glu Phe Leu Gln Gly Ile 65 70 75 80 ctc cgg act gag tgg aat tgg gac cct ctc att gtc agc gac tgg tac 288 Leu Arg Thr Glu Trp Asn Trp Asp Pro Leu Ile Val Ser Asp Trp Tyr                 85 90 95 ggc acc tac acc act att gat gcc atc aaa gcc ggc ctt gat ctc gag 336 Gly Thr Tyr Thr Thr Ile Asp Ala Ile Lys Ala Gly Leu Asp Leu Glu             100 105 110 atg ccg ggc gtt tca cga tat cgc ggc aaa tac atc gag tct gct ctg 384 Met Pro Gly Val Ser Arg Tyr Arg Gly Lys Tyr Ile Glu Ser Ala Leu         115 120 125 cag gcc cgt ttg ctg aag cag tcc act atc gat gag cgc gct cgc cgc 432 Gln Ala Arg Leu Leu Lys Gln Ser Thr Ile Asp Glu Arg Ala Arg Arg     130 135 140 gtg ctc agg ttc gcc cag aag gcc agc cat ctc aag gtc tcc gag gta 480 Val Leu Arg Phe Ala Gln Lys Ala Ser His Leu Lys Val Ser Glu Val 145 150 155 160 gag caa ggc cgt gac ttc cca gag gat cgc gtc ctc aac cgt cag atc 528 Glu Gln Gly Arg Asp Phe Pro Glu Asp Arg Val Leu Asn Arg Gln Ile                 165 170 175 tgc ggc agc agc att gtc cta ctg aag aat gag aac tcc atc tta cct 576 Cys Gly Ser Ser Ile Val Leu Leu Lys Asn Glu Asn Ser Ile Leu Pro             180 185 190 ctc ccc aag tcc gtc aag aag gtc gcc ctt gtt ggt tcc cac gtg cgt 624 Leu Pro Lys Ser Val Lys Lys Val Ala Leu Val Gly Ser His Val Arg         195 200 205 cta ccg gct atc tcg gga gga ggc agc gcc tct ctt gtc cct tac tat 672 Leu Pro Ala Ile Ser Gly Gly Gly Ser Ala Ser Leu Val Pro Tyr Tyr     210 215 220 gcc ata tct cta tac gat gcc gtc tct gag gta cta gcc ggt gcc acg 720 Ala Ile Ser Leu Tyr Asp Ala Val Ser Glu Val Leu Ala Gly Ala Thr 225 230 235 240 atc acg cac gag gtc ggt gcc tat gcc cac caa atg ctg ccc gtc atc 768 Ile Thr His Glu Val Gly Ala Tyr Ala His Gln Met Leu Pro Val Ile                 245 250 255 gac gca atg atc agc aac gcc gta atc cac ttc tac aac gac ccc atc 816 Asp Ala Met Ile Ser Asn Ala Val Ile His Phe Tyr Asn Asp Pro Ile             260 265 270 gat gtc aaa gac aga aag ctc ctt ggc agt gag aac gta tcg tcg aca 864 Asp Val Lys Asp Arg Lys Leu Leu Gly Ser Glu Asn Val Ser Ser Thr         275 280 285 tcg ttc cag ctc atg gat tac aac aac atc cca acg ctc aac aag gcc 912 Ser Phe Gln Leu Met Asp Tyr Asn Asn Ile Pro Thr Leu Asn Lys Ala     290 295 300 atg ttc tgg ggt act ctc gtg ggc gag ttt atc cct acc gcc acg gga 960 Met Phe Trp Gly Thr Leu Val Gly Glu Phe Ile Pro Thr Ala Thr Gly 305 310 315 320 att tgg gaa ttt ggc ctc agt gtc ttt ggc act gcc gac ctt tat att 1008 Ile Trp Glu Phe Gly Leu Ser Val Phe Gly Thr Ala Asp Leu Tyr Ile                 325 330 335 gat aat gag ctc gtg att gaa aat aca aca cat cag acg cgt gga acc 1056 Asp Asn Glu Leu Val Ile Glu Asn Thr Thr His Gln Thr Arg Gly Thr             340 345 350 gcc ttt ttc gga aag gga acg acg gaa aaa gtc gct acc agg agg atg 1104 Ala Phe Phe Gly Lys Gly Thr Thr Glu Lys Val Ala Thr Arg Arg Met         355 360 365 gtg gcc ggc agc acc tac aag ctg cgt ctc gag ttt ggg tct gcc aac 1152 Val Ala Gly Ser Thr Tyr Lys Leu Arg Leu Glu Phe Gly Ser Ala Asn     370 375 380 acg acc aag atg gag acg acc ggt gtt gtc aac ttt ggc ggc ggt gcc 1200 Thr Thr Lys Met Glu Thr Thr Gly Val Val Asn Phe Gly Gly Gla Ala 385 390 395 400 gta cac ctg ggt gcc tgt ctc aag gtc gac cca cag gag atg att gcg 1248 Val His Leu Gly Ala Cys Leu Lys Val Asp Pro Gln Glu Met Ile Ala                 405 410 415 cgg gcc gtc aag gcc gca gcc gat gcc gac tac acc atc atc tgc acg 1296 Arg Ala Val Lys Ala Ala Ala Asp Ala Asp Tyr Thr Ile Ile Cys Thr             420 425 430 gga ctc agc ggc gag tgg gag tct gag ggt ttt gac cgg cct cac atg 1344 Gly Leu Ser Gly Glu Trp Glu Ser Glu Gly Phe Asp Arg Pro His Met         435 440 445 gac ctg ccc cct ggt gtg gac acc atg atc tcg caa gtt ctt gac gcc 1392 Asp Leu Pro Pro Gly Val Asp Thr Met Ile Ser Gln Val Leu Asp Ala     450 455 460 gct ccc aat gct gta gtc gtc aac cag tca ggc acc cca gtg aca atg 1440 Ala Pro Asn Ala Val Val Val Asn Gln Ser Gly Thr Pro Val Thr Met 465 470 475 480 agc tgg gct cat aaa gca aag gcc att gtg cag gct tgg tat ggt ggt 1488 Ser Trp Ala His Lys Ala Lys Ala Ile Val Gln Ala Trp Tyr Gly Gly                 485 490 495 aac gag aca ggc cac gga atc tcc gat gtg ctc ttt ggc aac gtc aac 1536 Asn Glu Thr Gly His Gly Ile Ser Asp Val Leu Phe Gly Asn Val Asn             500 505 510 ccg tcg ggg aaa ctc tcc cta tcg tgg cca gtc gat gtg aag cac aac 1584 Pro Ser Gly Lys Leu Ser Leu Ser Trp Pro Val Asp Val Lys His Asn         515 520 525 cca gca tat ctc aac tac gcc agc gtt ggt gga cgg gtc ttg tat ggc 1632 Pro Ala Tyr Leu Asn Tyr Ala Ser Val Gly Gly Arg Val Leu Tyr Gly     530 535 540 gag gat gtt tac gtt ggc tac aag ttc tac gac aaa acg gag agg gag 1680 Glu Asp Val Tyr Val Gly Tyr Lys Phe Tyr Asp Lys Thr Glu Arg Glu 545 550 555 560 gtt ctg ttt cct ttt ggg cat ggc ctg tct tac gct acc ttc aag ctc 1728 Val Leu Phe Pro Phe Gly His Gly Leu Ser Tyr Ala Thr Phe Lys Leu                 565 570 575 cca gat tct acc gtg agg acg gtc ccc gaa acc ttc cac ccg gac cag 1776 Pro Asp Ser Thr Val Arg Thr Val Pro Glu Thr Phe His Pro Asp Gln             580 585 590 ccc aca gta gcc att gtc aag atc aag aac acg agc agt gtc ccg ggc 1824 Pro Thr Val Ala Ile Val Lys Ile Lys Asn Thr Ser Ser Val Pro Gly         595 600 605 gcc cag gtc ctg cag tta tac att tcg gcc cca aac tcg cct aca cat 1872 Ala Gln Val Leu Gln Leu Tyr Ile Ser Ala Pro Asn Ser Pro Thr His     610 615 620 cgc ccg gtc aag gag ctg cac gga ttc gaa aag gtg tat ctt gaa gct 1920 Arg Pro Val Lys Glu Leu His Gly Phe Glu Lys Val Tyr Leu Glu Ala 625 630 635 640 ggc gag gag aag gag gta caa ata ccc att gac cag tac gct act agc 1968 Gly Glu Glu Lys Glu Val Gln Ile Pro Ile Asp Gln Tyr Ala Thr Ser                 645 650 655 ttc tgg gac gag att gag agc atg tgg aag agc gag agg ggc att tat 2016 Phe Trp Asp Glu Ile Glu Ser Met Trp Lys Ser Glu Arg Gly Ile Tyr             660 665 670 gat gtg ctt gta gga ttc tcg agt cag gaa atc tcg ggc aag ggg aag 2064 Asp Val Leu Val Gly Phe Ser Ser Gln Glu Ile Ser Gly Lys Gly Lys         675 680 685 ctg att gtg cct gaa acg cga ttc tgg atg ggg ctg tag 2103 Leu Ile Val Pro Glu Thr Arg Phe Trp Met Gly Leu     690 695 700 <210> 92 <211> 700 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 92 Met Ile Leu Gly Cys Glu Ser Thr Gly Val Ile Ser Ala Val Lys His 1 5 10 15 Phe Val Ala Asn Asp Gln Glu His Glu Arg Arg Ala Val Asp Cys Leu             20 25 30 Ile Thr Gln Arg Ala Leu Arg Glu Val Tyr Leu Arg Pro Phe Gln Ile         35 40 45 Val Ala Arg Asp Ala Arg Pro Gly Ala Leu Met Thr Ser Tyr Asn Lys     50 55 60 Val Asn Gly Lys His Val Ala Asp Ser Ala Glu Phe Leu Gln Gly Ile 65 70 75 80 Leu Arg Thr Glu Trp Asn Trp Asp Pro Leu Ile Val Ser Asp Trp Tyr                 85 90 95 Gly Thr Tyr Thr Thr Ile Asp Ala Ile Lys Ala Gly Leu Asp Leu Glu             100 105 110 Met Pro Gly Val Ser Arg Tyr Arg Gly Lys Tyr Ile Glu Ser Ala Leu         115 120 125 Gln Ala Arg Leu Leu Lys Gln Ser Thr Ile Asp Glu Arg Ala Arg Arg     130 135 140 Val Leu Arg Phe Ala Gln Lys Ala Ser His Leu Lys Val Ser Glu Val 145 150 155 160 Glu Gln Gly Arg Asp Phe Pro Glu Asp Arg Val Leu Asn Arg Gln Ile                 165 170 175 Cys Gly Ser Ser Ile Val Leu Leu Lys Asn Glu Asn Ser Ile Leu Pro             180 185 190 Leu Pro Lys Ser Val Lys Lys Val Ala Leu Val Gly Ser His Val Arg         195 200 205 Leu Pro Ala Ile Ser Gly Gly Gly Ser Ala Ser Leu Val Pro Tyr Tyr     210 215 220 Ala Ile Ser Leu Tyr Asp Ala Val Ser Glu Val Leu Ala Gly Ala Thr 225 230 235 240 Ile Thr His Glu Val Gly Ala Tyr Ala His Gln Met Leu Pro Val Ile                 245 250 255 Asp Ala Met Ile Ser Asn Ala Val Ile His Phe Tyr Asn Asp Pro Ile             260 265 270 Asp Val Lys Asp Arg Lys Leu Leu Gly Ser Glu Asn Val Ser Ser Thr         275 280 285 Ser Phe Gln Leu Met Asp Tyr Asn Asn Ile Pro Thr Leu Asn Lys Ala     290 295 300 Met Phe Trp Gly Thr Leu Val Gly Glu Phe Ile Pro Thr Ala Thr Gly 305 310 315 320 Ile Trp Glu Phe Gly Leu Ser Val Phe Gly Thr Ala Asp Leu Tyr Ile                 325 330 335 Asp Asn Glu Leu Val Ile Glu Asn Thr Thr His Gln Thr Arg Gly Thr             340 345 350 Ala Phe Phe Gly Lys Gly Thr Thr Glu Lys Val Ala Thr Arg Arg Met         355 360 365 Val Ala Gly Ser Thr Tyr Lys Leu Arg Leu Glu Phe Gly Ser Ala Asn     370 375 380 Thr Thr Lys Met Glu Thr Thr Gly Val Val Asn Phe Gly Gly Gla Ala 385 390 395 400 Val His Leu Gly Ala Cys Leu Lys Val Asp Pro Gln Glu Met Ile Ala                 405 410 415 Arg Ala Val Lys Ala Ala Ala Asp Ala Asp Tyr Thr Ile Ile Cys Thr             420 425 430 Gly Leu Ser Gly Glu Trp Glu Ser Glu Gly Phe Asp Arg Pro His Met         435 440 445 Asp Leu Pro Pro Gly Val Asp Thr Met Ile Ser Gln Val Leu Asp Ala     450 455 460 Ala Pro Asn Ala Val Val Val Asn Gln Ser Gly Thr Pro Val Thr Met 465 470 475 480 Ser Trp Ala His Lys Ala Lys Ala Ile Val Gln Ala Trp Tyr Gly Gly                 485 490 495 Asn Glu Thr Gly His Gly Ile Ser Asp Val Leu Phe Gly Asn Val Asn             500 505 510 Pro Ser Gly Lys Leu Ser Leu Ser Trp Pro Val Asp Val Lys His Asn         515 520 525 Pro Ala Tyr Leu Asn Tyr Ala Ser Val Gly Gly Arg Val Leu Tyr Gly     530 535 540 Glu Asp Val Tyr Val Gly Tyr Lys Phe Tyr Asp Lys Thr Glu Arg Glu 545 550 555 560 Val Leu Phe Pro Phe Gly His Gly Leu Ser Tyr Ala Thr Phe Lys Leu                 565 570 575 Pro Asp Ser Thr Val Arg Thr Val Pro Glu Thr Phe His Pro Asp Gln             580 585 590 Pro Thr Val Ala Ile Val Lys Ile Lys Asn Thr Ser Ser Val Pro Gly         595 600 605 Ala Gln Val Leu Gln Leu Tyr Ile Ser Ala Pro Asn Ser Pro Thr His     610 615 620 Arg Pro Val Lys Glu Leu His Gly Phe Glu Lys Val Tyr Leu Glu Ala 625 630 635 640 Gly Glu Glu Lys Glu Val Gln Ile Pro Ile Asp Gln Tyr Ala Thr Ser                 645 650 655 Phe Trp Asp Glu Ile Glu Ser Met Trp Lys Ser Glu Arg Gly Ile Tyr             660 665 670 Asp Val Leu Val Gly Phe Ser Ser Gln Glu Ile Ser Gly Lys Gly Lys         675 680 685 Leu Ile Val Pro Glu Thr Arg Phe Trp Met Gly Leu     690 695 700 <210> 93 <211> 1496 <212> DNA <213> Artificial Sequence <220> <223> Maize optimized CBHI <400> 93 tgcagtccgc ctgcaccctc cagtccgaga cccacccgcc gctcacctgg cagaagtgct 60 cctccggcgg cacctgcacc cagcagaccg gctccgtggt gatcgacgcc aactggcgct 120 ggacccacgc caccaactcc tccaccaact gctacgacgg caacacctgg tcctccaccc 180 tctgcccgga caacgagacc tgcgccaaga actgctgcct cgacggcgcc gcctacgcct 240 ccacctacgg cgtgaccacc tccggcaact ccctctccat cggcttcgtg acccagtccg 300 cccagaagaa cgtgggcgcc cgcctctacc tcatggcctc cgacaccacc taccaggagt 360 tcaccctcct cggcaacgag ttctccttcg acgtggacgt gtcccagctc ccgtgcggcc 420 tcaacggcgc cctctacttc gtgtccatgg acgccgacgg cggcgtgtcc aagtacccga 480 ccaacaccgc cggcgccaag tacggcaccg gctactgcga ctcccagtgc ccgcgcgacc 540 tcaagttcat caacggccag gccaacgtgg agggctggga gccgtcctcc aacaacgcca 600 acaccggcat cggcggccac ggctcctgct gctccgagat ggacatctgg gaggccaact 660 ccatctccga ggccctcacc ccgcacccgt gcaccaccgt gggccaggag atctgcgagg 