CN1198778A - Aspergillus arabinofuranosidase - Google Patents

Abstract

The invention discloses an enzyme capable of degrading arabinoxylan. Additionally disclosed are the nucleotide sequences encoding this enzyme and the promoter controlling the expression of this enzyme.

Description

Aspergillus arabinofuranosidase

The present invention relates to a kind of enzyme, in addition, the nucleotide sequence of this kind of enzyme that the present invention relates to encode.The present invention also relates to a promotor, this promotor is used to control the expression of the described nucleotide sequence of the described enzyme of coding.

Specifically, enzyme of the present invention is a kind of arabinofuranosidase with araboxylan degrading activity.

Known hope organism-as filamentous fungus (as black aspergillus (AspergillusNiger)) or even some tissues of a kind of farm crop in instruct the expression of interested gene (" GOI ").Product albumen matter or enzyme can be useful for organism itself.For example, may wish to produce the crop protein product of forming by the amino acid of optimizing, to improve the nutritive value of crop.For example, this crop can be made more useful feed.

Perhaps, may wish separated product protein or enzyme, use this protein or enzyme to prepare then, for example, food compositions.From this point, this product albumen matter or enzyme can be compositions of food compositions, and perhaps it can be used to prepare food compositions, comprises the character or the outward appearance that change food compositions.Even may wish to use organism, as a kind of filamentous fungus or a kind of farm crop, for identical purpose is expressed the non-plant gene.

Also may wish to use a kind of organism,, express mammalian genes as a kind of filamentous fungus or a kind of farm crop.The example of latter's product comprises Interferon, rabbit, Regular Insulin, blood factor, and plasminogen activator.Also wish to use microorganism, as filamentous fungus, by using promoters active in this microorganism, by the GOI preparing product.

The fruits and vegetables cell walls is made up of polysaccharide mostly, and main component is a pectin, Mierocrystalline cellulose and xyloglucan (R.R.Selvendran and J.A.Robertson, IFR reports (IFRReport), 1989).Multiple cell walls model had been proposed, make great efforts to add intensity and flexible essential property (P.Albersheim, U.S.'s science (Sci.Am.), 232,81-95,1975, P.Albersheim, plant biochemistry (Plant Biochem.) third edition (Bonner and Varner), Ac.Pross, 1976; T Hayashi plant physiology and molecular biology of plants year summary (Ann.Rev.Plant Physiol ﹠amp; Plant Mol Biol, 40,139-168,1989).

The composition of plant cell wall is complicated and various.Find polysaccharide mainly with long chain cellulose (principal organ's composition of plant cell wall), hemicellulose (containing multiple β-xylan chain) and pectin substance are (by galacturonic glycan (galacturonan) and rhamnosyl galacturonic glycan (thamnogalacturonan); Arabinan; Form with Polygalactan and arabogalactan) form.From the angle of foodstuffs industry, pectin substance, particularly arabinan have become most important composition in the plant cell wall (Whitaker, J.R. (1984), enzyme microbial technique (Enzyme Microb.Technol.), 6,341).

A kind of form of vegetable polysaccharides is an arabinan.The narration of relevant arabinan can be referring to EP-A-0506190.According to document report, arabinan is made up of the main chain an of α who is connected with another-(1 → 5) group.Side chain is, and to be α-(1 → 2) under α-(1 → 3) or some situation be connected with main α-(1 → 5)-L-arabinan main chain.For example in apple, 1/3rd of pectinose total amount is present in side chain.The molecular weight of arabinan generally is about 15kDa.

Arabinan is generically and collectively referred to as the enzyme liberating of arabinase.On this point, to be a kind of enzyme discharge the pectinose residue from the arabinan main chain or from the arabinan side chain that contains of other hemicellulose backbone structure such as arabogalactan to the arabinan degrading activity, the ability of monomer or oligomer perhaps or even by the terminal arbinofuranose base unit of cracking monoterpene base α-L-arbinofuranose base glucoside and 1 → 6 key between the middle glucosyl unit discharges the monomeric ability of pectinose.

The activity of arabinan degrading enzyme comprises among the EP-A-0506190: the ability of a) cracking (1 → 2)-α-L-arabinose glycosidic bond; The ability of b) cracking (1 → 3)-α-L-arabinose glycosidic bond; The ability of c) cracking (1 → 5)-α-pectinose glycosidic bond; D) ability of 1 → 6 key between the terminal arbinofuranose base unit of cracking monoterpene base α-L-arbinofuranose base glucoside and the middle glucosyl unit.

Known arabinan degrading enzyme produces by each kind of plant and microorganism.In these microorganisms, fungi such as those Aspergillus, corticium, Rhodotorula (Kaii A. (1984) carbohydrate chemistry biological chemistry progress (Adv Carbohydr.Chem.Biochem.), 42,383), Dichotomitus (Brillouet etc., 1985, carbohydrate compound research (Carbohydrate Research, 144,113), Ascomycetes and Basidomycetes (Sydow, G, (1977) DDR patent application No.124812).

Another kind of vegetable polysaccharides is an xylan, and its main monosaccharide unit is a wood sugar.Xylan is the abundant composition of hemicellulose.Main hemicellulose is a kind of araboxylan in monocotyledons, and its pectinose side chain is connected with backbone of xylose residues.

Araboxylan is the carbohydrate of finding in the cereal cell walls.About the narration of araboxylan and its enzyme liberating can be referring to Voragen etc. (1992, araboxylan, the feature of xylan and zytase (Characterisation of Cereal Arabinoxylans, Xylans and Xylanases), the 51-67 page or leaf, J.Visser writes, and Elsevier Science Press publishes).

Araboxylan generally contains by β-1, the wood sugar main chain that the 4-key links together.This wood sugar main chain is replaced by the L-arabinose residue, and by α-1 key and xylose residues 2 or 3 are connected this L-arabinose residue.Described xylose residues can be single the replacement or two replacement.Except that replacing with pectinose, xylose residues also can be by acyl group, and glucuronic acid and various other carbohydrate replace.The pectinose residue can be further by phenolic acid such as forulic acid and coumaric acid replacement.The source that degree that replaces and kind depend on concrete araboxylan.

Found araboxylan in the cereal cell walls, they are parts of second layer cell walls.About 3%-that araboxylan constitutes whole meal flour is water miscible (WSP) partly wherein, and part is water-insoluble (WIP).

Although in fact the amount of araboxylan is the about 3% of wheat, the importance of araboxylan part is increasing.This is because the cereal araboxylan plays the hydro-colloid effect, because them and water formation gel-like structure.For example, although in fact araboxylan only accounts for 3% of flour dry weight, the araboxylan in the flour is at most in the dough rubbed of combination 30% of water.When the araboxylan combination water, it increases the viscosity of levigated cereal, and viscosity is to cereal being become be not easy to eat.

Can handle the rheological property of several systems that are used for levigate cereal with the enzyme of degraded araboxylan.In modern bakeshop, in order to reduce the required energy of face that processing is rubbed, also in order to make bread that more volume be arranged, the viscosity of the face that reduces to rub is good.This point normally enzyme of the wood sugar main chain by using the araboxylan of degrading realizes.

For the object of the invention only is referred to as Arabinoxylan degrading enzymes from the enzyme of the xylan backbone cracking pectinose side chain of araboxylan.

In the feed based on cereal, after feed was absorbed, the araboxylan in the cereal can improve the viscosity of liquid in the animal intestine.This is a problem, because this causes the uncomfortable sensation of animal, as maldigestion.Also reduced the nutritive value of feed.Can avoid these problems by the enzyme (as zytase) that in feed, adds the degraded araboxylan, avoiding maldigestion, and improve the nutritive value of feed.But the enzyme (particularly some zytases) of some degraded araboxylan requires to have unsubstituted main chain, so their activity is restricted.

About the further discussion of araboxylan can be referring to " xylan and zytase " (Xylans and Xylanases) (1992, J.Visser writes, and Elsevier Science Press publishes).

According to Kormelink etc., 1991 (Kormelink, F.J.M.Searle-Van LeeuwenM.J.F., Wood.T.M., Voragen, A.G.J. (1991), " from Aspergillus awamori purifying and the Arabic glycosyl furanose of sign (1; 4)-β-D-araboxylan lytic enzyme ", " microorganism, microbial technique are used) " (Appl.Microbiol.Biotechnol.25:753-758) described, a kind of Arabinoxylan degrading enzymes is the Arabic glycosyl furanose lytic enzyme (AXH) of (1,4)-β-D-araboxylan.But this piece document does not provide the sequencing data of this kind of enzyme or the nucleotide sequence of coding this kind of enzyme, or is used for the sequencing data of the promotor of this kind of enzyme.

Clearly, be useful preferably by the araboxylan this point of degrading with recombinant DNA technology.

The present invention attempts to provide a kind of enzyme with araboxylan degrading activity; Preferably wherein this kind of enzyme can make in some or specific cells or tissue, for example only a kind of organism, is generally a filamentous fungus, is preferably Aspergillus, as black aspergillus, perhaps or even in the specific cells of a kind of plant or the tissue makes.

The present invention also attempts to provide a kind of GOI of this kind of enzyme of encoding, and this GOI can be preferably expresses in specific cells or tissue, as a kind of organism, be generally filamentous fungus, be preferably Aspergillus,, perhaps or even in some or specific cells of a kind of plant or the tissue express as black aspergillus.

In addition, the present invention attempts to provide a kind of can instruct the GOI expression promoter, for example preferably at some specific cells or tissue, for example only a kind of organism, be generally filamentous fungus, be preferably Aspergillus, as black aspergillus, the nucleotide sequence of code book invention enzyme perhaps or even in the specific cells of a kind of plant or the tissue.Preferably in Aspergillus, use this promotor, wherein by the product of GOI coding from host organisms is secreted on every side matrix.

And, the present invention attempts to provide the structure that comprises described GOI and/or promotor, carrier, plasmid, cell, tissue, organ and organism, and preferred expression method in specific cells or tissue, for example only a kind of organism, generally be filamentous fungus,, perhaps or even in the specific cells of a kind of plant or the tissue express preferably in Aspergillus.

According to a first aspect of the invention, provide a kind of enzyme that obtains from Aspergillus, wherein to have following feature: MW be 33 to this enzyme, 270D ± 50D; The pI value is approximately 3.7; The araboxylan degrading activity; Optimal pH from about 2.5 to about 7.0 (more particularly about 3.3 to about 4.6, more particularly is approximately 4); Optimum temperuture is about 40 ℃ to about 60 ℃ (more particularly about 45 ℃ to about 55 ℃ more particularly are approximately 50 ℃); And wherein this kind of enzyme can get off pectinose cracking from the wood sugar skeleton of araboxylan.

According to a second aspect of the invention, provide enzyme, perhaps its variant, homologue, or fragment with sequence shown in SEQ.I.D.No.1.

According to a third aspect of the invention we, provide by nucleotide sequence shown in the SEQ.I.D No.2 or its variant, homologue, or fragment, or with the enzyme of its complementary sequence encoding.

The nucleotide sequence of coding enzyme of the present invention is provided according to a forth aspect of the invention.

According to a fifth aspect of the invention, provide nucleotide sequence, or its variant with the sequence shown in the SEQ.I.D.No.2, homologue or fragment, or with its complementary sequence.

According to a sixth aspect of the invention, provide promotor, or its variant with sequence shown in the SEQ.I.D.No.3, homologue or fragment, or with its complementary sequence.

According to a seventh aspect of the invention, provide terminator, or its variant with nucleotide sequence shown in the SEQ.I.D.No.13, homologue or fragment, or with its complementary sequence.

According to an eighth aspect of the invention, provide signal sequence, or its variant with nucleotide sequence shown in the SEQ.I.D.No.14, homologue or fragment, or with its complementary sequence.

According to a ninth aspect of the invention, provide by using the method for a kind of promoter expression GOI, wherein said promotor is a promotor of the present invention.

The application of enzyme liberating araboxylan of the present invention is provided according to the tenth aspect of the invention.

According to an eleventh aspect of the invention, provide the composition (combination) of the enzyme of degraded araboxylan, said composition is made up of enzyme of the present invention and zytase.

According to a twelfth aspect of the invention, be provided for expressing the enzyme of araboxylan of degrading, perhaps be used to control it and express, or be used to control the plasmid NCIMB40703 that another GOI expresses, or the nucleotide sequence that can from then on obtain.

According to a thirteenth aspect of the invention, provide signal sequence with sequence shown in the SEQ.I.D.No15, or its variant, homologue or fragment.

According to a fourteenth aspect of the invention, provide enzyme of the present invention to slow down or prevent maldigestion and/or improve absorptivity and/or have additional nutrients the medicine taken in or the purposes of food in preparation.

According to a fifteenth aspect of the invention, provide the arabinofuranosidase (arabinofuranosidase) with araboxylan degrading activity, it has the immunoreactivity with the arabinofuranosidase antibody that resists the purifying with sequence shown in the SEQ.I.D.No1.

According to a sixteenth aspect of the invention, provide the arabinofuranosidase promotor, wherein this promotor is by the intermediate product inductive of xylose metabolism.

According to a seventeenth aspect of the invention, provide the substrate method of viscosity that reduces branching, wherein the side chain of enzyme liberating substrate and the main chain of the substrate of not degrading.

According to a further aspect in the invention, provide the purposes of enzyme of the present invention as viscosity modifier.

According to another aspect of the invention, provide enzyme of the present invention to reduce the purposes of pectin viscosity.

Others of the present invention comprise the construction that is made of the aforementioned aspect of the present invention, carrier, plasmid, cell, tissue, organ and transgenic organism.

Others of the present invention comprise expressing or making to be expressed or transforms arbitrary nucleotide sequence, construction, plasmid, carrier, cell, tissue, organ or organism, with and the method for product.

Additional aspects of the present invention comprise uses described promotor at substratum such as meat soup or express GOI in transgenic organism.

Of the present invention comprising on the one hand again, comprise animal-feed with described enzyme preparation or processing food.

Preferred described enzyme is by nucleotide sequence shown in the SEQ.I.D.No.2, or its variant, and homologue or fragment are perhaps with its complementary sequence encoding.

Preferred described nucleotide sequence has sequence shown in the SEQ.I.D.No.2, or its variant, homologue or its fragment or with its complementary sequence.

Preferred described nucleotide sequence and promotor are operatively connected.

Preferred described promotor comprises sequence C CAAT.

Preferred described promotor is to have sequence shown in the SEQ.I.D.No.3, or its variant, homologue or fragment, or with the promotor of its complementary sequence.

Preferred described promotor comprises the sequence from the 100bps in Xma111 to BamH1 site.

Preferred promotor of the present invention and GOI are operatively connected.

Preferred described GOI comprises nucleotide sequence of the present invention.

Preferred transgenic organism is a fungi.

Preferred transgenic organism is a filamentous fungus, is more preferably Aspergillus.

Preferred transgenic organism is a kind of plant.

Preferably in application, described enzyme and zytase, preferred endo-xylanase (endoxylanase) is used in combination.

The highly preferred embodiment of each side of the present invention does not comprise any natural enzyme in the physical environment, natural promoter or natural nucleus glycoside acid sequence.

Preferably, at arbitrary plasmid, carrier, as expression vector or conversion carrier, cell, tissue, organ, in organism or the transgenic organism, described promotor and at least a GOI combination exist.

Preferred described promotor and GOI are inserted in the genome of transgenic organism with being stabilized.

Preferred transgenic organism is a filamentous fungus, and preferred Aspergillus is more preferably black aspergillus.Transgenic organism even can be a kind of plant is as unifacial leaf or dicotyledons.

Highly preferred embodiment is a kind of enzyme that obtains from aspergillus, and wherein this enzyme has following characteristics, and MW is 33,270D ± 50D; The pI value is approximately 3.7; The pectinose sill gathers the enzyme liberating activity; Optimal pH from about 2.5 to about 7.0 (more particularly about 3.3 to about 4.6, more specifically is about 4); Optimum temperuture is about 40 ℃ to about 60 ℃ (more especially about 45 ℃ to about 55 ℃, more specifically for being approximately 50 ℃); Wherein said enzyme can be from cracking pectinose on the wood sugar main chain of araboxylan; Wherein said enzyme has sequence shown in the SEQ.I.D.No.1, or its variant, homologue or fragment.

Another highly preferred embodiment is a kind of enzyme that obtains from aspergillus, and wherein to have following feature: MW be 33 to this enzyme, 270D ± 50D; The pI value is approximately 3.7; The pectinose sill gathers the enzyme liberating activity; Optimal pH from about 2.5 to about 7.0 (more particularly about 3.3 to about 4.6, more specifically is about 4); Optimum temperuture is about 40 ℃ to about 60 ℃ (more especially about 45 ℃ to about 55 ℃, more specifically for being approximately 50 ℃); Wherein said enzyme can be from cracking pectinose on the wood sugar main chain of araboxylan; Wherein said enzyme has nucleotide sequence shown in the SEQ.I.D.No.2 or its variant, homologue or fragment coding, or by with its complementary sequence encoding.

The invention has the advantages that the arabinofuranosidase that it provides a kind of preparation to have the araboxylan degrading activity, and the method for the nucleotide sequence of coding this kind of enzyme.In addition, the invention provides and to control that or other nucleotide sequence expression promoter.

Other advantage is that enzyme of the present invention can influence levigated cereal, as the viscosity of the dough of rubbing, and makes it be easy to processing, as for example obtaining bigger loaf volume.

It also is advantageous that enzyme of the present invention is used for feed, because its degraded araboxylan, thereby improved the nutritive value of feed.In addition, it has reduced the viscosity of araboxylan in the animal intestine, thereby slows down or prevented maldigestion.

It is useful especially that enzyme of the present invention and zytase are used in combination, because enzyme of the present invention and xylan have surprised beat all synergy each other.

From this aspect, enzyme of the present invention has improved the degradation effect of zytase, and zytase has improved the degradation effect of enzyme of the present invention, and zytase has improved the degradation effect of enzyme of the present invention.The activity of believing zytase has been enhanced, and has less substituent polysaccharide substrate because enzyme of the present invention provides.

Therefore, the invention provides a kind of enzyme with araboxylan degrading activity, wherein this enzyme can make in some or specific cells or tissue, for example only preparation in the specific cells of organism or tissue, this organism is generally filamentous fungus, preferred Aspergillus, for example this enzyme of black aspergillus even can in a kind of plant, prepare.

More particularly, enzyme of the present invention can be from the wood sugar main chain of araboxylan cracking pectinose specifically.

Arabinofuranosidase of the present invention is different from arabinofuranosidase of the prior art.From this aspect, their not abilities of branching arabinan of degrading of the EP-A-0506190 those disclosed enzyme of the arabinofuranosidase of description of the Prior Art-for example-it is characterized in that, and use right-nitrophenyl Arabinoside to measure.

The arabinofuranosidase of the present invention nonbranched arabinan of not degrading, and have only minimum activity for the nitrophenyl Arabinoside.On the contrary, arabinofuranosidase of the present invention is used to the araboxylan of degrading.Therefore say that arabinofuranosidase of the present invention and the isolating arabinofuranosidase of prior art are diverse.

The present invention also provides the GOI of the described enzyme of coding, and this GOI can be preferably expresses in specific cells or tissue, and as expressing in some or specific cells or the tissue of organism, this organism is generally filamentous fungus, is preferably Aspergillus, as black aspergillus.This GOI even can in a kind of plant, express.

In addition, the invention provides and a kind ofly can instruct the GOI expression promoter, nucleotide sequence as code book invention enzyme, preferred only expression in some specific cells of organism or tissue, generally this organism is a filamentous fungus, preferred Aspergillus, black aspergillus for example, perhaps or even in the specific cells of a kind of plant or the tissue express, preferably use described promotor in aspergillus, wherein the product of GOI coding is from host organisms is secreted on every side matrix.Even can tailor promotor (if necessary) in plant, to express GOI.

The present invention also provides the construction that comprises described GOI and/or promotor, carrier, plasmid, cell, tissue, organ and organism are expressed the method for GOI, preferably only express in the specific cells of organism or tissue, for example, this organism is generally filamentous fungus, preferably in Aspergillus, perhaps or even in specific cells of plant or the tissue expresses.

With the related term of enzyme " variant ", " homologue ", or " fragment " comprises any replacement of (or a plurality of) amino acid for sequence, variation, modify, displacement, disappearance or interpolation, condition is that the gained aminoacid sequence has the araboxylan degrading activity, the identical activity that preferably has enzyme shown in the sequence table (SEQ.I.D.No.1 or 12) at least, specifically, term " homologue " comprises the homology of relevant structure and/or function, and condition is that the gained enzyme has the araboxylan degrading activity.About sequence homology, preferably with SEQ.I.D.No.1 at least 75% shown in the sequence table, more preferably at least 85%, more preferably at least 90% homology.More preferably with SEQ.I.D.No.1 at least 95% shown in the sequence table, more preferably at least 98% homology.

The related term of nucleotide sequence " variant " with the described enzyme of coding, " homologue ", or " fragment " comprises any replacement of (or a plurality of) nucleic acid for sequence, variation, modify, displacement, disappearance or interpolation, condition is the nucleotide sequence coded enzyme with araboxylan degrading activity of gained, the identical activity that preferably has enzyme shown in the sequence table (SEQ.I.D.No.2 or 12) at least, specifically, term " homologue " comprises the homology about structure and/or function, and condition is the nucleotide sequence coded enzyme with araboxylan degrading activity of gained.The homology of relevant sequence, preferably with the SEQ.I.D.No.2 at least 75% shown in the sequence table, more preferably at least 85%, more preferably at least 90% homology.More preferably with more preferably at least 98% homology of SEQ.I.D.No.2 shown in the sequence table at least 95%.

The term relevant " variant " with promotor, " homologue ", or " fragment " comprises any replacement of (or a plurality of) nucleic acid for sequence, variation, modify, displacement, disappearance or add, condition are that the gained nucleotides sequence is listed in expression system-for example have in cell transformed or the transgenic organism according to the present invention ability that works as promotor.Specifically, term " homologue " comprises the homology relevant with structure and/or function, and condition is that the gained nucleotide sequence has the ability that works as promotor.About sequence homology, preferably with SEQ.I.D.No.3 at least 75% shown in the sequence table, more preferably at least 85%, more preferably at least 90% homology.More preferably with SEQ.I.D.No.3 at least 95% shown in the sequence table, more preferably at least 98% homology.

The term " variant " relevant with terminator or signal nucleotide sequence, " homologue " or " fragment " comprises any replacement of (or several) nucleic acid for sequence, variation, modify, displacement, disappearance or add, condition is the gained nucleotide sequence, in expression system-for example have respectively in cell transformed or the transgenic organism according to the present invention work or encode as the terminator ability of aminoacid sequence with the ability that works as signal sequence.Specifically, term " homologue " comprises the homology of relevant structure and/or function, and condition is the ability that the gained nucleotide sequence has each conduct or coding terminator or signal sequence.About sequence homology, preferably with SEQ.I.D.No.13 shown in the sequence table and 14 (respectively) at least 75%, more preferably at least 85%, more preferably at least 90% homology.More preferably with SEQ.I.D.No.13 shown in the sequence table and 14 (respectively) at least 95%, more preferably at least 98% homology.

The term " variant " relevant with the signal aminoacid sequence, " homologue ", or " fragment " comprises any replacement of (or a plurality of) amino acid for sequence, variation, modify, to be institute's calling sequence have the ability that works as signal sequence in expression system-for example according to transformant of the present invention or transgenic organism for displacement, disappearance or add, condition.Specifically, term " homologue " comprises the homology of relevant structure and/or function, and condition is the ability that the gained nucleotide sequence has the conduct or the signal of encoding respectively.About sequence homology, be preferable over SEQ.I.D.No.15 at least 75% shown in the sequence table, more preferably at least 85%, more preferably at least 90% homology.More preferably with SEQ.I.D.No.15 at least 95% shown in the sequence table, more preferably at least 98% homology.

Above-mentioned term is the synonym of sequence allelic variation.

Term " complementation " refers to that the present invention also comprises can be respectively and the nucleotide sequence of the nucleotide sequence hybridization of encoding sequence or promoter sequence.