720 gcgacggctg cggcggcacc tactccgaca accgctacgg cggcacctgc gacccggacg 780 gctgcgactg gaacccgtac cgcctcggca acacctcctt ctacggcccg ggctcctcct 840 tcaccctcga caccaccaag aagctcaccg tggtgaccca gttcgagacc tccggcgcca 900 tcaaccgcta ctacgtgcag aacggcgtga ccttccagca gccgaacgcc gagctcggct 960 cctactccgg caacgagctc aacgacgact actgcaccgc cgaggaggcc gagttcggcg 1020 gctcctcctt ctccgacaag ggcggcctca cccagttcaa gaaggccacc tccggcggca 1080 tggtgctcgt gatgtccctc tgggacgact actacgccaa catgctctgg ctcgactcca 1140 cctacccgac caacgagacc tcctccaccc cgggcgccgt gcgcggctcc tgctccacct 1200 cctccggcgt gccggcccag gtggagtccc agtccccgaa cgccaaggtg accttctcca 1260 acatcaagtt cggcccgatc ggctccaccg gcaacccgtc cggcggcaac ccgccgggcg 1320 gcaacccgcc gggcaccacc accacccgcc gcccggccac caccaccggc tcctccccgg 1380 gcccgaccca gtcccactac ggccagtgcg gcggcatcgg ctactccggc ccgaccgtgt 1440 gcgcctccgg caccacctgc caggtgctca acccgtacta ctcccagtgc ctctag 1496 <210> 94 <211> 1365 <212> DNA <213> Artificial Sequence <220> <223> Maize optimized CBHII <400> 94 atggtgccgc tcgaggagcg ccaggcctgc tcctccgtgt ggggccagtg cggcggccag 60 aactggtccg gcccgacctg ctgcgcctcc ggctccacct gcgtgtactc caacgactac 120 tactcccagt gcctcccggg cgccgcctcc tcctcctcct ccacccgcgc cgcctccacc 180 acctcccgcg tgtccccgac cacctcccgc tcctcctccg ccaccccgcc gccgggctcc 240 accaccaccc gcgtgccgcc ggtgggctcc ggcaccgcca cctactccgg caacccgttc 300 gtgggcgtga ccccgtgggc caacgcctac tacgcctccg aggtgtcctc cctcgccatc 360 ccgtccctca ccggcgccat ggccaccgcc gccgccgccg tggccaaggt gccgtccttc 420 atgtggctcg acaccctcga caagaccccg ctcatggagc agaccctcgc cgacatccgc 480 accgccaaca agaacggcgg caactacgcc ggccagttcg tggtgtacga cctcccggac 540 cgcgactgcg ccgccctcgc ctccaacggc gagtactcca tcgccgacgg cggcgtggcc 600 aagtacaaga actacatcga caccatccgc cagatcgtgg tggagtactc cgacatccgc 660 accctcctcg tgatcgagcc ggactccctc gccaacctcg tgaccaacct cggcaccccg 720 aagtgcgcca acgcccagtc cgcctacctc gagtgcatca actacgccgt gacccagctc 780 aacctcccga acgtggccat gtacctcgac gccggccacg ccggctggct cggctggccg 840 gccaaccagg acccggccgc ccagctcttc gccaacgtgt acaagaacgc ctcctccccg 900 cgcgccctcc gcggcctcgc caccaacgtg gccaactaca acggctggaa catcacctcc 960 ccgccgtcct acacccaggg caacgccgtg tacaacgaga agctctacat ccacgccatc 1020 ggcccgctcc tcgccaacca cggctggtcc aacgccttct tcatcaccga ccagggccgc 1080 tccggcaagc agccgaccgg ccagcagcag tggggcgact ggtgcaacgt gatcggcacc 1140 ggcttcggca tccgcccgtc cgccaacacc ggcgactccc tcctcgactc cttcgtgtgg 1200 gtgaagccgg gcggcgagtg cgacggcacc tccgactcct ccgccccgcg cttcgactcc 1260 cactgcgccc tcccggacgc cctccagccg gccccgcagg ccggcgcctg gttccaggcc 1320 tacttcgtgc agctcctcac caacgccaac ccgtccttcc tctag 1365 <210> 95 <211> 1317 <212> DNA <213> Artificial Sequence <220> <223> Maize optimized EGLI <400> 95 atgcagcagc cgggcacctc caccccggag gtgcacccga agctcaccac ctacaagtgc 60 accaagtccg gcggctgcgt ggcccaggac acctccgtgg tgctcgactg gaactaccgc 120 tggatgcacg acgccaacta caactcctgc accgtgaacg gcggcgtgaa caccaccctc 180 tgcccggacg aggccacctg cggcaagaac tgcttcatcg agggcgtgga ctacgccgcc 240 tccggcgtga ccacctccgg ctcctccctc accatgaacc agtacatgcc gtcctcctcc 300 ggcggctact cctccgtgtc cccgcgcctc tacctcctcg actccgacgg cgagtacgtg 360 atgctcaagc tcaacggcca ggagctctcc ttcgacgtgg acctctccgc cctcccgtgc 420 ggcgagaacg gctccctcta cctctcccag atggacgaga acggcggcgc caaccagtac 480 aacaccgccg gcgccaacta cggctccggc tactgcgacg cccagtgccc ggtgcagacc 540 tggcgcaacg gcaccctcaa cacctcccac cagggcttct gctgcaacga gatggacatc 600 ctcgagggca actcccgcgc caacgccctc accccgcact cctgcaccgc caccgcctgc 660 gactccgccg gctgcggctt caacccgtac ggctccggct acaagtccta ctacggcccg 720 ggcgacaccg tggacacctc caagaccttc accatcatca cccagttcaa caccgacaac 780 ggctccccgt ccggcaacct cgtgtccatc acccgcaagt accagcagaa cggcgtggac 840 atcccgtccg cccagccggg cggcgacacc atctcctcct gcccgtccgc ctccgcctac 900 ggcggcctcg ccaccatggg caaggccctc tcctccggca tggtgctcgt gttctccatc 960 tggaacgaca actcccagta catgaactgg ctcgactccg gcaacgccgg cccgtgctcc 1020 tccaccgagg gcaacccgtc caacaccctc gccaacaacc cgaacaccca cgtggtgttc 1080 tccaacatcc gctggggcga catcggctcc accaccaact ccaccgcccc gccgccgccg 1140 ccggcctcct ccaccacctt ctccaccacc cgccgctcct ccaccacctc ctcctccccg 1200 tcctgcaccc agacccactg gggccagtgc ggcggcatcg gctactccgg ctgcaagacc 1260 tgcacctccg gcaccacctg ccagtactcc aacgactact actcccagtg cctctag 1317 <210> 96 <211> 1401 <212> DNA <213> Artificial Sequence <220> <223> Maize optimized BGLII <400> 96 atgctcccga aggacttcca gtggggcttc gccaccgccg cctaccagat cgagggcgcc 60 gtggaccagg acggccgcgg cccgtccatc tgggacacct tctgcgccca gccgggcaag 120 atcgccgacg gctcctccgg cgtgaccgcc tgcgactcct acaaccgcac cgccgaggac 180 atcgccctcc tcaagtccct cggcgccaag tcctaccgct tctccatctc ctggtcccgc 240 atcatcccgg agggcggccg cggcgacgcc gtgaaccagg ccggcatcga ccactacgtg 300 aagttcgtgg acgacctcct cgacgccggc atcaccccgt tcatcaccct cttccactgg 360 gacctcccgg agggcctcca ccagcgctac ggcggcctcc tcaaccgcac cgagttcccg 420 ctcgacttcg agaactacgc ccgcgtgatg ttccgcgccc tcccgaaggt gcgcaactgg 480 atcaccttca acgagccgct ctgctccgcc atcccgggct acggctccgg caccttcgcc 540 ccgggccgcc agtccacctc cgagccgtgg accgtgggcc acaacatcct cgtggcccac 600 ggccgcgccg tgaaggccta ccgcgacgac ttcaagccgg cctccggcga cggccagatc 660 ggcatcgtgc tcaacggcga cttcacctac ccgtgggacg ccgccgaccc ggccgacaag 720 gaggccgccg agcgccgcct cgagttcttc accgcctggt tcgccgaccc gatctacctc 780 ggcgactacc cggcctccat gcgcaagcag ctcggcgacc gcctcccgac cttcaccccg 840 gaggagcgcg ccctcgtgca cggctccaac gacttctacg gcatgaacca ctacacctcc 900 aactacatcc gccaccgctc ctccccggcc tccgccgacg acaccgtggg caacgtggac 960 gtgctcttca ccaacaagca gggcaactgc atcggcccgg agacccagtc cccgtggctc 1020 cgcccgtgcg ccgccggctt ccgcgacttc ctcgtgtgga tctccaagcg ctacggctac 1080 ccgccgatct acgtgaccga gaacggcacc tccatcaagg gcgagtccga cctcccgaag 1140 gagaagatcc tcgaggacga cttccgcgtg aagtactaca acgagtacat ccgcgccatg 1200 gtgaccgccg tggagctcga cggcgtgaac gtgaagggct acttcgcctg gtccctcatg 1260 gacaacttcg agtgggccga cggctacgtg acccgcttcg gcgtgaccta cgtggactac 1320 gagaacggcc agaagcgctt cccgaagaag tccgccaagt ccctcaagcc gctcttcgac 1380 gagctcatcg ccgccgccta g 1401 <210> 97 <211> 2103 <212> DNA <213> Artificial Sequence <220> <223> Maize optimized CEL3D <400> 97 atgatcctcg gctgcgagtc caccggcgtg atctccgccg tgaagcactt cgtggccaac 60 gaccaggagc acgagcgccg cgccgtggac tgcctcatca cccagcgcgc cctccgcgag 120 gtgtacctcc gcccgttcca gatcgtggcc cgcgacgccc gcccgggcgc cctcatgacc 180 tcctacaaca aggtgaacgg caagcacgtg gccgactccg ccgagttcct ccagggcatc 240 ctccgcaccg agtggaactg ggacccgctc atcgtgtccg actggtacgg cacctacacc 300 accatcgacg ccatcaaggc cggcctcgac ctcgagatgc cgggcgtgtc ccgctaccgc 360 ggcaagtaca tcgagtccgc cctccaggcc cgcctcctca agcagtccac catcgacgag 420 cgcgcccgcc gcgtgctccg cttcgcccag aaggcctccc acctcaaggt gtccgaggtg 480 gagcagggcc gcgacttccc ggaggaccgc gtgctcaacc gccagatctg cggctcctcc 540 atcgtgctcc tcaagaacga gaactccatc ctcccgctcc cgaagtccgt gaagaaggtg 600 gccctcgtgg gctcccacgt gcgcctcccg gccatctccg gcggcggctc cgcctccctc 660 gtgccgtact acgccatctc cctctacgac gccgtgtccg aggtgctcgc cggcgccacc 720 atcacccacg aggtgggcgc ctacgcccac cagatgctcc cggtgatcga cgccatgatc 780 tccaacgccg tgatccactt ctacaacgac ccgatcgacg tgaaggaccg caagctcctc 840 ggctccgaga acgtgtcctc cacctccttc cagctcatgg actacaacaa catcccgacc 900 ctcaacaagg ccatgttctg gggcaccctc gtgggcgagt tcatcccgac cgccaccggc 960 atctgggagt tcggcctctc cgtgttcggc accgccgacc tctacatcga caacgagctc 1020 gtgatcgaga acaccaccca ccagacccgc ggcaccgcct tcttcggcaa gggcaccacc 1080 gagaaggtgg ccacccgccg catggtggcc ggctccacct acaagctccg cctcgagttc 1140 ggctccgcca acaccaccaa gatggagacc accggcgtgg tgaacttcgg cggcggcgcc 1200 gtgcacctcg gcgcctgcct caaggtggac ccgcaggaga tgatcgcccg cgccgtgaag 1260 gccgccgccg acgccgacta caccatcatc tgcaccggcc tctccggcga gtgggagtcc 1320 gagggcttcg accgcccgca catggacctc ccgccgggcg tggacaccat gatctcccag 1380 gtgctcgacg ccgccccgaa cgccgtggtg gtgaaccagt ccggcacccc ggtgaccatg 1440 tcctgggccc acaaggccaa ggccatcgtg caggcctggt acggcggcaa cgagaccggc 1500 cacggcatct ccgacgtgct cttcggcaac gtgaacccgt ccggcaagct ctccctctcc 1560 tggccggtgg acgtgaagca caacccggcc tacctcaact acgcctccgt gggcggccgc 1620 gtgctctacg gcgaggacgt gtacgtgggc tacaagttct acgacaagac cgagcgcgag 1680 gtgctcttcc cgttcggcca cggcctctcc tacgccacct tcaagctccc ggactccacc 1740 gtgcgcaccg tgccggagac cttccacccg gaccagccga ccgtggccat cgtgaagatc 1800 aagaacacct cctccgtgcc gggcgcccag gtgctccagc tctacatctc cgccccgaac 1860 tccccgaccc accgcccggt gaaggagctc cacggcttcg agaaggtgta cctcgaggcc 1920 ggcgaggaga aggaggtgca gatcccgatc gaccagtacg ccacctcctt ctgggacgag 1980 atcgagtcca tgtggaagtc cgagcgcggc atctacgacg tgctcgtggg cttctcctcc 2040 caggagatct ccggcaaggg caagctcatc gtgccggaga cccgcttctg gatgggcctc 2100 tag 2103 <210> 98 <211> 420 <212> DNA <213> Zea mays <220> <223> Q protein promoter <400> 98 gggctggtaa attacttggg agcaatggta tgcaaatcct ttgcatgtac gcaaaactag 60 ctagttgtca caagttgtat atcgattcgt cgcgtttcaa caactcatgc aacattacaa 120 acaagtaaca caatattaca aagttagttt catacaaagc aagaaaagga caataatact 180 tgacatgtaa agtgaagctt attatacttc ctaatccaac acaaaacaaa aaaaagttgc 240 acaaaggtcc aaaaatccac atcaaccatt aacctatacg taaagtgagt gatgagtcac 300 attatccaac aaatgtttat caatgtggta tcatacaagc attgacatcc cataaatgca 360 agaaattgtg ccaacaaagc tataagtaac cctcatatgt atttgcactc atgcatcaca 420 <210> 99 <211> 1188 <212> DNA <213> artificial sequence <220> <223> synthetic ferulic acid esterase <400> 99 atggccgcct ccctcccgac catgccgccg tccggctacg accaggtgcg caacggcgtg 60 ccgcgcggcc aggtggtgaa catctcctac ttctccaccg ccaccaactc cacccgcccg 120 gcccgcgtgt acctcccgcc gggctactcc aaggacaaga agtactccgt gctctacctc 180 ctccacggca tcggcggctc cgagaacgac tggttcgagg gcggcggccg cgccaacgtg 240 atcgccgaca acctcatcgc cgagggcaag atcaagccgc tcatcatcgt gaccccgaac 300 accaacgccg ccggcccggg catcgccgac ggctacgaga acttcaccaa ggacctcctc 360 aactccctca tcccgtacat cgagtccaac tactccgtgt acaccgaccg cgagcaccgc 420 gccatcgccg gcctctctat gggcggcggc cagtccttca acatcggcct caccaacctc 480 gacaagttcg cctacatcgg cccgatctcc gccgccccga acacctaccc gaacgagcgc 540 ctcttcccgg acggcggcaa ggccgcccgc gagaagctca agctcctctt catcgcctgc 600 ggcaccaacg actccctcat cggcttcggc cagcgcgtgc acgagtactg cgtggccaac 660 aacatcaacc acgtgtactg gctcatccag ggcggcggcc acgacttcaa cgtgtggaag 720 ccgggcctct ggaacttcct ccagatggcc gacgaggccg gcctcacccg cgacggcaac 780 accccggtgc cgaccccgtc cccgaagccg gccaacaccc gcatcgaggc cgaggactac 840 gacggcatca actcctcctc catcgagatc atcggcgtgc cgccggaggg cggccgcggc 900 atcggctaca tcacctccgg cgactacctc gtgtacaagt ccatcgactt cggcaacggc 960 gccacctcct tcaaggccaa ggtggccaac gccaacacct ccaacatcga gcttcgcctc 1020 aacggcccga acggcaccct catcggcacc ctctccgtga agtccaccgg cgactggaac 1080 acctacgagg agcagacctg ctccatctcc aaggtgaccg gcatcaacga cctctacctc 1140 gtgttcaagg gcccggtgaa catcgactgg ttcaccttcg gcgtgtag 1188 <210> 100 <211> 395 <212> PRT <213> artificial sequence <220> <223> synthetic ferulic acid esterase <400> 100 Met Ala Ala Ser Leu Pro Thr Met Pro Pro Ser Gly Tyr Asp Gln Val 1 5 10 15 Arg Asn Gly Val Pro Arg Gly Gln Val Val Asn Ile Ser Tyr Phe Ser             20 25 30 Thr Ala Thr Asn Ser Thr Arg Pro Ala Arg Val Tyr Leu Pro Pro Gly         35 40 45 Tyr Ser Lys Asp Lys Lys Tyr Ser Val Leu Tyr Leu Leu His Gly Ile     50 55 60 Gly Gly Ser Glu Asn Asp Trp Phe Glu Gly Gly Gly Arg Ala Asn Val 65 70 75 80 Ile Ala Asp Asn Leu Ile Ala Glu Gly Lys Ile Lys Pro Leu Ile Ile                 85 90 95 Val Thr Pro Asn Thr Asn Ala Ala Gly Pro Gly Ile Ala Asp Gly Tyr             100 105 110 Glu Asn Phe Thr Lys Asp Leu Leu Asn Ser Leu Ile Pro Tyr Ile Glu         115 120 125 Ser Asn Tyr Ser Val Tyr Thr Asp Arg Glu His Arg Ala Ile Ala Gly     130 135 140 Leu Ser Met Gly Gly Gly Gln Ser Phe Asn Ile Gly Leu Thr Asn Leu 145 150 155 160 Asp Lys Phe Ala Tyr Ile Gly Pro Ile Ser Ala Ala Pro Asn Thr Tyr                 165 170 175 Pro Asn Glu Arg Leu Phe Pro Asp Gly Gly Lys Ala Ala Arg Glu Lys             180 185 190 Leu Lys Leu Leu Phe Ile Ala Cys Gly Thr Asn Asp Ser Leu Ile Gly         195 200 205 Phe Gly Gln Arg Val His Glu Tyr Cys Val Ala Asn Asn Ile Asn His     210 215 220 Val Tyr Trp Leu Ile Gln Gly Gly Gly His Asp Phe Asn Val Trp Lys 225 230 235 240 Pro Gly Leu Trp Asn Phe Leu Gln Met Ala Asp Glu Ala Gly Leu Thr                 245 250 255 Arg Asp Gly Asn Thr Pro Val Pro Thr Pro Ser Pro Lys Pro Ala Asn             260 265 270 Thr Arg Ile Glu Ala Glu Asp Tyr Asp Gly Ile Asn Ser Ser Ser Ile         275 280 285 Glu Ile Ile Gly Val Pro Pro Glu Gly Gly Arg Gly Ile Gly Tyr Ile     290 295 300 Thr Ser Gly Asp Tyr Leu Val Tyr Lys Ser Ile Asp Phe Gly Asn Gly 305 310 315 320 Ala Thr Ser Phe Lys Ala Lys Val Ala Asn Ala Asn Thr Ser Asn Ile                 325 330 335 Glu Leu Arg Leu Asn Gly Pro Asn Gly Thr Leu Ile Gly Thr Leu Ser             340 345 350 Val Lys Ser Thr Gly Asp Trp Asn Thr Tyr Glu Glu Gln Thr Cys Ser         355 360 365 Ile Ser Lys Val Thr Gly Ile Asn Asp Leu Tyr Leu Val Phe Lys Gly     370 375 380 Pro Val Asn Ile Asp Trp Phe Thr Phe Gly Val 385 390 395 <210> 101 <211> 1188 <212> DNA <213> artificial sequence <220> <223> plasmid 13036 <400> 101 atggccgcct ccctcccgac catgccgccg tccggctacg accaggtgcg caacggcgtg 60 ccgcgcggcc aggtggtgaa catctcctac ttctccaccg ccaccaactc cacccgcccg 120 gcccgcgtgt acctcccgcc gggctactcc aaggacaaga agtactccgt gctctacctc 180 ctccacggca tcggcggctc cgagaacgac tggttcgagg gcggcggccg cgccaacgtg 240 atcgccgaca acctcatcgc cgagggcaag atcaagccgc tcatcatcgt gaccccgaac 300 accaacgccg ccggcccggg catcgccgac ggctacgaga acttcaccaa ggacctcctc 360 aactccctca tcccgtacat cgagtccaac tactccgtgt acaccgaccg cgagcaccgc 420 gccatcgccg gcctctctat gggcggcggc cagtccttca acatcggcct caccaacctc 480 gacaagttcg cctacatcgg cccgatctcc gccgccccga acacctaccc gaacgagcgc 540 ctcttcccgg acggcggcaa ggccgcccgc gagaagctca agctcctctt catcgcctgc 600 ggcaccaacg actccctcat cggcttcggc cagcgcgtgc acgagtactg cgtggccaac 660 aacatcaacc acgtgtactg gctcatccag ggcggcggcc acgacttcaa cgtgtggaag 720 ccgggcctct ggaacttcct ccagatggcc gacgaggccg gcctcacccg cgacggcaac 780 accccggtgc cgaccccgtc cccgaagccg gccaacaccc gcatcgaggc cgaggactac 840 gacggcatca actcctcctc catcgagatc atcggcgtgc cgccggaggg cggccgcggc 900 atcggctaca tcacctccgg cgactacctc gtgtacaagt ccatcgactt cggcaacggc 960 gccacctcct tcaaggccaa ggtggccaac gccaacacct ccaacatcga gcttcgcctc 1020 aacggcccga acggcaccct catcggcacc ctctccgtga agtccaccgg cgactggaac 1080 acctacgagg agcagacctg ctccatctcc aaggtgaccg gcatcaacga cctctacctc 1140 gtgttcaagg gcccggtgaa catcgactgg ttcaccttcg gcgtgtag 1188 <210> 102 <211> 395 <212> PRT <213> artificial sequence <220> <223> plasmid 13036 <400> 102 Met Ala Ala Ser Leu Pro Thr Met Pro Pro Ser Gly Tyr Asp Gln Val 1 5 10 15 Arg Asn Gly Val Pro Arg Gly Gln Val Val Asn Ile Ser Tyr Phe Ser             20 25 30 Thr Ala Thr Asn Ser Thr Arg Pro Ala Arg Val Tyr Leu Pro Pro Gly         35 40 45 Tyr Ser Lys Asp Lys Lys Tyr Ser Val Leu Tyr Leu Leu His Gly Ile     50 55 60 Gly Gly Ser Glu Asn Asp Trp Phe Glu Gly Gly Gly Arg Ala Asn Val 65 70 75 80 Ile Ala Asp Asn Leu Ile Ala Glu Gly Lys Ile Lys Pro Leu Ile Ile                 85 90 95 Val Thr Pro Asn Thr Asn Ala Ala Gly Pro Gly Ile Ala Asp Gly Tyr             100 105 110 Glu Asn Phe Thr Lys Asp Leu Leu Asn Ser Leu Ile Pro Tyr Ile Glu         115 120 125 Ser Asn Tyr Ser Val Tyr Thr Asp Arg Glu His Arg Ala Ile Ala Gly     130 135 140 Leu Ser Met Gly Gly Gly Gln Ser Phe Asn Ile Gly Leu Thr Asn Leu 145 150 155 160 Asp Lys Phe Ala Tyr Ile Gly Pro Ile Ser Ala Ala Pro Asn Thr Tyr                 165 170 175 Pro Asn Glu Arg Leu Phe Pro Asp Gly Gly Lys Ala Ala Arg Glu Lys             180 185 190 Leu Lys Leu Leu Phe Ile Ala Cys Gly Thr Asn Asp Ser Leu Ile Gly         195 200 205 Phe Gly Gln Arg Val His Glu Tyr Cys Val Ala Asn Asn Ile Asn His     210 215 220 Val Tyr Trp Leu Ile Gln Gly Gly Gly His Asp Phe Asn Val Trp Lys 225 230 235 240 Pro Gly Leu Trp Asn Phe Leu Gln Met Ala Asp Glu Ala Gly Leu Thr                 245 250 255 Arg Asp Gly Asn Thr Pro Val Pro Thr Pro Ser Pro Lys Pro Ala Asn             260 265 270 Thr Arg Ile Glu Ala Glu Asp Tyr Asp Gly Ile Asn Ser Ser Ser Ile         275 280 285 Glu Ile Ile Gly Val Pro Pro Glu Gly Gly Arg Gly Ile Gly Tyr Ile     290 295 300 Thr Ser Gly Asp Tyr Leu Val Tyr Lys Ser Ile Asp Phe Gly Asn Gly 305 310 315 320 Ala Thr Ser Phe Lys Ala Lys Val Ala Asn Ala Asn Thr Ser Asn Ile                 325 330 335 Glu Leu Arg Leu Asn Gly Pro Asn Gly Thr Leu Ile Gly Thr Leu Ser             340 345 350 Val Lys Ser Thr Gly Asp Trp Asn Thr Tyr Glu Glu Gln Thr Cys Ser         355 360 365 Ile Ser Lys Val Thr Gly Ile Asn Asp Leu Tyr Leu Val Phe Lys Gly     370 375 380 Pro Val Asn Ile Asp Trp Phe Thr Phe Gly Val 385 390 395 <210> 103 <211> 1245 <212> DNA <213> artificial sequence <220> <223> plasmid 13038 <400> 103 atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccatg 60 gccgcctccc tcccgaccat gccgccgtcc ggctacgacc aggtgcgcaa cggcgtgccg 120 cgcggccagg tggtgaacat ctcctacttc tccaccgcca ccaactccac ccgcccggcc 180 cgcgtgtacc tcccgccggg ctactccaag gacaagaagt actccgtgct ctacctcctc 240 cacggcatcg gcggctccga gaacgactgg ttcgagggcg gcggccgcgc caacgtgatc 300 gccgacaacc tcatcgccga gggcaagatc aagccgctca tcatcgtgac cccgaacacc 360 aacgccgccg gcccgggcat cgccgacggc tacgagaact tcaccaagga cctcctcaac 420 tccctcatcc cgtacatcga gtccaactac tccgtgtaca ccgaccgcga gcaccgcgcc 480 atcgccggcc tctctatggg cggcggccag tccttcaaca tcggcctcac caacctcgac 540 aagttcgcct acatcggccc gatctccgcc gccccgaaca cctacccgaa cgagcgcctc 600 ttcccggacg gcggcaaggc cgcccgcgag aagctcaagc tcctcttcat cgcctgcggc 660 accaacgact ccctcatcgg cttcggccag cgcgtgcacg agtactgcgt ggccaacaac 720 atcaaccacg tgtactggct catccagggc ggcggccacg acttcaacgt gtggaagccg 780 ggcctctgga acttcctcca gatggccgac gaggccggcc tcacccgcga cggcaacacc 840 ccggtgccga ccccgtcccc gaagccggcc aacacccgca tcgaggccga ggactacgac 900 ggcatcaact cctcctccat cgagatcatc ggcgtgccgc cggagggcgg ccgcggcatc 960 ggctacatca cctccggcga ctacctcgtg tacaagtcca tcgacttcgg caacggcgcc 1020 acctccttca aggccaaggt ggccaacgcc aacacctcca acatcgagct tcgcctcaac 1080 ggcccgaacg gcaccctcat cggcaccctc tccgtgaagt ccaccggcga ctggaacacc 1140 tacgaggagc agacctgctc catctccaag gtgaccggca tcaacgacct ctacctcgtg 1200 ttcaagggcc cggtgaacat cgactggttc accttcggcg tgtag 1245 <210> 104 <211> 414 <212> PRT <213> artificial sequence <220> <223> plasmid 13038 aa <400> 104 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Met Ala Ala Ser Leu Pro Thr Met Pro Pro Ser Gly Tyr             20 25 30 Asp Gln Val Arg Asn Gly Val Pro Arg Gly Gln Val Val Asn Ile Ser         35 40 45 Tyr Phe Ser Thr Ala Thr Asn Ser Thr Arg Pro Ala Arg Val Tyr Leu     50 55 60 Pro Pro Gly Tyr Ser Lys Asp Lys Lys Tyr Ser Val Leu Tyr Leu Leu 65 70 75 80 His Gly Ile Gly Gly Ser Glu Asn Asp Trp Phe Glu Gly Gly Gly Arg                 85 90 95 Ala Asn Val Ile Ala Asp Asn Leu Ile Ala Glu Gly Lys Ile Lys Pro             100 105 110 Leu Ile Ile Val Thr Pro Asn Thr Asn Ala Ala Gly Pro Gly Ile Ala         115 120 125 Asp Gly Tyr Glu Asn Phe Thr Lys Asp Leu Leu Asn Ser Leu Ile Pro     130 135 140 Tyr Ile Glu Ser Asn Tyr Ser Val Tyr Thr Asp Arg Glu His Arg Ala 145 150 155 160 Ile Ala Gly Leu Ser Met Gly Gly Gly Gln Ser Phe Asn Ile Gly Leu                 165 170 175 Thr Asn Leu Asp Lys Phe Ala Tyr Ile Gly Pro Ile Ser Ala Ala Pro             180 185 190 Asn Thr Tyr Pro Asn Glu Arg Leu Phe Pro Asp Gly Gly Lys Ala Ala         195 200 205 Arg Glu Lys Leu Lys Leu Leu Phe Ile Ala Cys Gly Thr Asn Asp Ser     210 215 220 Leu Ile Gly Phe Gly Gln Arg Val His Glu Tyr Cys Val Ala Asn Asn 225 230 235 240 Ile Asn His Val Tyr Trp Leu Ile Gln Gly Gly Gly His Asp Phe Asn                 245 250 255 Val Trp Lys Pro Gly Leu Trp Asn Phe Leu Gln Met Ala Asp Glu Ala             260 265 270 Gly Leu Thr Arg Asp Gly Asn Thr Pro Val Pro Thr Pro Ser Pro Lys         275 280 285 Pro Ala Asn Thr Arg Ile Glu Ala Glu Asp Tyr Asp Gly Ile Asn Ser     290 295 300 Ser Ser Ile Glu Ile Ile Gly Val Pro Pro Glu Gly Gly Arg Gly Ile 305 310 315 320 Gly Tyr Ile Thr Ser Gly Asp Tyr Leu Val Tyr Lys Ser Ile Asp Phe                 325 330 335 Gly Asn Gly Ala Thr Ser Phe Lys Ala Lys Val Ala Asn Ala Asn Thr             340 345 350 Ser Asn Ile Glu Leu Arg Leu Asn Gly Pro Asn Gly Thr Leu Ile Gly         355 360 365 Thr Leu Ser Val Lys Ser Thr Gly Asp Trp Asn Thr Tyr Glu Glu Gln     370 375 380 Thr Cys Ser Ile Ser Lys Val Thr Gly Ile Asn Asp Leu Tyr Leu Val 385 390 395 400 Phe Lys Gly Pro Val Asn Ile Asp Trp Phe Thr Phe Gly Val                 405 410 <210> 105 <211> 1425 <212> DNA <213> artificial sequence <220> <223> plasmid 13039 <400> 105 atgctggcgg ctctggccac gtcgcagctc gtcgcaacgc gcgccggcct gggcgtcccg 60 gacgcgtcca cgttccgccg cggcgccgcg cagggcctga ggggggcccg ggcgtcggcg 120 gcggcggaca cgctcagcat gcggaccagc gcgcgcgcgg cgcccaggca ccagcaccag 180 caggcgcgcc gcggggccag gttcccgtcg ctcgtcgtgt gcgccagcgc cggcgccatg 240 gccgcctccc tcccgaccat gccgccgtcc ggctacgacc aggtgcgcaa cggcgtgccg 300 cgcggccagg tggtgaacat ctcctacttc tccaccgcca ccaactccac ccgcccggcc 360 cgcgtgtacc tcccgccggg ctactccaag gacaagaagt actccgtgct ctacctcctc 420 cacggcatcg gcggctccga gaacgactgg ttcgagggcg gcggccgcgc caacgtgatc 480 gccgacaacc tcatcgccga gggcaagatc aagccgctca tcatcgtgac cccgaacacc 540 aacgccgccg gcccgggcat cgccgacggc tacgagaact tcaccaagga cctcctcaac 600 tccctcatcc cgtacatcga gtccaactac tccgtgtaca ccgaccgcga gcaccgcgcc 660 atcgccggcc tctctatggg cggcggccag tccttcaaca tcggcctcac caacctcgac 720 aagttcgcct acatcggccc gatctccgcc gccccgaaca cctacccgaa cgagcgcctc 780 ttcccggacg gcggcaaggc cgcccgcgag aagctcaagc tcctcttcat cgcctgcggc 840 accaacgact ccctcatcgg cttcggccag cgcgtgcacg agtactgcgt ggccaacaac 900 