The term relevant with the present invention " Nucleotide " comprises genomic dna, cDNA, synthetic DNA, and RNA.Preferred its refers to DNA, more preferably refers to be used for the cDNA of encoding sequence of the present invention.

Term " construction "-itself and term be as " conjugate ", and " cartridge clip " and " hybrid " are synonym-the comprise GOI that directly or indirectly links to each other with promotor.An example that indirectly connects is that a suitable interval group is arranged, an intron sequences for example, and for example ShI intron or ADH intron play intermediation between promotor and GOI.For the term " fusion " relevant with the present invention that comprises direct or indirect connection also is the same.Most preferably these terms do not comprise what the gene of codase normally linked with the wild type gene promotor in each case, and the natural combination of the situation of both under its physical environment.Highly preferred embodiment is to be operatively connected described or a kind of GOI with a kind of or described promotor.

Described construction even can comprise or express a marker, this marker makes selection be transferred to for example filamentous fungus, preferred Aspergillus, black aspergillus for example, perhaps plant, preferred cereal, for example corn selects desired gene constructs to become possibility in the rice, barley etc.There is operable multiple marker, the marker (in particular for plant) of those coding mannose-6-phosphate isomerases or the G418 resistance of those markers of antibiotics resistance-for example is provided for example, Totomycin, bleomycin, kantlex and gentamicin resistance.

Term " carrier " comprises expression vector and conversion carrier.

Term " expression vector " refers in the energy body or the construction of vivoexpression.

Term " conversion carrier " refers to and can transfer to the construction of another species-for example transfer to filamentous fungus from intestinal bacteria (E.coli) plasmid, preferred Aspergillus from species.It also can be to transfer to the construction that edaphic bacillus is transferred to a kind of plant from the E.coli plasmid.

Term " tissue " comprises tissue itself and organ.

The term relevant with the present invention " organism " comprises the nucleotide sequence that may comprise promotor of the present invention and/or code book invention enzyme and/or from any organism of the product that wherein obtains, and wherein said promotor can make to be expressed when GOI and/or nucleotides sequence broomrape wherein of the present invention exist in organism and can be expressed.

Preferred organism is a filamentous fungus, preferred Aspergillus, more preferably black aspergillus.

The term relevant with the present invention " transgenic organism " comprises the nucleotide sequence that comprises promotor of the present invention and/or code book invention enzyme and/or from any organism of the product that wherein obtains, GOI is expressed wherein described in vivo promotor and/or nucleotides sequence wherein of the present invention is listed in and can be expressed.Preferred promoter and/or nucleotide sequence are inserted in the genome of organism.

Preferred transgenic organism is a filamentous fungus, preferred Aspergillus, more preferably black aspergillus.

Therefore, transgenic organism of the present invention comprises and includes arbitrary promotor of the present invention, the nucleotide sequence of code book invention enzyme, construction of the present invention, carrier of the present invention, plasmid of the present invention, cell of the present invention, the present invention organizes or its product, perhaps comprises a kind of organism of their combination.For example, transgenic organism can comprise the GOI under the promotor control of the present invention, preferred extraneous nucleotide sequence.Transgenic organism also can comprise the nucleotide sequence of the code book invention enzyme under the promotor control, and described promotor can be a promotor of the present invention.

In highly preferred embodiment, transgenic organism does not comprise the combination of the nucleotide sequence of promotor of the present invention and code book invention enzyme, and wherein said promotor and nucleotide sequence are natural for this organism, and are in their natural surroundings.Therefore in highly preferred embodiment, the present invention do not comprise be in when being under its control that also is in the natural promoter under its natural surroundings its natural surroundings according to natural nucleotide coding sequence of the present invention.In addition, in highly preferred embodiment, the present invention do not comprise be in its natural surroundings and by also be in physical environment, it is in that natural nucleotide encoding sequence under the control that also is in the natural promoter under its natural surroundings expresses according to natural enzyme of the present invention.

Term " promotor " is the common meaning in this area, for example-and RNA polymerase combination site in the Jacob-Mond genetic expression theory.

On the one hand, promotor of the present invention can be expressed GOI, and it can be the nucleotide sequence of code book invention enzyme.

On the other hand, nucleotide sequence according to the present invention is in and makes under the control of nucleotide sequence expression promoter.On this point, if the not necessarily identical promotor of promotor with promotor of the present invention.From this respect, promotor can be a kind of cell or tissue specificity promoter.For example, if organism is a kind of plant, then promotor can be at stem, and seedling influences the nucleotide sequence expression promoter in one or more of root and leaf texture.

For example, the promotor of nucleotide sequence of the present invention can be that α-Amy 1 promotor (is also referred to as Amy 1 promotor in addition, Amy 637 promotors or α-Amy 637 promotors), referring to the common unsettled UK patent application No.9421292.5 of application on October 21st, 1994.Described promotor comprises sequence shown in Figure 1.

Perhaps the promotor of nucleotide sequence of the present invention can be that α-Amy 3 promotors (are also referred to as Amy 3 promotors in addition, Amy 351 promotors or α-Amy 351 promotors), referring to the careful and unsettled UK patent application No.9421286.7 of application on October 21st, 1994.Described promotor comprises sequence shown in Figure 2.

Preferred promoter is a promotor of the present invention.

Except that above-mentioned nucleotide sequence, promotor, promotor particularly of the present invention can comprise in addition to be guaranteed or is increased in the character of expressing in the suitable host.For example described character can be conserved regions, as Pribnow Box or TATA casket.Described promotor also can comprise other sequence of influence (for example keep, strengthen, improve) GOI expression level.Other for example suitable sequence comprises Sh 1-intron or ADH intron.Other sequence comprises can induce the temperature of element-for example, compound, and light or stress reaction can be induced element.

Also can exist and strengthen the suitable element of transcribing or translating.An example of latter's element is TMV 5 ' signal sequence, referring to Sleat gene 217[1987], 217-225; With Dawson molecular biology of plants (Dawson Plant Mol, Biol) 23[1993] 97).

In addition, the present invention also relates to the nucleotide sequence and/or the combination of elements of promotor and/or coded protein or enzyme.For example, the present invention relates to and may be the combination that the GOI according to a nucleotide sequence of the present invention is operatively connected according to promotor of the present invention, with the another one promotor, as the combination of a kind of tissue-specific promoter of being operatively connected with identical or different GOI.

The present invention also relates to express with promotor the nucleotide sequence purposes of coding enzyme according to the present invention, wherein the part of promotor is an inactivation, but wherein this promotor still can play promotor.The part inactivation of promotor is good in some cases.

Specifically, for Amy 351 promotors of mentioning in early time, a part that can the inactivation promotor, thus the promotor of part inactivation in more single-minded mode as only in a kind of particular tissue type or organ, expressing GOI.

Inactivation is divided in term " inactivation " finger, and meaning is that the expression pattern of promotor is modified, but wherein the promotor of part inactivation still plays the promotor effect.But as mentioned above, the promotor of modified can be expressed GOI in the particular organization of at least a (but not being whole) former promotor.A kind of such promotor is aforesaid Amy 351 promotors.

The example of part inactivation comprises the folding pattern that changes promoter sequence, perhaps changes it and nucleotide sequence partly makes up, and like this, the part of nucleotide sequence is by for example RNA polymerase identification.The method of another preferred part inactivation promotor is that it is truncated to its fragment.Another kind method is at least a portion sudden change that makes this sequence, thereby RNA polymerase can not make up this part or another part.

Another kind of modification is that the combination of proteins site is regulated in sudden change, for example known effect of the checking CreA protein that applies the carbon degradation production from filamentous fungus, thereby remove natural promoter catastaticly check effect.

Term " GOI " is meant interested any gene with reference to the present invention.GOI can be to be external or any Nucleotide of itself for the postgraduate of institute object (filamentous fungus for example, preferred Aspergillus, or a kind of plant).The typical example of GOI comprises the protein modified in metabolism and the catabolic process and the encoding gene of enzyme.A kind of reagent that GOI can encode and introduce or improve pathogen resistance.GOI can also be an antisense construct thing of modifying the expression of the natural transcript that exists in the related tissue.The GOI filamentous fungus of can also encoding, the non-natural protein of preferred Aspergillus, a kind of compound of perhaps encoding the animal or human being beneficial to.

For example, GOI can encode pharmaceutically active protein matter or enzyme, for example, treatment compound Regular Insulin, Interferon, rabbit, human serum albumin, human growth factor and thrombin any.From this aspect, but cell transformed or organism can prepare institute's phase compound of receiving amount, and this compound obtains from this cell or organism easily.GOI also can be a kind of protein that gives a kind of food or nutritive value of crops.Typical example comprises can suppress the vegetable-protein that antinutritional factor vegetable-protein that generates and the amino acid with expectation are more formed (for example than non-transgenic plant higher lysine content being arranged).GOI can also encode and can be used for the enzyme of food-processing, for example rennin, thaumatin and alpha-galactosidase.GOI can be the encoding gene of any harmful toxins, it can be an antisense transcript, as for Rhizoma Solani tuber osi protein (patatin) or α-Dian Fenmei, the antisense transcript of ADP-glucose Pyrophosphate phosphohydrolase (for example referring to EP-A-0455316), the antisense transcript of a kind of proteolytic ferment antisense or dextranase.

GOI can be coding for alpha-diastatic nucleotide sequence, and this is us in the theme of the common unsettled UK patent application 9413439.2 of application on July 4th, 1994, and sequence provides in Fig. 3.GOI can be coding for alpha-diastatic nucleotide sequence, and this is us in the theme of the common unsettled UK patent application 9421290.9 of application on October 21st, 1994, and sequence provides in Fig. 4.GOI can be the nucleotide sequence of coding ADP-glucose Pyrophosphate phosphohydrolase, and this is our theme in the careful and unsettled PCT patent application PCT/EP94/01082 of application on April 7th, 1994, and its sequence provides in Fig. 5 and Fig. 6.GOI can be any nucleotide sequence of coding for alpha-glucan hydrolase, and this has explanation at us in the careful and unsettled PCT patent application PCT/EP94/03397 of application on October 15th, 1994, and its sequence provides in Fig. 7-10.

In a preferred embodiment, GOI is the nucleotide sequence of coding enzyme according to the present invention.

As mentioned above, a kind of preferred host living beings is an Aspergillus, as black aspergillus.Transgenosis aspergillus of the present invention can make according to the explanation of following document: Rambosek, J, and Leach, J.1987 (the recombinant DNA in the filamentous fungus: improve and explore, CRC biotechnology comment summary (Recombinant DNA in filamentous Fungi:Progress and Bospects CRCCrit.Rev Biotechnol.) 6:357-393), Davis R.W.1994 (MartinelliS.D., Kinghorn J.R. (writing) " aspergillus: 50 years industrial microorganism progress " (Aspergillus:50 years on Progress in industrial microbiology), vol 29, Elsevier Amsterdam 1994,525-560, " allogeneic gene expression and protein secreting in the aspergillus (Heterologous gene expression and protein secretion inAspergillus)); Ballance; D.J.1991 (Leong; S.A.Berka R; M. (writing) " molecule technological mycology; system of filamentous fungus and application " (Molecular IndustrialMycology, Systems and Application for Filamentous Fungi.) Marcel DekkerInc NewYork 1991.pp1-29, " summary of conversion system of filamentous fungus and fungal gene structure " (Transformation systems for Filamentous Fungi and an Overview ofFungal Gene Structure), with Turner (Martinelli S D. G.1994, Kinghorn J.R. (writing) " aspergillus: 50 years industrial microorganism progress ", vol 29, Elsevier Amster-dam 1994, pp641-666, " carrier of genetic manipulation " (Vectors for genetic manipulation)), and following explanation has been summed up and has been produced according to those explanations of transgenosis aspergillar of the present invention.

Be close in the century, industrial widely-used filamentous fungus produces organic compound and enzyme.Traditional Japanese Koji and fermented soybean have used the Aspergillus centuries.Used black aspergillus production organic acid, particularly citric acid and produced the various enzymes that are used for industry in this century.

Filamentous fungus is widely used in industry two major causes.At first, filamentous fungus can produce a large amount of extracellular productses, for example enzyme and organic compound, for example microbiotic or organic acid.Secondly, filamentous fungus can grow on cheap substrate, cereal for example, chaff, beet pulp etc.Same cause makes filamentous fungus become interesting organism according to heterogenous expression of the present invention.

In order to make transgenosis aspergillus,, GOI (for example a kind of amylase or SEQ.I.D.No.2) prepares expression constructs by being inserted into the construction that expression designs in filamentous fungus.

Worked out the construction that several types is used for heterogenous expression.These constructions comprise according to of the present invention in fungi promoters active (if perhaps GOI coding according to enzyme of the present invention and another promotor of needs).The example of the promotor except that the present invention comprises the fungal promoters that is used for highly expressing extracellular enzyme, for example, and glucoamylase promotor or α-Dian Fenmei promotor.This GOI can merge with instructing this GOI encoded protein matter excretory signal sequence (signal sequence for example of the present invention or another suitable sequence).Usually use fungic origin signal sequence, signal sequence for example of the present invention.Activated terminator stops expression system, terminator for example of the present invention in fungi.

Worked out the expression system in the fungi of another type, wherein GOI merges with the less or major part of the fungal gene of coding stabilizing protein.This can stablize the GOI encoded protein.In such system, a cleavage site of being discerned by the differential protein lytic enzyme can be inserted between mycoprotein and the GOI encoded protein, like this, the fusion rotein of generation can be cut by the differential protein lytic enzyme in this site, thereby discharges GOI encoded protein matter (" POI ").As an example, can insert the site of in some aspergillus, finding at least as the KEX-2 of peptase identification.Such fusion causes cutting in the body, thereby protects POI and produce POI, and does not produce than larger fusion protein.

Reported heterogenous expression coding bacterium in aspergillus, fungi, several genes of vertebrates and plant protein.If GOI does not merge with signal sequence, then protein can deposit in the born of the same parents.Protein will be in tenuigenin accumulation, and usually not by glycosylation, this is useful for some bacterioproteins.If GOI is equipped with a signal sequence, then protein will be outside born of the same parents accumulation.

With regard to producing stability and host strain modification, when some heterologous proteins were secreted in the fungus culture medium, they were not very stable, and most of fungies produce several extracellular protein lytic enzymes of degraded heterologous protein.For fear of this problem, used the host of some special fungal bacterial strains of the proteolysis production of enzyme with reduction as heterologous production.

For transforming filamentous fungus, (Ballance 1991, above) to have worked out some conversion schemes for a lot of filamentous funguss.Wherein the basis of a lot of schemes is preparation protoplastiss and uses PEG and C _a ²⁺Ion is inserted into DNA in the protoplastis.The protoplast regeneration of Zhuan Huaing then, and select the fungi that transforms with various selected marker things.The marker that is used for transformation is some nutrient defect type mark things, argB for example, and trpC, niaD, and pyrG, antibiotic resistance markers, as the benomyl resistance, hygromycin resistance and phleomycin resistance.A kind of transformation marker thing that is in daily use is the amdS gene of Aspergillus nidulans (A.nidulans), and this gene of high copy number makes fungal growth with acrylamide as only nitrogen source.

Although do not disclose enzyme of the present invention among EP-B-0470145 and the CA-A-2006454, the encode nucleotide sequence and the promotor of the present invention of this enzyme, but these two pieces of documents provide some useful background notes to the technology of these types, can be applied to preparation transgenic plant of the present invention.Some such background notes are included in the following note.

The ultimate principle of the structure of the plant that genetically engineered changes is to insert genetic information in Plant Genome, thereby obtains to insert the stable maintenance of material.

Inserting genetic information has several technology, and two main principles are directly to introduce genetic information and use carrier system to introduce genetic information.The summary of general technology can be referring to Potrykus (plant physiology and molecular biology of plants year summary (Annu Rev Plant Physiol Plant MolBiol) [1991] 42:205-225) and Christou (agricultural-food-industry (Agro-Food-Industry) Hi-Tech, March/April, 1994, article 17-27).

Therefore, on the one hand, the present invention relates to a kind of carrier system, it carries promotor of the present invention or nucleotide sequence or construction, and this promotor or nucleotide sequence or construction can be inserted into an organism, a kind of plant for example, genome in.

Described carrier system can comprise a carrier, but also can comprise two carriers.Under the situation that two carriers are arranged, it has been generally acknowledged that this carrier system is binary vector system (binary vectorsystem).The binary vector system describes in further detail in (1980) such as Gynheung An, binary vector, and plant molecular biology manual A3,1-19 (Binary Vectors, PlantMolecular Biology Manual A3,1-19).

Be to use from the Ti-plasmids of Agrobacterium tumefaciens (Agrobacteriumtumefaciens) or from the Ri plasmid of hair root edaphic bacillus (Agrobacteriumrhizogenes) with the basis of a kind of system that is widely used of a kind of given promotor or nucleotide sequence or construction transformed plant cells; An etc.; (1986); plant physiology (Plant Physiol) 81; 301-305; with Butcher D.N. etc.; (1980); phytopathologist's tissue culture method (Tissue Culture Methods for Plant Pathologists; write: D.S.Ingrams and J.P.Helgeson, 203-208).

Constructed and be applicable to several different Ti and the Ri plasmid that makes up above-mentioned plant or vegetable cell construction.The example of nonrestrictive such Ti-plasmids is pGV3850.

Promotor of the present invention, or nucleotide sequence or construction should preferably be inserted in the Ti-plasmids between the end sequence of T-DNA or adjacent T-DNA sequence, thereby avoiding the sequence fragmentation on these next-door neighbour T-DNA borders, is essential because at least one in these districts demonstrates for the T-DNA insertion Plant Genome that will modify.

Should be appreciated that from top explanation, if described organism is a kind of plant, carrier system then of the present invention preferably comprises at least one carrier system of the boundary portion branch of necessary sequence of infection plant (for example Vir district) and T-DNA sequence, wherein said boundary member be positioned at genetic constructs with carrier on.

And described carrier system is Agrobacterium tumefaciens Ti-plasmid or hair root edaphic bacillus Ri-plasmid or its derivative preferably, because these plasmids are known, and is widely used in the transgenic plant structure.Have a lot of carrier systems, they are based on these plasmid or derivatives thereofs.

In transgenic plant make up, promotor of the present invention or nucleotide sequence or construction can at first be structured in a kind of microorganism, carrier can duplicate in this microorganism, and its easy operation, the example of a kind of useful microorganism is E.coli, but also can use other microorganism with above-mentioned character.Behind the carrier in E.coli, having made up carrier system as defined above, if desired, it is transferred in the suitable edaphic bacillus bacterial strain, for example transfer in the Agrobacterium tumefaciens.Therefore, the Ti-plasmid that preferably will be loaded with promotor of the present invention or nucleotide sequence or structure is transferred to suitable edaphic bacillus bacterial strain, in A.tumefaciens (Agrobacterium tumefaciens), thereby obtain being loaded with promotor of the present invention, the edaphic bacillus cell of nucleotide sequence or structure, wherein DNA then is transferred in the vegetable cell that will modify.

In CA-A-2006454, reported and to have obtained to comprise the E.coli dubbing system and make a large amount of cloning vectors of selecting transformant to become possible marker.These carriers comprise for example pBR 322, pUC series, M13 mp series, pA CYC 184 etc.

In this way, Nucleotide of the present invention or structure or promotor can be inserted into restriction site suitable in the carrier.The plasmid that comprises is used for transforming at E.coli.The E.coli cell is cultivated the back results in suitable nutritional medium, and cracking.Reclaim plasmid then.Normally used analytical procedure is the sequencing analysis, restricted enzyme cutting analysis, and electrophoresis also has biological chemistry and molecular biology method.After the operation of per step, the dna sequence dna of use can restriction enzyme digestion and is connected with next dna sequence dna.Each sequence can be cloned in identical or different plasmid.

In plant, after each insertion method of the desired promotor of the present invention or construction or nucleotide sequence, may need to exist and/or insert other dna sequence dnas.If for example used Ti-or the Ri-plasmid in the vegetable cell for conversion, the right margin at least of this Ti-and Ri-plasmid T-DNA then, and usually be that right margin and left margin (as the flanking region that inserts gene) can be connected.Use T-DNA for transformed plant cells and furtherd investigate, and in EP-A-120516, description is arranged; Hoekema, in: The Binary Plant VectorSystem offset drukkerij Kanters B.B., Alblasserdam, 1985, the V chapters; Fraley etc., plant science comment (Crit Rev, Plant Sci.), 4:1-46; With An etc., EMBOJ (1985) 4:277-284.

With edaphic bacillus direct infection plant tissue is a simple technology, be widely used, and at " phytopathologist's the tissue culture method " of (1980) such as Butcher D.N., write: D.S.Ingrams and J.P.Helgeson have description among the 203-208.This technology further specify (Annu RevPlant Mol Biol) [1991] 42:205-225 referring to Potrykus (plant physiology molecular biology of plants year summary)) and Christou (agricultural-food-industry, Hi-Tech, March/April, 1994,17-27).With this technology, can be at some part or the tissue of plant, promptly at the leaf of plant, root, stem, or infection plant on the part at other position.

Generally, and will cut out the edge of a knife by infected plant, for example use the razor cutting plants for the edaphic bacillus direct infection plant tissue that carries described promotor and/or described GOI, perhaps with pin with the plant acanthopore, perhaps with the sand paper plant that rubs.Inoculate wound with edaphic bacillus then, afterwards, postvaccinal plant or plant part are grown on suitable medium, make it grow into maturation plant.

After vegetable cell was fabricated, these cells can and keep according to known tissue culture method growth, were for example adding for example amino acid of essential growth factor, plant hormone, culturing cell in the suitable medium matter of VITAMIN etc.

The plant that cell transformed regeneration genetically engineered is modified can carry out from cell or tissue culture regenerated known method with plant, for example by select the branch of conversion with microbiotic, with by containing suitable nutritive ingredient, the substratum of plant hormone etc. is uploaded to be commissioned to train and is supported branch and carry out.

For further specifying of Plant Transformation referring to EP-A-0449375.

In a word, the invention provides arabinofuranosidase with araboxylan degrading activity and the nucleotide sequence of encoding this kind of enzyme.In addition, provide and can control that, or another nucleotide sequence expression promoter.It comprises the terminator and the signal sequence of expressing for identical nucleotide sequence in addition.

Following sample is deposited in the The NationalCollections of Industrial and Marine Bacteria Limited of depositary institution (NCIMB) of approval according to budapest treaty, Great Britain and Northern Ireland United Kingdom, Scotland, Aberdeen, No. 23, Machar Drive, AB21RY, January 16 nineteen ninety-five:

The E.coli{ that comprises plasmid pB53.1 is that E.coli DH 5 α-pB53.1} preserving number is NCIMB 40703.

Mode by embodiment illustrates the present invention now.

The following examples are carried out with reference to the accompanying drawings, wherein;

Fig. 1-the 10th, the promotor of early stage patent application and GOI sequence are useful for each side of the present invention.

Figure 11 is the main body of preservation thing NCIMB 40703, the plasmid figure of plasmid pB53.1;

Figure 12 is the outline diagram that promotor of the present invention is lacked;

Figure 13 is pXP-AMY plasmid figure;

Figure 14 is pXP-XssAMY plasmid figure;

Figure 15 is diagram;

Figure 16 is the HP-TLC curve;

Figure 17 is the HP-TLC curve;

Figure 18 is the HPLC collection of illustrative plates;

Figure 19 is the viscosity diagram;

Figure 20 is active diagram;

Figure 21 is active diagram;

Figure 22 is active diagram;

The particularly application of recombinant DNA technology is discussed in following narration.The generality explanation of recombinant DNA technology can be referring to Sambrook, J., Fritsch, E.F.Maniatis T (writing) " molecular cloning, laboratory manual " (Molecular Cloning A laboratory manual.) second edition, Cold Spring Harbour Laboratory Press New York 1989.