atcaaccacg tgtactggct catccagggc ggcggccacg acttcaacgt gtggaagccg 960 ggcctctgga acttcctcca gatggccgac gaggccggcc tcacccgcga cggcaacacc 1020 ccggtgccga ccccgtcccc gaagccggcc aacacccgca tcgaggccga ggactacgac 1080 ggcatcaact cctcctccat cgagatcatc ggcgtgccgc cggagggcgg ccgcggcatc 1140 ggctacatca cctccggcga ctacctcgtg tacaagtcca tcgacttcgg caacggcgcc 1200 acctccttca aggccaaggt ggccaacgcc aacacctcca acatcgagct tcgcctcaac 1260 ggcccgaacg gcaccctcat cggcaccctc tccgtgaagt ccaccggcga ctggaacacc 1320 tacgaggagc agacctgctc catctccaag gtgaccggca tcaacgacct ctacctcgtg 1380 ttcaagggcc cggtgaacat cgactggttc accttcggcg tgtag 1425 <210> 106 <211> 474 <212> PRT <213> artificial sequence <220> <223> plasmid 13039 aa <400> 106 Met Leu Ala Ala Leu Ala Thr Ser Gln Leu Val Ala Thr Arg Ala Gly 1 5 10 15 Leu Gly Val Pro Asp Ala Ser Thr Phe Arg Arg Gly Ala Ala Gln Gly             20 25 30 Leu Arg Gly Ala Arg Ala Ser Ala Ala Ala Asp Thr Leu Ser Met Arg         35 40 45 Thr Ser Ala Arg Ala Ala Pro Arg His Gln His Gln Gln Ala Arg Arg     50 55 60 Gly Ala Arg Phe Pro Ser Leu Val Val Cys Ala Ser Ala Gly Ala Met 65 70 75 80 Ala Ala Ser Leu Pro Thr Met Pro Pro Ser Gly Tyr Asp Gln Val Arg                 85 90 95 Asn Gly Val Pro Arg Gly Gln Val Val Asn Ile Ser Tyr Phe Ser Thr             100 105 110 Ala Thr Asn Ser Thr Arg Pro Ala Arg Val Tyr Leu Pro Pro Gly Tyr         115 120 125 Ser Lys Asp Lys Lys Tyr Ser Val Leu Tyr Leu Leu His Gly Ile Gly     130 135 140 Gly Ser Glu Asn Asp Trp Phe Glu Gly Gly Gly Arg Ala Asn Val Ile 145 150 155 160 Ala Asp Asn Leu Ile Ala Glu Gly Lys Ile Lys Pro Leu Ile Ile Val                 165 170 175 Thr Pro Asn Thr Asn Ala Ala Gly Pro Gly Ile Ala Asp Gly Tyr Glu             180 185 190 Asn Phe Thr Lys Asp Leu Leu Asn Ser Leu Ile Pro Tyr Ile Glu Ser         195 200 205 Asn Tyr Ser Val Tyr Thr Asp Arg Glu His Arg Ala Ile Ala Gly Leu     210 215 220 Ser Met Gly Gly Gly Gln Ser Phe Asn Ile Gly Leu Thr Asn Leu Asp 225 230 235 240 Lys Phe Ala Tyr Ile Gly Pro Ile Ser Ala Ala Pro Asn Thr Tyr Pro                 245 250 255 Asn Glu Arg Leu Phe Pro Asp Gly Gly Lys Ala Ala Arg Glu Lys Leu             260 265 270 Lys Leu Leu Phe Ile Ala Cys Gly Thr Asn Asp Ser Leu Ile Gly Phe         275 280 285 Gly Gln Arg Val His Glu Tyr Cys Val Ala Asn Asn Ile Asn His Val     290 295 300 Tyr Trp Leu Ile Gln Gly Gly Gly His Asp Phe Asn Val Trp Lys Pro 305 310 315 320 Gly Leu Trp Asn Phe Leu Gln Met Ala Asp Glu Ala Gly Leu Thr Arg                 325 330 335 Asp Gly Asn Thr Pro Val Pro Thr Pro Ser Pro Lys Pro Ala Asn Thr             340 345 350 Arg Ile Glu Ala Glu Asp Tyr Asp Gly Ile Asn Ser Ser Ser Ile Glu         355 360 365 Ile Ile Gly Val Pro Pro Glu Gly Gly Arg Gly Ile Gly Tyr Ile Thr     370 375 380 Ser Gly Asp Tyr Leu Val Tyr Lys Ser Ile Asp Phe Gly Asn Gly Ala 385 390 395 400 Thr Ser Phe Lys Ala Lys Val Ala Asn Ala Asn Thr Ser Asn Ile Glu                 405 410 415 Leu Arg Leu Asn Gly Pro Asn Gly Thr Leu Ile Gly Thr Leu Ser Val             420 425 430 Lys Ser Thr Gly Asp Trp Asn Thr Tyr Glu Glu Gln Thr Cys Ser Ile         435 440 445 Ser Lys Val Thr Gly Ile Asn Asp Leu Tyr Leu Val Phe Lys Gly Pro     450 455 460 Val Asn Ile Asp Trp Phe Thr Phe Gly Val 465 470 <210> 107 <211> 1263 <212> DNA <213> artificial sequence <220> <223> plasmid 13347 <400> 107 atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccatg 60 gccgcctccc tcccgaccat gccgccgtcc ggctacgacc aggtgcgcaa cggcgtgccg 120 cgcggccagg tggtgaacat ctcctacttc tccaccgcca ccaactccac ccgcccggcc 180 cgcgtgtacc tcccgccggg ctactccaag gacaagaagt actccgtgct ctacctcctc 240 cacggcatcg gcggctccga gaacgactgg ttcgagggcg gcggccgcgc caacgtgatc 300 gccgacaacc tcatcgccga gggcaagatc aagccgctca tcatcgtgac cccgaacacc 360 aacgccgccg gcccgggcat cgccgacggc tacgagaact tcaccaagga cctcctcaac 420 tccctcatcc cgtacatcga gtccaactac tccgtgtaca ccgaccgcga gcaccgcgcc 480 atcgccggcc tctctatggg cggcggccag tccttcaaca tcggcctcac caacctcgac 540 aagttcgcct acatcggccc gatctccgcc gccccgaaca cctacccgaa cgagcgcctc 600 ttcccggacg gcggcaaggc cgcccgcgag aagctcaagc tcctcttcat cgcctgcggc 660 accaacgact ccctcatcgg cttcggccag cgcgtgcacg agtactgcgt ggccaacaac 720 atcaaccacg tgtactggct catccagggc ggcggccacg acttcaacgt gtggaagccg 780 ggcctctgga acttcctcca gatggccgac gaggccggcc tcacccgcga cggcaacacc 840 ccggtgccga ccccgtcccc gaagccggcc aacacccgca tcgaggccga ggactacgac 900 ggcatcaact cctcctccat cgagatcatc ggcgtgccgc cggagggcgg ccgcggcatc 960 ggctacatca cctccggcga ctacctcgtg tacaagtcca tcgacttcgg caacggcgcc 1020 acctccttca aggccaaggt ggccaacgcc aacacctcca acatcgagct tcgcctcaac 1080 ggcccgaacg gcaccctcat cggcaccctc tccgtgaagt ccaccggcga ctggaacacc 1140 tacgaggagc agacctgctc catctccaag gtgaccggca tcaacgacct ctacctcgtg 1200 ttcaagggcc cggtgaacat cgactggttc accttcggcg tgtccgagaa ggacgaactc 1260 tag 1263 <210> 108 <211> 420 <212> PRT <213> artificial sequence <220> <223> plasmid 13347 <400> 108 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Met Ala Ala Ser Leu Pro Thr Met Pro Pro Ser Gly Tyr             20 25 30 Asp Gln Val Arg Asn Gly Val Pro Arg Gly Gln Val Val Asn Ile Ser         35 40 45 Tyr Phe Ser Thr Ala Thr Asn Ser Thr Arg Pro Ala Arg Val Tyr Leu     50 55 60 Pro Pro Gly Tyr Ser Lys Asp Lys Lys Tyr Ser Val Leu Tyr Leu Leu 65 70 75 80 His Gly Ile Gly Gly Ser Glu Asn Asp Trp Phe Glu Gly Gly Gly Arg                 85 90 95 Ala Asn Val Ile Ala Asp Asn Leu Ile Ala Glu Gly Lys Ile Lys Pro             100 105 110 Leu Ile Ile Val Thr Pro Asn Thr Asn Ala Ala Gly Pro Gly Ile Ala         115 120 125 Asp Gly Tyr Glu Asn Phe Thr Lys Asp Leu Leu Asn Ser Leu Ile Pro     130 135 140 Tyr Ile Glu Ser Asn Tyr Ser Val Tyr Thr Asp Arg Glu His Arg Ala 145 150 155 160 Ile Ala Gly Leu Ser Met Gly Gly Gly Gln Ser Phe Asn Ile Gly Leu                 165 170 175 Thr Asn Leu Asp Lys Phe Ala Tyr Ile Gly Pro Ile Ser Ala Ala Pro             180 185 190 Asn Thr Tyr Pro Asn Glu Arg Leu Phe Pro Asp Gly Gly Lys Ala Ala         195 200 205 Arg Glu Lys Leu Lys Leu Leu Phe Ile Ala Cys Gly Thr Asn Asp Ser     210 215 220 Leu Ile Gly Phe Gly Gln Arg Val His Glu Tyr Cys Val Ala Asn Asn 225 230 235 240 Ile Asn His Val Tyr Trp Leu Ile Gln Gly Gly Gly His Asp Phe Asn                 245 250 255 Val Trp Lys Pro Gly Leu Trp Asn Phe Leu Gln Met Ala Asp Glu Ala             260 265 270 Gly Leu Thr Arg Asp Gly Asn Thr Pro Val Pro Thr Pro Ser Pro Lys         275 280 285 Pro Ala Asn Thr Arg Ile Glu Ala Glu Asp Tyr Asp Gly Ile Asn Ser     290 295 300 Ser Ser Ile Glu Ile Ile Gly Val Pro Pro Glu Gly Gly Arg Gly Ile 305 310 315 320 Gly Tyr Ile Thr Ser Gly Asp Tyr Leu Val Tyr Lys Ser Ile Asp Phe                 325 330 335 Gly Asn Gly Ala Thr Ser Phe Lys Ala Lys Val Ala Asn Ala Asn Thr             340 345 350 Ser Asn Ile Glu Leu Arg Leu Asn Gly Pro Asn Gly Thr Leu Ile Gly         355 360 365 Thr Leu Ser Val Lys Ser Thr Gly Asp Trp Asn Thr Tyr Glu Glu Gln     370 375 380 Thr Cys Ser Ile Ser Lys Val Thr Gly Ile Asn Asp Leu Tyr Leu Val 385 390 395 400 Phe Lys Gly Pro Val Asn Ile Asp Trp Phe Thr Phe Gly Val Ser Glu                 405 410 415 Lys Asp Glu Leu             420 <210> 109 <211> 1296 <212> DNA <213> artificial sequence <220> <223> plasmid 11267 <400> 109 atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc caccagcgct 60 gcgcagtccg agccggagct gaagctggag tccgtggtga tcgtgtcccg ccacggcgtg 120 cgcgccccga ccaaggccac ccagctcatg caggacgtga ccccggacgc ctggccgacc 180 tggccggtga agctcggcga gctgaccccg cgcggcggcg agctgatcgc ctacctcggc 240 cactactggc gccagcgcct cgtggccgac ggcctcctcc cgaagtgcgg ctgcccgcag 300 tccggccagg tggccatcat cgccgacgtg gacgagcgca cccgcaagac cggcgaggcc 360 ttcgccgccg gcctcgcccc ggactgcgcc atcaccgtgc acacccaggc cgacacctcc 420 tccccggacc cgctcttcaa cccgctcaag accggcgtgt gccagctcga caacgccaac 480 gtgaccgacg ccatcctgga gcgcgccggc ggctccatcg ccgacttcac cggccactac 540 cagaccgcct tccgcgagct ggagcgcgtg ctcaacttcc cgcagtccaa cctctgcctc 600 aagcgcgaga agcaggacga gtcctgctcc ctcacccagg ccctcccgtc cgagctgaag 660 gtgtccgccg actgcgtgtc cctcaccggc gccgtgtccc tcgcctccat gctcaccgaa 720 atcttcctcc tccagcaggc ccagggcatg ccggagccgg gctggggccg catcaccgac 780 tcccaccagt ggaacaccct cctctccctc cacaacgccc agttcgacct cctccagcgc 840 accccggagg tggcccgctc ccgcgccacc ccgctcctcg acctcatcaa gaccgccctc 900 accccgcacc cgccgcagaa gcaggcctac ggcgtgaccc tcccgacctc cgtgctcttc 960 atcgccggcc acgacaccaa cctcgccaac ctcggcggcg ccctggagct gaactggacc 1020 ctcccgggcc agccggacaa caccccgccg ggcggcgagc tggtgttcga gcgctggcgc 1080 cgcctctccg acaactccca gtggattcag gtgtccctcg tgttccagac cctccagcag 1140 atgcgcgaca agaccccgct ctccctcaac accccgccgg gcgaggtgaa gctcaccctc 1200 gccggctgcg aggagcgcaa cgcccagggc atgtgctccc tcgccggctt cacccagatc 1260 gtgaacgagg cccgcatccc ggcctgctcc ctctaa 1296 <210> 110 <211> 431 <212> PRT <213> artificial sequence <220> <223> plasmid 11267 aa sequence <400> 110 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Ala Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val             20 25 30 Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln         35 40 45 Leu Met Gln Asp Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys     50 55 60 Leu Gly Glu Leu Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly 65 70 75 80 His Tyr Trp Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Pro Lys Cys                 85 90 95 Gly Cys Pro Gln Ser Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu             100 105 110 Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp         115 120 125 Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro     130 135 140 Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala Asn 145 150 155 160 Val Thr Asp Ala Ile Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe                 165 170 175 Thr Gly His Tyr Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn             180 185 190 Phe Pro Gln Ser Asn Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser         195 200 205 Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp     210 215 220 Cys Val Ser Leu Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu 225 230 235 240 Ile Phe Leu Leu Gln Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly                 245 250 255 Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His Asn             260 265 270 Ala Gln Phe Asp Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg         275 280 285 Ala Thr Pro Leu Leu Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro     290 295 300 Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe 305 310 315 320 Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu                 325 330 335 Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly             340 345 350 Glu Leu Val Phe Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp         355 