In these embodiments, enzyme of the present invention is called AbfC sometimes, and in addition, promotor of the present invention is called the AbfC promotor sometimes.The purifying of arabinofuranosidase

Black aspergillus 3M43 grows in the substratum that contains wheat bran and beet pulp.Fermenting broth is from the filtered solid part preparation of meat soup, spissated fermenting broth is loaded into uses 20mM Tris, on 25 * 100mm Q-SEPHAROSE (Pharmacia) high performance column that HCl pH7.5 balance is crossed, linear gradient is O '-500Mm NaCl, collects the elutriant fraction.Arabinofuranosidase comes out at 130-150Mm NaCl place wash-out.

Merge the fraction that contains arabinofuranosidase, with 50 * 200mm G-25 SEPHAROSE Superfine (Pharmacia) desalination.With distilled water wash-out pillar.

Concentrate enzyme with the High-Trap column spinner after the desalination, then the fraction of spissated desalination is carried out gel-filtration on 50 * 600mm SUPERPEX, 50 posts.Load sample adds 0.2M NaCl wash-out pillar with 0.2M phosphate buffered pH7.0, collects the elutriant fraction.

Merge the fraction and the desalination that contain arabinofuranosidase, and concentrate as mentioned above.The fraction that merges is loaded into 50mM and contains 1.5M (NH ₄) ₂SO ₄16 * 100mm Phenylsepharose high performance column (Pharmacia) of crossing of phosphate buffered saline buffer pH6.0 balance on.Use (NH ₄) ₂SO ₄The concentration that concentration changes from 1.5-0M, and divide fraction to collect elutriant.Merge the fraction that contains arabinofuranosidase.Use phast system's gel (Pharmacia) to estimate the purity of arabinofuranosidase by SDS-PAGE.Characterize

The molecular weight of the arabinofuranosidase of the mass spectroscopy purifying by using the laser desorption technology finds that the MW of arabinofuranosidase is 33,270D ± 50D.

Use wide spectrum pI test kit (Pharmacia) to measure the pI value.The pI value of arabinofuranosidase is approximately 3.7.

After SDS-PAGE analyzed, it was glycosylated that processing PAS reagent demonstrates arabinofuranosidase.According to I.Van Seuningen and M.Davril (1992) " electrophoresis " (Electrophoresis) 13, the method for pp97-99 is carried out PAS dyeing.

Measure the function of AbfC activity for water-soluble piperylene (WSP) concentration (mg/ml).The result provides in Figure 21.The result shows that when concentration of substrate is 8mg/ml WSP it is maximum that the AbfC activity reaches.The pH activity research

Be used for studying pH to the active influence of arabinofuranosidase of the present invention as the substrate in the 50mM sodium-citrate-phosphate buffer from the water-soluble piperylene 10mg/ml of wheat.The incubation time is 15 minutes.Find that arabinofuranosidase of the present invention has the wide pH optimum range (referring to Figure 20) from about 2.5 to about 7.0, more particularly from about 3.3 to about 4.6, more particularly about 4.The temperature activity research

Be used for studying temperature to the active influence of arabinofuranosidase of the present invention as the substrate in the 50mM sodium acetate of pH5.0 from the water-soluble piperylene 10mg/ml of wheat.The incubation time is 15 minutes.Find arabinofuranosidase of the present invention at about 40 ℃ to about 60 ℃,, optimum activity (Figure 22) is arranged during more preferably about 50 ℃ temperature more preferably at about 45 ℃ to about 55 ℃.This enzyme still has activity at about 10 ℃, and demonstrates residual activity at 70 ℃ and 80 ℃.The amino acid sequence analysis of arabinofuranosidase

Use Stone ﹠amp; The modification method of the method for Williams 1993 explanations, use Lys-C sequencing level endo-protease to digest described enzyme (Stone available from BoehringerMannheim, K.L. and Williams, K.R. (1993) " proteinic enzymic digestion separates with the HPLC peptide ", Matsudaira P. (writing), " being used for the protein of microsequencing and the experiment guide of peptide purification ", second edition, Science Press, San Diego, 1993, pp.45-73 (Enzymaticdigestion of Protein and HPLC Peptide Isolation, In:Matsudaira P. (Editor) .A Practical Guide to Protein and Peptide Purification forMicrosequencing.).

Cryodesiccated β-arabinofuranosidase (0.4mg) is dissolved in the 8M urea of 50 μ l, 0.4M NH ₄HCO ₃, among the pH8.4.Be full of N above ₂The back adds 5 μ l 45MmDTT, and protein is at 50 ℃ of N ₂Following sex change was also reduced 15 minutes.After being cooled to RT, add 5 μ l100Mm iodo-acetamides so that cysteine derivativeization 15 minutes, condition is RT, dark, N ₂50 μ l 50Mm Tricine and the 10mM EDTA that then add 90 μ l water and 5 μ g endo-protease Lys-C, the solution of pH8.0, digestion is at 37 ℃ of N ₂Under carried out 24 hours.The gained peptide is at VYDAC C18 post (0.46 * 15cm; 10 μ m; The SeparationsGroup; California) go up with the reversed-phase HPLC separation, use solvent orange 2 A: the 0.1%TFA aqueous solution, solvent B:0.1%TFA acetonitrile solution.Before using pulse liquid to be circulated in soon to measure sequence on the AppliedBiosystems 476A sequenator, with identical solvent system with the peptide of selection again at Develosil C18 post (0.46 * 10cm; 3 μ m) enterprising circumstances in which people get things ready for a trip spectrum.Peptide sequence below finding:

SEQ?I.D.No.4

SEQ?I.D.No.5

SEQ?I.D.No.6

SEQ?I.D.No.7

SEQ I.D.No.8 gene fragment PCR clone's separation

With Applied Biosystem dna synthesizer 392 type synthetic pcr primer things.At this on the one hand, from one of peptide sequence of finding, i.e. SEQ.I.D.No.5 synthetic pcr primer thing.This primer is: from the primer (oppositely) of EMTAQA

SEQ?ID?NO.9????GCY?TGN?GCN?GTC?ATY?TC

17mer????64mix

A primer from MIVEAIG

SEQ?ID?NO.10???ATG?ATH?GTN?GAR?GCN?ATH?GG

20mer 288mixPCR amplification is carried out with each the 100 μ l reaction solution (PERKIN ELMER) at use Amplitaq polysaccharase in these primers of 100pmol.Ensuing program is:

The step temperature-time

1???????94℃????2min

2???????94℃????1min

3???????55℃????2min

4???????72℃????2min

5???????72℃????5min

65 ℃ of SOAK step 2-4 repeat 40 circulations.PCR is reflected on the PERKIN ELMER DNA Thermal circulation instrument and carries out.

According to manufacturer explanation (Novagen), the fragment of 100bp amplification is separated and be cloned on pT7-Blue T one carrier.Separate black aspergillus (A.niger) genomic dna

1g refrigerated A.niger mycelium grinds in liquid nitrogen in mortar.Behind the vaporized nitrogen, the mycelium of grinding extracts with extraction damping fluid (100mM TrisHCl, pH8.0,0.50mM EDTA, 500mM NaCl, the 10mM beta-mercaptoethanol) 15ml that contains 1ml 20% sodium lauryl sulphate.Add 5ml 5M KAc after 10 minutes 65 ℃ of insulations, pH5.0 then is incubated 20 minutes on ice with mixture after the mixing.After the extraction, with centrifugal 20 minutes of mixture, supernatant liquor mixed to be settled out the DNA of extraction with 0.6 volume Virahol.Further after centrifugal 15 minutes, with the DNA resolution of precipitate in 0.7ml TE (10mM Tris, HCl, pH8.0,1mMEDTA), and with 75 μ l 3M NaAc, pH8.0 and 500 μ l isopropanol precipitatings.

After centrifugal, settling is with dry under 70%ETOH flushing and the vacuum.DNA is dissolved in 200 μ l TE and stores down at-20 ℃.The structure in storehouse

Partly digest 20 μ g genomic dnas with Tsp 509 I, provide terminal compatible end with EcoRI.The DNA of digestion separates on 1% sepharose and purifying 4-10kb fragment.Use λ ZAPII EcoRI/CIAP test kit (available from Stratagene) to make up the storehouse according to manufacturer's explanation.2 μ l joints (amounting to 5 μ l) are filled extract with the Gigapack Gold II from Stratagene and are adorned post.This storehouse comprises 650,000 independently clones.The screening in storehouse

At the dull and stereotyped upper flat plate inoculation of NZY 2 * 50000pfu, and carry out plaque at Hybond N film (Sheet) on (Amersham) and draw (plaquelift).Carrying out plaque in duplicate draws.Ready-to-go marker cassette and the usefulness of film available from pharmacia ³²The PCR clone hybridization of PdCTP (Amersham) mark.Have only when on two films, all detecting hybridization, just just to be regarded as and clone.Gene is carried out sequencing, and the sequence of discovery shows that all peptides that check order are all by sequence encoding that found.Sequence information SEQ.I.D.No.12 represents promoter sequence, enzyme encoding sequence, the aminoacid sequence of terminator sequence and signal sequence and enzyme of the present invention.Arabinofuranosidase is analyzed

From two of wheat-flour different araboxylan goods, insoluble piperylene of wheat (WIP) and wheat solubility piperylene (WSP) are degraded separately with arabinofuranosidase of the present invention, perhaps make up the endo-xylanase degraded from the A.niger purifying.Detect and carry out with 1% substrate among the 50Mm 50Mm sodium acetate buffer pH5.0.Being reflected at 30 ℃ carried out 32.5 hours.Reaction stops the ethanol sedimentation high molecular weight material by adding 3 volume ethanol.Sample is centrifugal and collect supernatant liquor, and is dry under the vacuum, and resuspending is in 0.5Ml distilled water.Sample diluted in water with 1: 1, (300 * 7.8mm cat29010) goes up analysis, uses Shimadzu C-R4A Chromatopac HPLC system at Chromopack Carbohydrate pb post, use Shimadzu RI D-6A RI-detector, carry out according to manufacturer's explanation.

Pillar xylotriose by 0.48mg/ml, 0.48mg/ml xylo-bioses, the standard calibration that 0.60mg/ml wood sugar and 0.58mg/ml L-arabinose are formed.Identify and quantitative each peak value with the software that equipment provides.Result's-discharge from the insoluble piperylene of wheat sugared substrate is the 1%WIP the 50Mm sodium acetate buffer pH5.0.

	Xylotriose	Xylo-bioses	Wood sugar	Pectinose
					There is not enzyme	??0.0	??0.0	??0.0	??0.0
abfC	??0.0	??0.0	??0.0	??0.11
					xyl	??0.09	??0.14	??0.0	??0.0
abfC+xyl	??0.37	??0.41	??0.0	??0.30

AbfC refers to enzyme of the present invention; Xyl refers to zytase mentioned above.Result's-discharge from wheat solubility piperylene sugared substrate is the 1%WIP the 50Mm sodium acetate buffer pH5.0.Numerical value is represented with mg/ml.

	Xylotriose	Xylo-bioses	Wood sugar	Pectinose
					There is not enzyme	?0.0	?0.0	??0.0	??0.0
abfC	?0.0	?0.0	??0.0	??0.30
					xyl	?0.08	?0.14	??0.0	??0.0
abfC+xyl	?0.42	?0.47	??0.0	??0.42

AbfC refers to enzyme of the present invention; XyC refers to zytase mentioned above.

Figure 17 provides AbfC enzyme and the synergistic HP-TLC curve of zytase A.Abbreviation below having used in the figure: water-soluble piperylene (WSP); Water-insoluble piperylene (WIP); The oat xylan is a substrate.Standard is: the x-wood sugar; x ₂-xylo-bioses; x ₃-xylotriose; The A-pectinose.

It is the HPLC analysis of the hydrolysate of substrate that Figure 18 provides with 1% oat spelt xylan.Figure 18 (a) and Figure 18 (b) demonstrate the product when using AbfC enzyme and zytase separately respectively.Figure 18 (c) demonstrates the product when AbfC enzyme and zytase are used in combination.

These experimental results provide two important discoveries.

The first, enzyme of the present invention discharges pectinose from araboxylan, particularly L-arabinose.

The second, enzyme of the present invention and endo-xylanase compound action are apparently higher than the adduction of its each self-applying.Therefore, these two kinds of enzymes are with the cooperative mode enzymic activity that interacts.Induce the AbfC gene: identify inductor

The adjusting of transcribing with the syzygy research black aspergillus AbfC encoding gene of the beta-Glucuronidase encoding gene that comprises AbfC promotor and E.coli (uid A).