360 365 Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys     370 375 380 Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu 385 390 395 400 Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly                 405 410 415 Phe Thr Gln Ile Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu             420 425 430 <210> 111 <211> 1314 <212> DNA <213> artificial sequence <220> <223> plasmid 11268 <400> 111 atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc caccagcgct 60 gcgcagtccg agccggagct gaagctggag tccgtggtga tcgtgtcccg ccacggcgtg 120 cgcgccccga ccaaggccac ccagctcatg caggacgtga ccccggacgc ctggccgacc 180 tggccggtga agctcggcga gctgaccccg cgcggcggcg agctgatcgc ctacctcggc 240 cactactggc gccagcgcct cgtggccgac ggcctcctcc cgaagtgcgg ctgcccgcag 300 tccggccagg tggccatcat cgccgacgtg gacgagcgca cccgcaagac cggcgaggcc 360 ttcgccgccg gcctcgcccc ggactgcgcc atcaccgtgc acacccaggc cgacacctcc 420 tccccggacc cgctcttcaa cccgctcaag accggcgtgt gccagctcga caacgccaac 480 gtgaccgacg ccatcctgga gcgcgccggc ggctccatcg ccgacttcac cggccactac 540 cagaccgcct tccgcgagct ggagcgcgtg ctcaacttcc cgcagtccaa cctctgcctc 600 aagcgcgaga agcaggacga gtcctgctcc ctcacccagg ccctcccgtc cgagctgaag 660 gtgtccgccg actgcgtgtc cctcaccggc gccgtgtccc tcgcctccat gctcaccgaa 720 atcttcctcc tccagcaggc ccagggcatg ccggagccgg gctggggccg catcaccgac 780 tcccaccagt ggaacaccct cctctccctc cacaacgccc agttcgacct cctccagcgc 840 accccggagg tggcccgctc ccgcgccacc ccgctcctcg acctcatcaa gaccgccctc 900 accccgcacc cgccgcagaa gcaggcctac ggcgtgaccc tcccgacctc cgtgctcttc 960 atcgccggcc acgacaccaa cctcgccaac ctcggcggcg ccctggagct gaactggacc 1020 ctcccgggcc agccggacaa caccccgccg ggcggcgagc tggtgttcga gcgctggcgc 1080 cgcctctccg acaactccca gtggattcag gtgtccctcg tgttccagac cctccagcag 1140 atgcgcgaca agaccccgct ctccctcaac accccgccgg gcgaggtgaa gctcaccctc 1200 gccggctgcg aggagcgcaa cgcccagggc atgtgctccc tcgccggctt cacccagatc 1260 gtgaacgagg cccgcatccc ggcctgctcc ctctccgaga aggacgagct gtaa 1314 <210> 112 <211> 437 <212> PRT <213> artificial sequence <220> <223> plasmid 11268 amino acid sequence <400> 112 Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Ala Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val             20 25 30 Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln         35 40 45 Leu Met Gln Asp Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys     50 55 60 Leu Gly Glu Leu Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly 65 70 75 80 His Tyr Trp Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Pro Lys Cys                 85 90 95 Gly Cys Pro Gln Ser Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu             100 105 110 Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp         115 120 125 Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro     130 135 140 Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala Asn 145 150 155 160 Val Thr Asp Ala Ile Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe                 165 170 175 Thr Gly His Tyr Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn             180 185 190 Phe Pro Gln Ser Asn Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser         195 200 205 Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp     210 215 220 Cys Val Ser Leu Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu 225 230 235 240 Ile Phe Leu Leu Gln Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly                 245 250 255 Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His Asn             260 265 270 Ala Gln Phe Asp Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg         275 280 285 Ala Thr Pro Leu Leu Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro     290 295 300 Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe 305 310 315 320 Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu                 325 330 335 Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly             340 345 350 Glu Leu Val Phe Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp         355 360 365 Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys     370 375 380 Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu 385 390 395 400 Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly                 405 410 415 Phe Thr Gln Ile Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu Ser             420 425 430 Glu Lys Asp Glu Leu         435  

Claims (234)

a) SEQ ID NOs: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, 59, 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108 and 110 or complements thereof, or SEQ ID NOs: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41 Any of, 43, 46, 48, 50, 52, 59, 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108 and 110 One complement and hybridize under low stringency hybridization conditions, α-amylase, pullulase, α-glucosidase, glucose isomerase, gulucoamylase, xylanase, protease, cellulase, glucanase, Encoding a polypeptide having beta glucosidase or phytase activity or b) SEQ ID NOs: 10, 13, 14, 15, 16, 18, 20, 24, 26, 27, 28, 29, 30, 33, 34, 35 Polypeptides comprising, 36, 38, 40, 42, 44, 45, 47, 49, 51, 62, 64, 66, 70, 80, 82, 84, 86, 88, 90, 92, 109 or 111 or Minutes encoding its enzymatically active fragments The polynucleotide. The fusion polypeptide of claim 1, wherein the polynucleotide comprises a first polypeptide having α-amylase, pullulanase, α-glucosidase, glucose isomerase or glucoamylase activity, and a second polypeptide. Isolated polynucleotides encoding. The isolated polynucleotide of claim 2, wherein the second peptide comprises a signal sequence peptide. The isolated polynucleotide of claim 3, wherein the signal sequence peptide targets the first polypeptide to vacuoles, cytoplasmic reticulum, chloroplasts, starch granules, seeds or cell walls of the plant. The isolated polynucleotide of claim 3, wherein the signal sequence is an N-terminal signal sequence from waxy, an N-terminal signal sequence from γ-Zane, a starch binding domain, or a C-terminal starch binding domain. The isolated polynucleotide of claim 1, wherein the polynucleotide hybridizes with the complement of any one of SEQ ID NOs: 2, 9 or 52 under low stringency hybridization conditions and encodes a polypeptide having α-amylase activity. The isolated polynucleotide of claim 1, wherein the polynucleotide hybridizes with the complement of any one of SEQ ID NOs: 4 or 25 under low stringency hybridization conditions and encodes a polypeptide having pullulanase activity. The isolated polynucleotide of claim 1, wherein the polynucleotide hybridizes with the complement of SEQ ID NO: 6 and encodes a polypeptide having α-glucosidase activity. The method of claim 1, wherein the polynucleotide hybridizes to the complement of any one of SEQ ID NOs: 19, 21, 37, 39, 41 or 43 under low stringency hybridization conditions and encodes a polypeptide having glucose isomerase activity. Isolated polynucleotides. The isolated polynucleotide of claim 1, wherein the polynucleotide hybridizes with the complement of any one of SEQ ID NOs: 46, 48, 50, or 59 under low stringency hybridization conditions and encodes a polypeptide having glucoamylase activity. An isolated polynucleotide comprising any one of SEQ ID NOs: 2 or 9 or its complement. An isolated polynucleotide comprising any of SEQ ID NO: 4 or 25 or the complement thereof. An isolated polynucleotide comprising SEQ ID NO: 6 or its complement. An isolated polynucleotide comprising any one of SEQ ID NOs: 19, 21, 37, 39, 41 or 43 or its complement. An isolated polynucleotide comprising any one of SEQ ID NOs: 46, 48, 50, or 59 or its complement. a) SEQ ID NOs: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41, 43, 46, 48, 50, 52, 59, 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108 or 110 or complements thereof, or SEQ ID NOs: 2, 4, 6, 9, 19, 21, 25, 37, 39, 41 Any of, 43, 46, 48, 50, 52, 59, 61, 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108 or 110 One complement and hybridize under low stringency hybridization conditions, α-amylase, pullulase, α-glucosidase, glucose isomerase, glucoamylase, xylanase, protease, cellulase, glucanase, beta Encoding a polypeptide having glucosidase or phytase activity or b) SEQ ID NOs: 10, 13, 14, 15, 16, 18, 20, 24, 26, 27, 28, 29, 30, 33, 34, 35, Polypeptides comprising 36, 38, 40, 42, 44, 45, 47, 49, 51, 62, 64, 66, 70, 80, 82, 84, 86, 88, 90, 92, 109 or 111 or Poles Encoding Enzymatically Active Fragments An expression cassette comprising a nucleotide. The expression cassette of claim 16, wherein said expression cassette is operably linked to a promoter. The expression cassette of claim 17, wherein the promoter is an inducible promoter. The expression cassette of claim 17, wherein the promoter is a tissue-specific promoter. The expression cassette of claim 19, wherein the promoter is an endosperm-specific promoter. The expression cassette of claim 20, wherein the endosperm-specific promoter is a corn γ-zein promoter or a corn ADP-gpp promoter. The expression cassette of claim 21, wherein the promoter comprises SEQ ID NO: 11 or SEQ ID NO: 12. 23. The expression cassette of claim 16, wherein the polynucleotide is oriented in the sense direction with respect to the promoter. The expression cassette of claim 16, wherein the polynucleotide of a) further encodes a signal sequence operably linked to a polypeptide encoded by the polynucleotide. The expression cassette of claim 24, wherein the signal sequence targets the operably linked polypeptide to vacuoles, cytoplasmic reticulum, chloroplasts, starch granules, seeds or cell walls of the plant. The expression cassette of claim 25, wherein the signal sequence is an N-terminal signal sequence from waxy or an N-terminal signal sequence from γ-Zane. The expression cassette of claim 25, wherein the signal sequence is a starch binding domain. The expression cassette of claim 16, wherein the polynucleotide of b) is operably linked to a tissue-specific promoter. The expression cassette of claim 28, wherein the tissue-specific promoter is a Zea mays γ-zein promoter or a Zea mays ADP-gpp promoter. An expression cassette comprising a polynucleotide comprising any one of SEQ ID NOs: 2 or 9 or its complement. An expression cassette comprising a polynucleotide comprising SEQ ID NO: 6 or its complement. An expression cassette comprising a polynucleotide comprising any one of SEQ ID NOs: 19, 21, 37, 39, 41 or 43 or its complement. An expression cassette comprising a polynucleotide comprising any one of SEQ ID NOs: 46, 48, 50 or 59 or its complement. An expression cassette comprising a polynucleotide comprising any of SEQ ID NO: 4 or 25 or the complement thereof. SEQ ID NOs: 10, 13, 14, 15, 16, 24, 26, 27, 28, 29, 30, 33, 34, 35, 36, 38, 40, 42, 44, 45, 47, 49, 51, 61, A polypeptide having an amino acid sequence of any of 63, 65, 79, 81, 83, 85, 87, 89, 91, 93, 94, 95, 96, 97, 99, 108 or 110 or an enzymatically active fragment thereof An expression cassette comprising a polynucleotide encoding. An expression cassette comprising a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 10, 13, 14, 15, 16, 33, 35 or 51 or a polynucleotide encoding an active fragment thereof having α-amylase activity. An expression cassette comprising a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 3, 24 or 34, or a polynucleotide encoding an active fragment thereof having pullulanase activity. An expression cassette comprising a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 5, 26 or 27 or a polynucleotide encoding an active fragment thereof having α-glucosidase activity. Expression cassette comprising a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 18, 20, 28, 29, 30, 38, 40, 42 or 44 or a polynucleotide encoding its active fragment having glucose isomerase activity . An expression cassette comprising a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 45, 47 or 49 or a polynucleotide encoding its active fragment having glucoamylase activity. A vector comprising an expression cassette according to claim 16. 