The converted product that GUS produces is grown on different carbon sources; And qualitative, quantitative is measured the ability to hydrolysis p-nitrophenol glucose aldehyde. result is as follows: carbon source is induced the GUS after 24 hours active (1%) (wood sugar 12.37 xylitols 1.49 arabinoses 6.66 arabites 5.30 glucose 0.70 cellobiose 0.95 wood sugar-oligomer 70 17.26 glucopyranosides 0.40 methyl-xylopyranose glucosides 24.2 xyloglucans (xyloglucan) the 1.00 pectin 0.27 arabogalactan 2.60 arabites+glucose 2.20 of unit/mg)

The result shows the AbfC promotor when at wood sugar, and wood sugar-oligomer 70, methyl-xylopyranoside, pectinose and arabitol exist to cultivate after 24 hours down to be connected.These researchs show that also methyl-wood sugar pyranoside is the natural and the strongest elicitor of described promotor.

The AbfC promotor is prevented by glucose consumingly, therefore locates under the effect of carbon catabolite repression.But different with disclosed promotor by pectinose and arabitol inductive furans arabinofuranosidase/xylosidase, AbfC promotor of the present invention is regulated strongly by the intermediate in the xylose metabolism.Therefore, the present invention also relates to a kind of furans arabinofuranosidase/xylosidase promotor, wherein said promotor is that the xylose metabolism intermediate is derivable.The different promoters disappearance is to the influence of the adjusting of AbfC genetic expression

Detect the needed possible upstream regulatory sequence of AbfC genetic expression in order to study the molecular level regulating effect, to experimentize.Being structured in this gene 5 ' upstream has a series of plasmids (referring to Figure 12) of disappearance.E coli uid A gene is used as reporter gene, and carries out qualitative GUS and analyze.

The result shows that the 590bp AbfC promotor of brachymemma comprises the inducibility and the required enough information of its regulating effect of AbfC gene.The 100bps sequence B amH1 site disappearance of promotor is from the feasible activity that has reduced this promotor of Xma111.Therefore this 100bps district is important for the genetic expression good horizontal.290bps disappearance before the ATG has identified that this district is important but insufficient for the activity of cancelling this promotor.All converted products that comprise the analysis that this promotor makes up manifest at the trial very shallow blueness (+GUS).This district is :-170 TCATCCAATAT as can be seen, this district comprises the CCAAT element, and is the target thing of the common transcription activator of inferring.This sequence is similar to the nucleoprotein combination site of finding in two kinds of starch inducible promoters, described two kinds of starch inducible promoters are: black aspergillus glucoamylase gene and aspergillus oryzae (Aspergillus oryzae) amylase gene, and Aspergillus nidulans (Aspergillusnidulans) amdS gene.Using the black aspergillus that transforms with AbfC promotor and AbfC signal sequence to produce heterologous protein black aspergillar transforms

The conversion scheme of black aspergillus (A.niger) is with the basis that is disclosed as of following document: Buxton, F.P., Gwynne D.I., Davis, R.W.1985 (" with the argB gene transformation black aspergillus of Aspergillus nidulans " (Transformation of Aspergillus niger using the arg Bgene of Aspergillus nidulans), " gene " (Gene), 37:207-214), Daboussi, M.J., Djeballi, A., Gerlinger, C., Blaiseau, P.L., Cassan, M., Lebrun, M.H., Parisot, D., Brygoo, and Y.1989 (" use the nitrate reductase gene transformation of Aspergillus nidulans several have a fungi " (Transformation ofseven species of filamentous fungi nsing the nitrate Neductose gene ofAspergillus nidulans), Curr.Genet.15:453-456) and Punt, P.J., van den Hondel, C.A.M.J.J.1992 (" serves as that the basis conversion has a fungi with hygromycin B and phleomycin resistance marker " (Transformation of filamentous fungi based onhygromycin B and Phlemycin resistance markers), " Enzymology method " (Meth, Enzym.) 216:447-457).

Purifying about protoplastis, (CBS 120.49 from the new N400 that forms of spore in 5-10ml water, Centraalbureau voor Schimmelcultures, Baarn) (grown 7 days)-PDA (potato dextrose agar (Potato Dextrose Agar)-fromDifco Lab.Detroit) plate upper punch washes spore.Fill the shake flask of 200mlPDC (potato glucose meat soup, Difco 0549-17-9, Difco Lab, Detroit) with this spore suspension inoculation, and under 30 ℃, shook (250rpm) 16-20 hour.

Use Miracloth paper results mycelium, and the mycelium that 3-4g is wet is transferred to and is filled 10ml STC (the 1.2M Sorbitol Powder that contains 75Mg lyase (Sigma L-2265) and 4500 unit lyticases (Sigma L-8012), 10mM Tris HCl pH7.5,50Mm CaCl ₂) aseptic accompanying in the Ti Shi ware.

Cultivate mycelium until the mycelium degraded and discharge protoplastis with enzyme.Filter the mycelium of degraded by aseptic 60 μ m order strainers then.In a swing-out rotor, gathered in the crops protoplastis in centrifugal 10 minutes with the speed of 2000rpm.Abandoning supernatant, and settling is dissolved in 8ml1.5M MgSO ₄In after, with 3000rpm centrifugal 10 minutes.

The upper strata band that will contain protoplastis with transfer pipet is transferred in another test tube, and adds 2ml0.6M KCl.Carefully add 5ml 30% sucrose at the top, test tube centrifugal 15 minutes with 3000rpm.

The protoplastis that is in interface zone is transferred in the new test tube and with 1vol.STC and is diluted.Solution centrifugal 10 minutes with 3000rpm.Settling is dissolved among the 1ml STC at last with STC flushing twice.The counting protoplastis, and before conversion, also will concentrate.

About transforming, with 100 μ l protoplastis solution (10 ⁶-10 ⁷Protoplastis) mix with the 10 μ l dna solutions that contain 5-10 μ g DNA, and incubation 25 minutes at room temperature.With 200 μ l, 200 μ l and 800 μ l carefully add 60%PEG-4000 in batches then.This mixture is incubation 20 minutes at room temperature.In mixture, add 3ml STC, and careful the mixing.This mixture centrifugal 10 minutes with 3000rpm.

Remove supernatant liquor, and protoplastis is solubilized in the residual supernatant liquor, add the 3-5ml top agarose, and protoplastis is coated on the option board fast.AbfC promotor and allogeneic gene expression

Expression vector pXP-Amy (Figure 13) comprises the 2.1kb α-Dian Fenmei encoding gene from Thermomyces lanuginosus that is cloned in AbfC promotor (2.1kb) downstream and zytase A terminator upstream.This carrier is used for the cotransformation experiment with hygromycin gene as selective marker, tests the functional of AbfC promotor.

Best converted product accumulates in the concussion flask with at least 1 grams per liter α-Dian Fenmei in the substratum.In 48 hours, detect the starch degradation activity then, and be that enzymic activity peak (Figure 15) is found in growth on sugar beet pulp and the wheat bran in the time of 4 days.AbfC signal sequence function in the protein secreting

Preparation comprises the expression constructs with the AbfC gene signal peptide that merges from the ripe α-Dian Fenmei translation of T.lanuginosus, and finds that this construction expresses in producing bacterial strain.About this point, translation fusion construct pXPXss-Amy (Figure 14) places transcribing under the control of AbfC promotor and zytase A termination signal.The insertion of endogenous signal peptide causes expressing the detectability of the raising of both cotransformation things of diastatic hygromycin resistance mark.This endogenous signal peptide instructs the extracellular amylase secretion.The proteic substrate specificity of AbfC

Measure the substrate specificity of the AbfC of purifying with the hemicellulose that comprises pectinose: from wheat, oat and larchen araboxylan, pectinose branching and that take off, arabogalactan, sugar beet pectin, and xyloglucan.

HPLC and HP-TLC the results are shown in Figure 16, the abbreviation below wherein having used: the water-soluble piperylene of WSP-, and WIP-water-insoluble piperylene, the AG-arabogalactan takes off B-A-and takes off an arabinan (de B-A-debranched arabinan).The standard substance that uses is: A-pectinose, X-wood sugar.

The result shows that pectinose is the hydrolysate of pectinose sill glycan.Do not have hydrolysate from arabogalactan, discharge on an arabinan of taking off or the xyloglucan.Pectinose is as the hydrolysate of branching arabinan and discharge.Therefore AbfC is a kind of 1,2/1,2 debranching factors, and it does not have activity to the L-arabinose base furanose residue that linearity 1,5 α-key of finding connects in taking off an arabinan and Arabic glycosyl Polygalactan.This enzyme also discharges a kind of product when pectin is used as substrate, believe that this product is the pectinose that comprises forulic acid or Arabic disaccharides.Reduce viscosity by AbfC

Result of study about substrate specificity shows that also enzyme of the present invention can be used for reducing the viscosity of feed.About this point, this enzyme can partly not removed the viscosity that the main chain of substrate reduces the branching substrate by removing branching.This viscosity modifier with known degraded substrate main chain is opposite.

Therefore, the present invention relates to a kind of branching substrate method of viscosity that reduces, wherein the branching of enzyme liberating substrate part but the main chain of the substrate of not degrading.

Particularly, the present invention relates to enzyme of the present invention as viscosity modifier.

About this point, the research that experimentizes reduces viscosity from the water-soluble piperylene composition of wheat-flour by the furans arabinofuranosidase/xylosidase.In this experiment, at 20 ℃, incubation is 20 hours under the pH5.5 condition with 100 μ lAbfC for the water-soluble piperylene of 6ml.

The result shows (referring to Figure 19), and enzyme of the present invention can be used for reducing the viscosity of pectin, particularly in beverage-viscosity of the pectin that for example in fruit juice, uses.

Therefore, the present invention relates to use enzyme of the present invention to reduce pectin viscosity.Produce antibody

By enzyme to rabbit injection purifying, and according to N Harboe and A Ingild (" immunity; separating immune globulin; estimation antibody titers " (Immunization, Isolation ofImmunoglobulins, Estimation of Antibody Titre), " quantitative immunoelectrophoresis, methods and applications handbook (A Manual of Quantitative Immunoelectrophoresis, Methods and Applications), NH Axelsen etc. writes, Universitetsforlaget, Oslo, 1973) and TG Cooper (" biochemical tools " (The Tools ofBiochemistry), John Wiley ﹠amp; Sons, New York, 1977) method separating immune globulin from antiserum(antisera) of describing produces the antibody of anti-enzyme of the present invention.Sum up

Although known black aspergillus produces the furans arabinofuranosidase/xylosidase, the invention provides a kind of new creative furans arabinofuranosidase/xylosidase, and the promotor of the sequence of this enzyme of encoding and this sequence.Important advantage of the present invention is that this kind of enzyme can a large amount production.

In addition, promotor of the present invention and adjusting sequence (for example signal sequence and terminator) can be used for expressing or can being used for the expression of organism GOI, for example express in black aspergillus.

Arabinofuranosidase of the present invention is different from the furans arabinofuranosidase/xylosidase of previously known.About this point, their arabinan of degrading of those enzymes among the EP-A-0506190 of previously described furans arabinofuranosidase/xylosidase-for example-it is characterized in that, and with the assay determination of p-nitrophenyl Arabinoside.

The furans arabinofuranosidase/xylosidase of the present invention arabinan of not degrading, and on the p-nitrophenyl Arabinoside, see minimum activity only.

On the contrary, arabinofuranosidase of the present invention is used to the araboxylan of degrading.Therefore, furans arabinofuranosidase/xylosidase of the present invention is quite different with existing isolating furans arabinofuranosidase/xylosidase.

More particularly, enzyme of the present invention can be from the wood sugar main chain of araboxylan cracking pectinose specifically.