41. A vector comprising an expression cassette according to any one of claims 30-40. A cell comprising an expression cassette according to claim 16. 41. A cell comprising an expression cassette according to any one of claims 30-40. 45. The cell of claim 44, wherein the cell is selected from the group consisting of Agrobacterium , monocotyledonous cells, dicotyledonous cells, Liliopsida cells, Panicoideae cells, corn cells and grain cells. 46. The cell of claim 45 which is corn cell or rice cell. 46. The cell of claim 45, wherein the cell is selected from the group consisting of Agrobacterium, monocotyledonous cells, dicotyledonous cells, Lilium ganglia cells, millet follicle cells, corn cells and grain cells. 48. The cell of claim 47 which is corn cell. A plant stably transformed with the vector according to claim 41. A plant stably transformed with the vector according to claim 42. Comprising an α-amylase having an amino acid sequence of any one of SEQ ID NOs: 1, 10, 13, 14, 15, 16, 33 or 35, or encoded by a polynucleotide comprising any of SEQ ID NOs: 2 or 9 Plant stably transformed with the vector. The plant of claim 51, wherein the α-amylase is superthermophilic. A plant having an amino acid sequence of any one of SEQ ID NOs: 24 or 34, or stably transformed with a vector comprising a pullulanase encoded by a polynucleotide comprising any one of SEQ ID NOs: 4 or 25. A plant having an amino acid sequence of any one of SEQ ID NOs: 26 or 27 or stably transformed with a vector comprising α-glucosidase encoded by a polynucleotide comprising SEQ ID NO: 6. 55. The plant of claim 54, wherein the α-glucosidase is superthermophilic. Poly having an amino acid sequence of any one of SEQ ID NOs: 18, 20, 28, 29, 30, 38, 40, 42 or 44, or comprising any of SEQ ID NOs: 19, 21, 37, 39, 41 or 43 A plant stably transformed with a vector comprising a glucose isomerase encoded by a nucleotide. The plant of claim 56, wherein the α-glucosidase is superthermophilic. Stably transformed with a vector comprising a glucose amylase having an amino acid sequence of any one of SEQ ID NOs: 45, 47 or 49 or encoded by a polynucleotide comprising any of SEQ ID NOs: 46, 48, 50 or 59 plant. 59. The plant of claim 58, wherein the glucose amylase is superthermophilic. Seed, fruit or grain of a plant according to claim 49. Seed, fruit or grain of a plant according to claim 50. Seed, fruit or grain of a plant according to claim 51. Seed, fruit or grain of a plant according to claim 53. Seed, fruit or grain of a plant according to claim 54. Seed, fruit or grain of a plant according to claim 56. Seed, fruit or grain of a plant according to claim 58. A transformed plant in which the genome is enhanced with recombinant polynucleotides encoding one or more processing enzymes operatively linked to a promoter sequence. 67. The plant of claim 67, which is a monocotyledonous plant. 69. The plant of claim 68, wherein the monocotyledonous plant is corn or rice. 67. The plant of claim 67, which is a dicotyledonous plant. 67. The plant of claim 67, which is a cereal plant or a commercially grown plant. The processing enzyme of claim 67, wherein the processing enzyme is α-amylase, glucoamylase, glucose isomerase, glucanase, β-amylase, α-glucosidase, isoamylase, pullulanase, neo-pululanase, iso- Pullulanase, amylopululase, cellulase, exo-1,4-β-cellobiohydrolase, exo-1,3-β-D-glucanase, β-glucosidase, endoglucan Agent, L-arabinase, α-arabinosidase, galactanase, galactosidase, mannase, mannosidase, xylanase, xylosidase, protease, glucanase, esterase, Plant selected from the group consisting of phytase and lipase. 73. The process of claim 72, wherein the processing enzyme is α-amylase, glucoamylase, glucose isomerase, β-amylase, α-glucosidase, isoamylase, pullulanase, neo-pululanase, iso-pululanase and A plant that is a starch-processing enzyme selected from the group consisting of amylopululase. 75. The plant of claim 73, wherein the enzyme is selected from α-amylase, glucoamylase, glucose isomerase, α-glucosidase, and pullulanase. 75. The plant of claim 74, wherein the enzyme is superthermophilic. 73. The plant of claim 72, wherein the enzyme is a non-starch degrading enzyme selected from the group consisting of protease, glucanase, xylanase, cellulase, β-glucosidase, esterase, phytase and lipase. 77. The plant of claim 76, wherein the enzyme is superthermophilic. 67. The plant of claim 67, wherein the enzyme accumulates in the vacuoles, cytoplasmic reticulum, chloroplasts, starch granules, seeds or cell walls of the plant. 79. The plant of claim 78, wherein the enzyme accumulates in the cytoplasmic net. 79. The plant of claim 78, wherein the enzyme accumulates in starch granules. 68. The plant of claim 67, wherein the genome is further enhanced with a second recombinant polynucleotide comprising a non-thermophilic enzyme. Enhances traits with recombinant polynucleotides encoding at least one processing enzyme selected from the group consisting of α-amylase, glucoamylase, glucose isomerase, α-glucosidase and pullulanase, wherein the genome is operably linked to a promoter sequence Switched plants. 83. The transformed plant of claim 82, wherein the processing enzyme is super thermophilic. 83. The transformed plant of claim 82, wherein the plant is corn or rice. Enhances traits with recombinant polynucleotides encoding at least one processing enzyme selected from the group consisting of α-amylase, glucoamylase, glucose isomerase, α-glucosidase and pullulanase, wherein the genome is operably linked to a promoter sequence Switched Corn Plants. 86. The transgenic corn plant according to claim 85, wherein the processing enzyme is super thermophilic. A transformed plant augmented with recombinant polynucleotide having SEQ ID NO: 2, 9 or 52, wherein the genome is operably linked to a promoter and signal sequence. A transformed plant augmented with recombinant polynucleotide having a sequence of SEQ ID NO: 4 or 25, wherein the genome is operably linked to a promoter and signal sequence. A transformed plant augmented with recombinant polynucleotide having the sequence of SEQ ID NO: 6, wherein the genome is operably linked to a promoter and signal sequence. A transformed plant whose genome has been enhanced with recombinant polynucleotides having the sequences of SEQ ID NOs: 19, 21, 37, 39, 41 or 43. A transformed plant whose genome has been enhanced with recombinant polynucleotides having the sequences of SEQ ID NOs: 46, 48, 50, or 59. The product of the transformed plant according to claim 82. 86. The product of a transformed plant according to claim 85. 92. The product of a transformed plant according to any one of claims 87-91. 95. The product of claim 92, which is seed, fruit or grain. 95. The product of claim 92, which is a processing enzyme, starch or sugar. A plant obtained from a plant according to claim 82. A plant obtained from a plant according to claim 85. 92. A plant obtained from the plant according to any one of claims 87-91. The plant of claim 97 which is a hybrid plant. 99. The plant of claim 98, which is a hybrid plant. 105. The plant of claim 99, which is a hybrid plant. 98. The plant of claim 97, which is a suburban plant. 99. The plant of claim 98, which is a suburban plant. 105. The plant of claim 99, which is a suburban plant. A starch composition comprising at least one processing enzyme which is a protease, glucanase, phytase, lipase, xylanase, cellulase, β-glucosidase or esterase. 107. The starch composition of claim 106, wherein the enzyme is superthermophilic. A grain comprising one or more processing enzymes that are α-amylase, pullulanase, α-glucosidase, glucoamylase or glucose isomerase. 109. The grain of claim 108, wherein the enzyme is superthermophilic. a) a grain comprising one or more non-starch processing enzymes, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding one or more enzymes, is subjected to starch granules under conditions that activate one or more such enzymes And yielding a mixture comprising the non-starch decomposition product; And b) separating the starch granules from the mixture. 119. The method of claim 110, wherein the enzyme is a protease, glucanase, phytase, lipase, xylanase, cellulase, β-glucosidase or esterase. 112. The method of claim 111, wherein the enzyme is superthermophilic. 118. The method of claim 110, wherein the grains are cracked grains. 118. The method of claim 110, wherein the grains are treated under low humidity conditions. 118. The method of claim 110, wherein the grains are treated under high humidity conditions. 111. The method of claim 110, wherein the grains are treated with sulfur dioxide. 118. The method of claim 110, further comprising separating the non-starch product from the mixture. Starch obtained by the method according to claim 110. Starch obtained by the method according to claim 112. A non-starch product obtained by the method according to claim 110. A non-starch product obtained by the method according to claim 112. The genome is enriched with an expression cassette encoding one or more starch-degrading or starch-isomerizing enzymes, and the transformed corn or portions thereof expressing in the endocrine are processed under conditions that activate one or more such enzymes. A method of producing ultra-high sugar corn, comprising converting polysaccharides in corn to sugar to yield hypersweet corn. 123. The method of claim 122, wherein the expression cassette further comprises a promoter operably linked to a polynucleotide encoding the enzyme. 123. The method of claim 123, wherein the promoter is a constitutive promoter. 123. The method of claim 123, wherein the promoter is a seed-specific promoter. 123. The method of claim 123, wherein the promoter is an endosperm-specific promoter. 123. The method of claim 123, wherein the enzyme is superthermia. 127. The method of claim 127, wherein the enzyme is α-amylase. 123. The method of claim 122, wherein the expression cassette further comprises a polynucleotide encoding a signal sequence operably linked to one or more enzymes. 129. The method of claim 129, wherein the signal sequence directs the superthermophilic enzyme to apoplasts. 129. The method of claim 129, wherein the signal sequence directs the super thermophilic enzyme to cytoplasmic reticulum. 123. The method of claim 122, wherein the enzyme comprises any of SEQ ID NOs: 13, 14, 15, 16, 33, or 35. The genome is enriched with an expression cassette encoding α-amylase, and the expression cassette is treated with transgenic maize or parts thereof expressing in endosperm under conditions that activate one or more enzymes to convert polysaccharides in maize to sugars for ultra high sugar content. A method of producing ultra-high sugar corn, including yielding corn. 134. The method of claim 133, wherein the enzyme is superthermophilic.  134. The method of claim 134, wherein the superthermophilic α-amylase comprises the amino acid sequence of any one of SEQ ID NOs: 10, 13, 14, 15, 16, 33, or 35, or an enzymatically active fragment thereof having α-amylase activity. 134. The expression cassette of claim 134, wherein the expression cassette hybridizes with any one of SEQ ID NOs: 2, 9, or 52, its complement, or any of SEQ ID NOs: 2, 9, or 52 under low stringency hybridization conditions and A method comprising a polynucleotide selected from polynucleotides encoding a polypeptide having. a) processing a plant part comprising starch granules and one or more processing enzymes obtained from a transformed plant whose genome has been enhanced with an expression cassette encoding one or more starch processing enzymes, under conditions which activate one or more enzymes, Accordingly processing the starch granules to form an aqueous solution comprising the hydrolyzed starch product; And b) collecting the aqueous solution comprising the hydrolyzed starch product. 137. The method of claim 137, wherein the hydrolyzed starch product comprises dextrin, maltooligosaccharides, sugars, and / or mixtures thereof. 138. The enzyme of claim 137, wherein the enzyme is α-amylase, α-glucosidase, glucoamylase, pullulanase, amylopululase, glucose isomerase, β-amylase, isoamylase, neopululase, iso- The pullulanase or a combination thereof. 138. The method of claim 137, wherein the one or more processing enzymes is superthermophilic. 139. The method of claim 139, wherein the one or more processing enzymes is superthermophilic. 138. The method of claim 137, wherein the genome of the plant part is further enhanced with an expression cassette encoding a non-superthermotropic starch processing enzyme. 142. The method of claim 142, wherein the non-superthermotropic starch processing enzyme is selected from the group consisting of amylase, glucoamylase, α-glucosidase, pullulanase, glucose isomerase, and combinations thereof. 138. The method of claim 137, wherein the one or more processing enzymes are expressed in endocrine.  138. The method of claim 137, wherein the plant portion is grain. 138. The method of claim 137, wherein the plant portion is from corn, wheat, barley, rye, oats, sugar cane or rice. 138. The method of claim 137, wherein the one or more processing enzymes is operably linked to a signal sequence that targets the promoter and enzyme to starch granules or cytoplasmic reticulum or cell wall. 137. The method of claim 137, further comprising separating the hydrolyzed starch product. 138. The method of claim 137, further comprising fermenting the hydrolyzed starch product. a) processing a plant part comprising starch granules and one or more starch processing enzymes, obtained from transformed plants whose genome has been enhanced with an expression cassette encoding one or more α-amylases, under conditions that activate one or more enzymes; Thereby processing the starch granules to form an aqueous solution comprising the hydrolyzed starch product; And b) collecting the aqueous solution comprising the hydrolyzed starch product. 151. The method of claim 150, wherein the α-amylase is superthermophilic. 151. The method of claim 151, wherein the superthermophilic α-amylase comprises the amino acid sequence of any one of SEQ ID NOs: 1, 10, 13, 14, 15, 16, 33, or 35, or a fragment thereof having α-amylase activity. 152. The expression cassette of claim 151, wherein the expression cassette hybridizes with any one of SEQ ID NOs: 2, 9, 46, or 52, complements thereof, or any of SEQ ID NOs: 2, 9, 46, or 52 under low stringency hybridization conditions -A polynucleotide selected from polynucleotides encoding a polypeptide having amylase activity. 151. The method of claim 150, wherein the genome of the transformed plant further comprises a polynucleotide encoding a non-thermic starch-processing enzyme. 151. The method of claim 150, further comprising treating the plant portion with a non-superthermotropic starch-processing enzyme. A transformed plant part comprising one or more starch-processing enzymes present in cells of the plant, obtained from transformed plants whose genome is enhanced with an expression cassette encoding one or more processing enzymes. 158. The enzyme of claim 156, wherein the enzyme is α-amylase, glucoamylase, glucose isomerase, β-amylase, α-glucosidase, isoamylase, pullulanase, neo-pululanase, iso-pululanase and amyl Plant part that is a starch-processing enzyme selected from the group consisting of lopululanase. 158. The plant part of claim 156, wherein the enzyme is superthermophilic. 158. The plant portion of claim 156, wherein the plant is corn. One or more non-starch processing enzymes present in the cell walls or cells of the plant, obtained from transformed plants whose genome has been enhanced with an expression cassette encoding one or more non-starch processing enzymes or one or more non-starch polysaccharide processing enzymes. Transformed plant portion comprising. 161. The plant part of claim 160, wherein the enzyme is superthermophilic. 161. The plant part of claim 160, wherein the non-starch processing enzyme is selected from the group consisting of protease, glucanase, xylanase, esterase, phytase, cellulase, β-glucosidase or lipase. 161. The plant part according to any one of claims 156 or 160, which is an ear, seed, fruit, grain, stover, bran or bagasse. Α- having an amino acid sequence of any one of SEQ ID NOs: 1, 10, 13, 14, 15, 16, 33 or 35, or encoded by a polynucleotide comprising any of SEQ ID NOs: 2, 9, 46 or 52 Transformed plant portion comprising amylase. A transformed plant part comprising an α-glucosidase having an amino acid sequence of any one of SEQ ID NOs: 5, 26 or 27, or encoded by a polynucleotide comprising the sequence of SEQ ID NO: 6. Encoded by a polynucleotide having an amino acid sequence of any one of SEQ ID NOs: 28, 29, 30, 38, 40, 42 or 44 or comprising any of SEQ ID NOs: 19, 21, 37, 39, 41 or 43 Transformed plant part comprising glucose isomerase. A transformed plant portion having a amino acid sequence of SEQ ID 45 or SEQ ID 47 or 49 or comprising a glucoamylase encoded by a polynucleotide comprising any of SEQ ID NOs: 46, 48, 50 or 59. A transformed plant part comprising a pullulanase encoded by a polynucleotide comprising any of SEQ ID NO: 4 or 25. 156. A method of converting starch in a transformed plant part according to 156, comprising activating a starch processing enzyme contained in the transformed plant part according to 156. 168. A method of converting starch into starch-derived product in said plant portion, comprising activating an enzyme comprised in the transformed plant portion according to any of claims 164-168. Starch, dextrins, maltooligosaccharides or sugars prepared according to the method according to claim 169. Starch, dextrins, maltooligosaccharides or sugars prepared according to the method according to claim 170. a) activating at least one enzyme with a transformed plant portion comprising at least one non-starch polysaccharide processing enzyme, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding at least one non-starch polysaccharide processing enzyme Treating under conditions such that the non-starch polysaccharide is decomposed to form an aqueous solution comprising oligosaccharides and / or sugars; And b) collecting the aqueous solution comprising oligosaccharides and / or sugars, A method of using a transformed plant part comprising one or more non-starch processing enzymes in the cell wall or cell of the plant part. 174. The method of claim 173, wherein the non-starch polysaccharide processing enzyme is a protease, glucanase, phytase, lipase, xylanase, cellulase, β-glucosidase or esterase. a) A transgenic seed comprising one or more proteases or lipases, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding one or more enzymes, is subjected to amino acids and fatty acids under conditions that activate one or more enzymes. Calculating an aqueous mixture comprising; And b) A method of using a transgenic seed comprising at least one processing enzyme, comprising collecting the aqueous mixture. 175. The method of claim 175, wherein the amino acids, fatty acids, or both are separated. 175. The method of claim 175, wherein the one or more proteases or lipases are hyperthermia. a) a plant portion comprising at least one polysaccharide processing enzyme, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding at least one polysaccharide processing enzyme, is subjected to conditions under which the at least one enzyme is activated to activate the polysaccharide Degrading to form oligosaccharides or fermentable sugars; And b) incubating the fermentable sugars under conditions that promote the conversion of the fermentable sugars or oligosaccharides to ethanol. 178. The method of claim 178, wherein the plant part is grain, fruit, seed, stem, wood, vegetable, or root. 178. The method of claim 178, wherein the plant portion is obtained from a plant selected from the group consisting of oats, barley, wheat, strawberries, grapes, rye, corn, rice, potatoes, sugar beets, sugar cane, pineapples, grasses and trees. 178. The method of claim 178, wherein the polysaccharide processing enzyme is α-amylase, glucoamylase, α-glucosidase, glucose isomerase, pullulanase or combinations thereof. 178. The method of claim 178, wherein the polysaccharide processing enzyme is superthermophilic. 178. The method of claim 178, wherein the polysaccharide processing enzyme is mesophilic. 181. The method of claim 181, wherein the polysaccharide processing enzyme is superthermophilic. a) a group consisting of α-amylase, glucoamylase, α-glucosidase, glucose isomerase or pullulanase or combinations thereof, obtained from a transformed plant whose genome has been enhanced with an expression cassette encoding one or more enzymes Treating the plant part comprising at least one enzyme selected from among the conditions of activating at least one enzyme, thereby degrading the polysaccharide to form a fermentable sugar; And b) incubating the fermentable sugar under conditions that promote the conversion of the fermentable sugar to ethanol. 185. The method of claim 185, wherein the one or more enzymes are hyperthermia. 185. The method of claim 185, wherein the one or more enzymes are mesophilic. 185. The polynucleotide of claim 185, wherein the α-amylase has an amino acid sequence of any one of SEQ ID NOs: 1, 10, 13, 14, 15, 16, 33, or 35 or comprises any of SEQ ID NOs: 2 or 9 How is it encrypted? 185. The method of claim 185, wherein the α-glucosidase has an amino acid sequence of any one of SEQ ID NOs: 5, 26 or 27, or is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 6. The method of claim 185, wherein the glucose isomerase has an amino acid sequence of any one of SEQ ID NOs: 28, 29, 30, 38, 40, 42, or 44, or any of SEQ ID NOs: 19, 21, 37, 39, 41, or 43. A method of encoding by a polynucleotide comprising one. 185. The method of claim 185, wherein the glucoamylase has the amino acid sequence of SEQ ID NO: 45 or is encoded by a polynucleotide comprising any of SEQ ID NOs: 46, 48, or 50. 185. The method of claim 185, wherein the pullulanase has the amino acid sequence of SEQ ID NO: 24 or 34 or is encoded by a polynucleotide comprising any of SEQ ID NO: 4 or 25. a) a plant part comprising at least one non-starch processing enzyme, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding at least one enzyme, is subjected to conditions under which the at least one enzyme is activated, Degrading the starch polysaccharides into oligosaccharides and fermentable sugars; And b) incubating the fermentable sugar under conditions that promote the conversion of the fermentable sugar to ethanol. 199. The method of claim 193, wherein the non-starch processing enzyme is a protease, glucanase, phytase, lipase, xylanase, cellulase, β-glucosidase or esterase. a) a group consisting of α-amylase, glucoamylase, α-glucosidase, glucose isomerase or pullulanase or combinations thereof, obtained from a transformed plant whose genome has been enhanced with an expression cassette encoding one or more enzymes Treating the plant part comprising at least one enzyme selected from among the conditions of activating at least one enzyme, thereby degrading the polysaccharide to form a fermentable sugar; And b) incubating the fermentable sugar under conditions that promote the conversion of the fermentable sugar to ethanol. 199. The method of claim 195, wherein the one or more enzymes are superthermophilic. a) a plant part comprising at least one starch processing enzyme, obtained from a transformed plant whose genome is enhanced with an expression cassette encoding at least one enzyme, is subjected to conditions under which the at least one enzyme is activated, thereby Processing the starch granules with sugar to form a high sugar content product; And b) processing the high sugar product into a starch food product, the method comprising producing a high sugar starch food product without the addition of additional sweetener. 197. The method of claim 197, wherein the starch food is formed from a high sugar content product and water. 197. The method of claim 197, wherein the starch food contains malt, flavor, vitamin, mineral, colorant, or any combination thereof. 197. The method of claim 197, wherein the one or more enzymes is superthermophilic. 199. The method of claim 197, wherein the enzyme is α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. 197. The method of claim 197, wherein the plant is selected from the group consisting of soybean, rye, oats, barley, wheat, corn, rice, and sugar cane. 197. The method of claim 197, wherein the starch food is cereal food. 197. The method of claim 197, wherein the starch food is a breakfast food. 197. The method of claim 197, wherein the starch food is ready to eat food. 197. The method of claim 197, wherein the starch food is baked food. 197. The method of claim 197, wherein the processing is baking, boiling, heating, steaming, electric discharge, or any combination thereof. a) starches comprising one or more starch processing enzymes, obtained from a transformed plant whose genome is enriched with an expression cassette encoding one or more enzymes, are subjected to conditions under which one or more enzymes are activated, thereby digesting the starch Forming sugar to form high sugar starch; And b) adding a high sugar starch to the product to produce a high sugar starch containing product, thereby sweetening the starch-containing product without the addition of sweeteners. 208. The method of claim 208, wherein the transformed plant is selected from the group consisting of corn, soybean, rye, oats, barley, wheat, rice, and sugar cane. 208. The method of claim 208, wherein the one or more enzymes are superthermophilic. 208. The method of claim 208, wherein the at least one enzyme is α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. A starch food obtained by the method according to claim 197. A high sugar starch containing product obtained by the method according to claim 208. Fruits or vegetables comprising one or more polysaccharide processing enzymes obtained from a transformed plant whose genome has been enhanced with an expression cassette encoding one or more polysaccharide processing enzymes are processed under conditions that activate one or more enzymes and thus A method of sweetening a polysaccharide-containing fruit or vegetable, comprising processing the polysaccharides in the vegetable to form a sugar and yielding a high sugar or fruit. 214. The method of claim 214, wherein the fruit or vegetable is selected from the group consisting of potatoes, tomatoes, bananas, pumpkins, peas and beans. 214. The method of claim 214, wherein the one or more enzymes are superthermophilic. 214. The method of claim 214, wherein the enzyme is α-amylase, α-glucosidase, glucoamylase, pullulanase, glucose isomerase, or any combination thereof. 156. A process for preparing an aqueous solution comprising sugar, comprising treating the starch granules obtained from the plant portion according to claim 156 under conditions activating one or more enzymes to yield an aqueous solution comprising sugar. a) processing a plant part comprising starch granules and at least one starch processing enzyme, obtained from a transformed plant whose genome has been enhanced with an expression cassette encoding at least one starch processing enzyme, under conditions which activate the at least one enzyme, Thereby processing the starch granules to form an aqueous solution comprising dextrin or sugar; And b) collecting the aqueous solution comprising the starch derived product, wherein the starch derived product is prepared from the grain without wet or dry milling the grain prior to recovery of the starch-derived product. 219. The method of claim 219, wherein the one or more starch processing enzymes is superthermophilic. 84. An α-amylase, comprising culturing the transformed plant according to claim 82 and separating therefrom the α-amylase, glucoamylase, glucose isomerase, α-glucosidase and pullulanase, A method of separating glucoamylase, glucose isomerase, α-glucosidase and pullulanase. 228. The method of claim 221, wherein the α-amylase, glucoamylase, glucose isomerase, α-glucosidase and pullulanase are superthermophilic. a) mixing the transformed grains with water; b) heating the mixture; c) separating the solids from the dextrin syrup prepared in step (b) and d) collecting maltodextrin, the method of producing maltodextrin. 237. The method of claim 223, wherein the transformed grain comprises one or more starch processing enzymes. 224. The method of claim 224, wherein the starch processing enzyme is α-amylase, glucoamylase, α-glucosidase, and glucose isomerase. 255. The method of claim 225, wherein the one or more starch processing enzymes is superthermophilic. 225. Maltodextrin prepared by a method according to any one of claims 223 to 226. 225. A maltodextrin composition prepared by the method of any of claims 223-226. a) processing a plant part comprising starch granules and at least one starch processing enzyme, obtained from a transformed plant whose genome has been enriched with an expression cassette encoding at least one processing enzyme, under conditions which activate one or more enzymes, Processing the starch granules accordingly to form an aqueous solution comprising dextrin or sugar; And b) a process for producing dextrin or sugar from the grains without mechanically crushing the grains prior to harvesting the starch-derived product, comprising collecting an aqueous solution comprising sugars and / or dextrins. 228. The method of claim 229, wherein the starch processing enzyme is α-amylase, glucoamylase, α-glucosidase, and glucose isomerase. a) processing a plant part comprising starch granules and at least one starch processing enzyme, obtained from a transformed plant whose genome has been enriched with an expression cassette encoding at least one processing enzyme, under conditions which activate one or more enzymes, Processing the starch granules accordingly to form an aqueous solution comprising dextrin or sugar; And b) collecting the aqueous solution comprising the fermentable sugars. 235. The method of claim 231, wherein the starch processing enzyme is α-amylase, glucoamylase, α-glucosidase, and glucose isomerase. Corn plants stably transformed with a vector comprising superthermophilic α-amylase. A corn plant stably transformed with a vector comprising a polynucleotide sequence encoding α-amylase that is greater than 60% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 51.

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