Enzyme of the present invention is useful, because it can improve method and improvement food and the feed itself for preparing food and feed.For example, enzyme of the present invention can be added in the animal-feed that is rich in araboxylan.Contain for example barley of cereal when what join monogastric animal (for example poultry or pig), wheat, corn, rye or oat or cereal by product be the feed of wheat bran or corn bran when (comprising silage) for example, this kind of enzyme can obviously improve the fragmentation of plant cell wall, makes animal utilize plant nutrient better.The result has improved growth velocity and/or feed conversion.And Arabinoxylan degrading enzymes can be used for reducing containing the viscosity of the feed of arabinan.If preferred the immersion in advance or wet food then can add Arabinoxylan degrading enzymes of the present invention before handling feed or silage.

Special advantage is that enzyme of the present invention can make up zytase, and especially endo-xylanase uses.

Another possible application of enzyme of the present invention is pulp and paper industry.Often report uses zytase from the Mierocrystalline cellulose of hemicellulose main chain with the hemicellulose residue is removed xylogen and terpene is good, and this is a process wood in the papermaking, a basic step of wood pulp or timber derived products.In the zytase treatment step, add the degraded that Arabinoxylan degrading enzymes produced according to the invention can help to contain arabinan hemicellulose main chain, therefore help improving and more effectively removing xylogen and terpene.Using Arabinoxylan degrading enzymes comprises in the hemicellulose main chain in the cork processing of glucuronic acid beneficial especially therein.

Enzyme of the present invention also is useful because its with the cooperative mode effect (seeing The above results) of endo-xylanase.

Of the present invention other change do not exceed under the scope of the invention situation apparent to those skilled in the art.Sequence table SEQ ID NO:1 enzyme sequence

(i) sequence signature:

(A) length: 296 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:1:Lys Cys Ser Leu Pro Ser1 5Ser Tyr Ser Trp Ser Ser Thr Asp Ala Leu Ala Thr Pro Lys Ser Gly

10??????????????????15??????????????????20Trp?Thr?Ala?Leu?Lys?Asp?Phe?Thr?Asp?Val?Val?Ser?Asp?Gly?Lys?His

25??????????????????30??????????????????35Ile?Val?Tyr?Ala?Ser?Thr?Thr?Asp?Glu?Ala?Gly?Asn?Tyr?Gly?Ser?Met

40??????????????????45??????????????????50Thr?Phe?Gly?Ala?Phe?Ser?Glu?Trp?Ser?Asn?Met?Ala?Ser?Ala?Ser?Lys?55??????????????????60??????????????????65??????????????????70Thr?Ala?Thr?Pro?Tyr?Asn?Ala?Val?Ala?Pro?Thr?Leu?Phe?Tyr?Phe?Lys

75??????????????????80??????????????????85Pro?Lys?Ser?Ile?Trp?Val?Leu?Ala?Tyr?Gln?Trp?Gly?Ser?Ser?Thr?Phe

90??????????????????95?????????????????100Thr?Tyr?Arg?Thr?Ser?Gln?Asp?Pro?Thr?Asn?Val?Asn?Gly?Trp?Ser?Ser

105?????????????????110?????????????????115Glu?Lys?Ala?Leu?Phe?Thr?Gly?Lys?Leu?Ser?Asp?Ser?Ser?Thr?Gly?Ala

120?????????????????125?????????????????130Ile?Asp?Gln?Thr?Val?Ile?Gly?Asp?Asp?Thr?Asn?Met?Tyr?Leu?Phe?Phe135?????????????????140?????????????????145?????????????????150Ala?Gly?Asp?Asn?Gly?Lys?Ile?Tyr?Arg?Ser?Ser?Met?Ser?Ile?Asp?Glu

155?????????????????160?????????????????165Phe?Pro?Gly?Ser?Phe?Gly?Ser?Gln?Tyr?Glu?Glu?Ile?Leu?Ser?Gly?Ala

170?????????????????175?????????????????180Thr?Asn?Asp?Leu?Phe?Glu?Ala?Val?Gln?Val?Tyr?Thr?Val?Asp?Gly?Gly

185?????????????????190?????????????????195Glu?Gly?Asn?Ser?Lys?Tyr?Leu?Met?Ile?Val?Glu?Ala?Ile?Gly?Ser?Thr

200?????????????????205?????????????????210Gly?His?Arg?Tyr?Phe?Arg?Ser?Phe?Thr?Ala?Ser?Ser?Leu?Gly?Gly?Glu215?????????????????220?????????????????225?????????????????230Trp?Thr?Ala?Gln?Ala?Ala?Ser?Glu?Asp?Lys?Pro?Phe?Ala?Ala?Lys?Pro

235?????????????????240?????????????????245Thr?Val?Ala?Pro?Pro?Gly?Pro?Lys?Thr?Leu?Ala?Met?Val?Thr?Trp?Phe

250?????????????????255?????????????????260Ala?Thr?Thr?Leu?Ile?Lys?Pro??*

265 270SEQ ID NO：2AAA TGC TCT CTT CCA TCG TCC TAT AGT TGG AGT TCA ACC GAT GCT CTCGCA ACT CCT AAG TCA GGA TGG ACC GCA CTG AAG GAC TTT ACT GAT GTTGTC TCT GAC GGC AAA CAT ATC GTC TAT GCG TCC ACT ACT GAT GAA GCGGGA AAC TAT GGC TCG ATG ACC TTT GGC GCT TTC TCA GAG TGG TCG AACATG GCA TCT GCT AGC AAG ACA GCC ACC CCC TAC AAT GCC GTG GCT CCTACC CTG TTC TAC TTC AAG CCG AAA AGC ATC TGG GTT CTG GCC TAC CAATGG GGC TCC AGC ACA TTC ACC TAC CGC ACC TCC CAA GAT CCC ACC AATGTC AAC GGC TGG TCG TCG GAG AAG GCG CTT TTC ACC GGA AAA CTC AGCGAC TCA AGC ACC GGT GCC ATT GAC CAG ACG GTG ATT GGC GAC GAT ACGAAT ATG TAT CTC TTC TTT GCT GGC GAC AAC GGC AAG ATC TAC CGA TCCAGC ATG TCC ATC GAT GAA TTT CCC GGA AGC TTC GGC AGC CAG TAC GAGGAA ATT CTG AGT GGT GCC ACC AAC GAC CTA TTC GAG GCG GTC CAA GTGTAC ACG GTT GAC GGC GGC GAG GGC AAC AGC AAG TAC CTC ATG ATC GTTGAG GCG ATC GGG TCC ACT GGA CAT CGT TAT TTC CGC TCC TTC ACG GCCAGC AGT CTC GGT GGA GAG TGG ACA GCC CAG GCG GCA AGT GAG GAT AAACCC TTC GCA GCA AAG CCA ACA GTG GCG CCA CCT GGA CCG AAG ACA TTAGCC ATG GTG ACT TGG TTC GCA ACA ACC CTG ATC AAA CCA TGASEQ ID NO：3CTGCAGAAGA TGGCAGTCGC CACAGCCGAT CACCCGATCC ATACTGGATG TTGTAACTTG 60GAGACAGCCT GCAGATGCTC TGATGAAGGT CTGCAAATAG TTCCTGGACC TCGATAGTGA 120AGTATACCGA TTCGTCAATG TTGTATATCC AGCCACTTTG AAAGTACCAA CTTTTAGTTC 180GATTGATCAG AATACTTTTG GTGTGTAACA TTGACAAGCC AAATTATCAA TCTCTTCTAC 240CGGTAAGGTG TCAACTACCC GGCCGAAAGT ACCGGAAGGT CGTGGTGTTT TAAGGTGAAA 300CAACTATCAG GGCGGCAATG TGTCAAAGTA GAACCAGTTT GCTTAGCGCC ATTAGGATCC 360ACGCCTAGAC CCTTGATGCC CGGGAGTTAT CCGTCCTGTC ACAGCAATTA TTTCCCCGAG 420TCTACTGCCG AAGAACAGCC ATTGTGGCGT ACTCACGGAA TTACCCACTG TGTAGGGTAG 480TCTTGAACGC CGTTCTAGAC ACGGCAACGC TCCGGTGGAC GATCGTTTCT GGCTAATGTA 540CTCCGTAGTT TAGGCAGCAT GCTGATCATC TTCCCCCTAG GGAAAGGCCC CTGAATAGTG 600CGCCAAAATG AGCTTGAGCA AAGGAATGTT CTTTCTAAGC CAAAGTGAGG GAAATAACCA 660AGCAGCCCAC TTTTATCCGA AACGTTTCTG GTGTCATCCA ATATGGATAA ATCCCGATTG 720TTCTTCTGCA CATATCTCTA TTGTCATAAG TGCAACTACA TATATTTGAA CATGGTTTGG 780TCCTCTTTCC AAGTTATTCG TTCTCCGTGA CCAGCGATTT CAGCCATTGA TTCTTTTGTT 840TCTTTCCCCG CGGATAAACT CATACGAAGSEQ ID NO：4：

(i) sequence signature:

(A) length: 20 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: peptide

(v) clip types: N-end

(xi) sequence description: SEQ ID NO:4:Lys Cys Ser Leu Pro Ser Ser Tyr Ser Trp Ser Ser Thr Asp Ala Leu1 5 10 15Ala Thr Pro Lys

The information of 20SEQ ID NO:5:

(i) sequence signature:

(A) length: 41 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: peptide

(v) clip types: intramolecularly

(xi) sequence description: SEQ ID NO:5:Tyr Leu Met Ile Val Glu Ala Ile Gly Ser Thr Gly His Arg Tyr Phe1 5 10 15Arg Ser Phe Thr Ala Ser Ser Leu Gly Gly Glu Met Thr Ala Gln Ala

20??????????????????25??????????????????30Ala?Ser?Glu?Asp?Lys?Pro?Phe?Xaa?Gly

The information of 35 40SEQ ID NO:6:

(i) sequence signature:

(A) length: 25 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: peptide

(v) clip types: intramolecularly

(xi) sequence description: SEQ ID NO:6:Ser Ile Trp Val Leu Ala Tyr Gln Trp Gly Ser Ser Thr Phe Thr Tyr1 5 10 15Arg Thr Ser Gln Asp Pro Thr Asn Val

The information of 20 25SEQ ID NO:7:

(i) sequence signature:

(A) length: 30 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: peptide

(v) clip types: intramolecularly

(xi) sequence description: SEQ ID NO:7:Asp Ile Val Tyr Ala Ser Thr Thr Asp Glu Ala Gly Asn Tyr Gly Ser1 5 10 15Met Thr Phe Gly Ala Phe Ser Glu Xaa Ser Asn Met Ala Ser

The information of 20 25 30SEQ ID NO:8:

(i) sequence signature:

(A) length: 41 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: peptide

(v) clip types: intramolecularly

(xi) sequence description: SEQ ID NO:8:Ile Iyr Arg Ser Ser Met Ser Ile Asp Glu Phe Pro Gly Ser Phe Gly1 5 10 15Ser Gln Tyr Glu Glu Ile Leu Ser Gly Ala Thr Asn Asp Leu Phe Glu

20??????????????????25??????????????????30Ala?Val?Gln?Val?Tyr?Thr?Val?Asp?Gly

The information of 35 40SEQ ID NO:9:

(i) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: other nucleic acid

(A) illustrate :/desc=" OLIGONUCLEOTIDE "

(xi) sequence description: the information of SEQ ID NO:6:GCYTGNGCNG TCATYTC 17SEQ ID NO:10:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: other nucleic acid

(A) illustrate :/desc=" OLIGONUCLEOTIDE "

(xi) sequence description: the information of SEQ ID NO:10:ATG ATH GTN GAR GCN ATH GG 20SEQ ID NO:11:

(i) sequence signature:

(A) length: 89 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ii) molecule type: other nucleic acid

(A) illustrate :/desc=" PCR fragment "

(xi) sequence description: the information of SEQ ID NO:11:ATGATTGTGG AGGCGATCGG GTCCACTGGA CATCGTTATT TCCGCTCCTT CACGGCCAGC 60AGTCTCGGTG GAGAGATGAC CGCACAGGC 89SEQ ID NO:12:

(i) sequence signature:

(A) length: 2555 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(vi) originate:

(A) organism: black aspergillus

(B) bacterial strain: 3M43

(ix) feature:

(A) title/keyword: CDS

(B) position: 870..1757

(ix) feature:

(A) title/keyword: sig_peptide

(B) position: 870..947

(ix) feature:

(A) title/keyword: mat_peptide

(B) position: 948..1754

( xi ) ：SEQ ID NO：12：CGGTAAGGTG TCAACTACCC GGCCGAAAGT ACCGGAAGGT CGTGGTGTTT TAAGGTGAAA 300CAACTATCAG GGCGGCAATG TGTCAAAGTA GAACCAGTTT GCTTAGCGCC ATTAGGATCC 360ACGCCTAGAC CCTTGATGCC CGGGAGTTAT CCGTCCTGTC ACAGCAATTA TTTCCCCGAG 420TCTACTGCCG AAGAACAGCC ATTGTGGCGT ACTCACGGAA TTACCCACTG TGTAGGGTAG 480TCTTGAACGC CGTTCTAGAC ACGGCAACGC TCCGGTGGAC GATCGTTTCT GGCTAATGTA 540CTCCGTAGTT TAGGCAGCAT GCTGATCATC TTCCCCCTAG GGAAAGGCCC CTGAATAGTG 600CGCCAAAATG AGCTTGAGCA AAGGAATGTT CTTTCTAAGC CAAAGTGAGG GAAATAACCA 660AGCAGCCCAC TTTTATCCGA AACGTTTCTG GTGTCATCCA ATATGGATAA ATCCCGATTG 720TTCTTCTGCA CATATCTCTA TTGTCATAAG TGCAACTACA TATATTTGAA CATGGTTTGG 780TCCTCTTTCC AAGTTATTCG TTCTCCGTGA CCAGCGATTT CAGCCATTGA TTCTTTTGTT 840TCTTTCCCCG CGGATAAACT CATACGAAG ATG AAG TTC TTC AAT GCC AAA GGC 893

Met?Lys?Phe?Phe?Asn?Ala?Lys?Gly

-26?-25?????????????????-20AGC?TTG?CTG?TCA?TCA?GGA?ATC?TAC?CTC?ATT?GCA?TTA?ACC?CCC?TTT?GTT??????941Ser?Leu?Leu?Ser?Ser?Gly?Ile?Tyr?Leu?Ile?Ala?Leu?Thr?Pro?Phe?Val

-15?????????????????-10??????????????????-5AAC?GCC?AAA?TGC?TCT?CTT?CCA?TCG?TCC?TAT?AGT?TGG?AGT?TCA?ACC?GAT??????989Asn?Ala?Lys?Cys?Ser?Leu?Pro?Ser?Ser?Tyr?Ser?Trp?Ser?Ser?Thr?Asp

1???????????????5??????????????????10GCT?CTC?GCA?ACT?CCT?AAG?TCA?GGA?TGG?ACC?GCA?CTG?AAG?GAC?TTT?ACT?????1037Ala?Leu?Ala?Thr?Pro?Lys?Ser?Gly?Trp?Thr?Ala?Leu?Lys?Asp?Phe?Thr?15??????????????????20??????????????????25??????????????????30GAT?GTT?GTC?TCT?GAC?GGC?AAA?CAT?ATC?GTC?TAT?GCG?TCC?ACT?ACT?GAT?????1085Asp?Val?Val?Ser?Asp?Gly?Lys?His?Ile?Val?Tyr?Ala?Ser?Thr?Thr?Asp

35??????????????????40??????????????????45GAA?GCG?GGA?AAC?TAT?GGC?TCG?ATG?ACC?TTT?GGC?GCT?TTC?TCA?GAG?TGG?????1133Glu?Ala?Gly?Asn?Tyr?Gly?Ser?Met?Thr?Phe?Gly?Ala?Phe?Ser?Glu?Trp

50??????????????????55??????????????????60TCG?AAC?ATG?GCA?TCT?GCT?AGC?AAG?ACA?GCC?ACC?CCC?TAC?AAT?GCC?GTG?????1181Ser?Asn?Met?Ala?Ser?Ala?Ser?Lys?Thr?Ala?Thr?Pro?Tyr?Asn?Ala?Val

65??????????????????70??????????????????75GCT?CCT?ACC?CTG?TTC?TAC?TTC?AAG?CCG?AAA?AGC?ATC?TGG?GTT?CTG?GCC?????1229Ala?Pro?Thr?Leu?Phe?Tyr?Phe?Lys?Pro?Lys?Ser?Ile?Trp?Val?Leu?Ala

80??????????????????85??????????????????90TAC?CAA?TGG?GGC?TCC?AGC?ACA?TTC?ACC?TAC?CGC?ACC?TCC?CAA?GAT?CCC?????1277Tyr?Gln?Trp?Gly?Ser?Ser?Thr?Phe?Thr?Tyr?Arg?Thr?Ser?Gln?Asp?Pro?95?????????????????100?????????????????105?????????????????110ACC?AAT?GTC?AAC?GGC?TGG?TCG?TCG?GAG?AAG?GCG?CTT?TTC?ACC?GGA?AAA?????1325Thr?Asn?Val?Asn?Gly?Trp?Ser?Ser?Glu?Lys?Ala?Leu?Phe?Thr?Gly?Lys

115?????????????????120?????????????????125CTC?AGC?GAC?TCA?AGC?ACC?GGT?GCC?ATT?GAC?CAG?ACG?GTG?ATT?GGC?GAC?????1373Leu?Ser?Asp?Ser?Ser?Thr?Gly?Ala?Ile?Asp?Gln?Thr?Val?Ile?Gly?Asp

130?????????????????135?????????????????140GAT?ACG?AAT?ATG?TAT?CTC?TTC?TTT?GCT?GGC?GAC?AAC?GGC?AAG?ATC?TAC?????1421Asp?Thr?Asn?Met?Tyr?Leu?Phe?Phe?Ala?Gly?Asp?Asn?Gly?Lys?Ile?Tyr

145?????????????????150?????????????????155CGA?TCC?AGC?ATG?TCC?ATC?GAT?GAA?TTT?CCC?GGA?AGC?TTC?GGC?AGC?CAG?????1469Arg?Ser?Ser?Met?Ser?Ile?Asp?Glu?Phe?Pro?Gly?Ser?Phe?Gly?Ser?Gln

160?????????????????165?????????????????170TAC?GAG?GAA?ATT?CTG?AGT?GGT?GCC?ACC?AAC?GAC?CTA?TTC?GAG?GCG?GTC?????1517Tyr?Glu?Glu?Ile?Leu?Ser?Gly?Ala?Thr?Asn?Asp?Leu?Phe?Glu?Ala?Val175?????????????????180?????????????????185?????????????????190CAA?GTG?TAC?ACG?GTT?GAC?GGC?GGC?GAG?GGC?AAC?AGC?AAG?TAC?CTC?ATG?????1565Gln?Val?Tyr?Thr?Val?Asp?Gly?Gly?Glu?Gly?Asn?Ser?Lys?Tyr?Leu?Met

195?????????????????200?????????????????205ATC?GTT?GAG?GCG?ATC?GGG?TCC?ACT?GGA?CAT?CGT?TAT?TTC?CGC?TCC?TTC?????1613Ile?Val?Glu?Ala?Ile?Gly?Ser?Thr?Gly?His?Arg?Tyr?Phe?Arg?Ser?Phe

210?????????????????215?????????????????220ACG?GCC?AGC?AGT?CTC?GGT?GGA?GAG?TGG?ACA?GCC?CAG?GCG?GCA?AGT?GAG?????1661Thr?Ala?Ser?Ser?Leu?Gly?Gly?Glu?Trp?Thr?Ala?Gln?Ala?Ala?Ser?Glu

225?????????????????230?????????????????235GAT?AAA?CCC?TTC?GCA?GCA?AAG?CCA?ACA?GTG?GCG?CCA?CCT?GGA?CCG?AAG?????1709Asp?Lys?Pro?Phe?Ala?Ala?Lys?Pro?Thr?Val?Ala?Pro?Pro?Gly?Pro?Lys

240 245 250ACA TTA GCC ATG GTG ACT TGG TTC GCA ACA ACC CTG ATC AAA CCA TGA 1757Thr Leu Ala Met Val Thr Trp Phe Ala Thr Thr Leu Ile Lys Pro *255 260 265 270CTGTCGATCC TTGCAACCTC CAGTTGCTCT ATCAGGGCCA TGACCCCCAA CAGCAGTGGC 1817GACTACAACC TCTTGCCATG GAAGCCGGGC GTCCTTACCT TGAAGCAGTG ACGAGCTTAT 1877CTTTAGTTGC AGATCGTGTT TCTCCTTTCT TCTTCAAGTA GTTTTAGTGG TGGAAGACAG 1937CAGAAGGTGG TCATCATCTT AGGCTCAGTT GGGGTGGGCT CCTGCCACGT TTTGTCCATA 1997GGCTAGTAAT TTGCACGGAA TTCAGTTCAT TGGCAAGGAG TGCGGTACGA ATACCTGTTT 2057TCACAATAGC AATTAGGCCC AGTAGTTATA CTACGTACTG GAATTGAGTA CTCGTAGTAG 2117CAAGATTGTT TGCCTCAGAG GGAATGGCCG ACACGTGAGC AAGTCACCTT CATCAGCTAG 2177TCGCGTTCCA CATAGACAAT GGTCCAGCTC CAGAGTGGAA TTTGGGCTAC TTTGAACGAT 2237GGCCGATTGA ATCGCGCGTC TCCTCAATTG TATTTAACCA CAATAGGCCA GGTATTGGCA 2297TTCACTCTCC GCCTTTGCGG GTGCCGGCAC GAGATGTCTC CTGAAGAAAC TAGGCAACGA 2357GCAGACTGTG GATATGGGAG ATGGTTGACG ATGTGCTTCT TGGTAAATTT GAAGCCTCCA 2417GGGCCTCTAG AAAGGCGGGA ATTTAAATCT CAAGTGCCCT AACGTGTCCG ACCACGGTGT 2477TGATCATCAT TCATTGAATC GGATAACAGT CTTGGTTCGG AAACTGAACA GGCGGCTCTT 2537GAATGACACT CTGGATCC 2555SEQ ID NO：13：CTGTCGATCC TTGCAACCTC CAGTTGCTCT ATCAGGGCCA TGACCCCCAA CAGCAGTGGC 60GACTACAACC TCTTGCCATG GAAGCCGGGC GTCCTTACCT TGAAGCAGTG ACGAGCTTAT 120CTTTAGTTGC AGATCGTGTT TCTCCTTTCT TCTTCAAGTA GTTTTAGTGG TGGAAGACAG 180CAGAAGGTGG TCATCATCTT AGGCTCAGTT GGGGTGGGCT CCTGCCACGT TTTGTCCATA 240GGCTAGTAAT TTGCACGGAA TTCAGTTCAT TGGCAAGGAG TGCGGTACGA ATACCTGTTT 300TCACAATAGC AATTAGGCCC AGTAGTTATA CTACGTACTG GAATTGAGTA CTCGTAGTAG 360CAAGATTGTT TGCCTCAGAG GGAATGGCCG ACACGTGAGC AAGTCACCTT CATCAGCTAG 420TCGCGTTCCA CATAGACAAT GGTCCAGCTC CAGAGTGGAA TTTGGGCTAC TTTGAACGAT 480GGCCGATTGA ATCGCGCGTC TCCTCAATTG TATTTAACCA CAATAGGCCA GGTATTGGCA 540TTCACTCTCC GCCTTTGCGG GTGCCGGCAC GAGATGTCTC CTGAAGAAAC TAGGCAACGA 600GCAGACTGTG GATATGGGAG ATGGTTGACG ATGTGCTTCT TGGTAAATTT GAAGCCTCCA 660GGGCCTCTAG AAAGGCGGGA ATTTAAATCT CAAGTGCCCT AACGTGTCCG ACCACGGTGT 720TGATCATCAT TCATTGAATC GGATAACAGT CTTGGTTCGG AAACTGAACA GGCGGCTCTT 780GAATGACACT CTGGATCC 798SEQ ID NO：14：ATG AAG TTC TTC AAT GCC AAA GGC AGC TTG CTG TCA TCA GGA ATC TAC 48CTC ATT GCA TTA ACC CCC TTT GTT AAC GCC 78SEQ ID NO：15

(i) sequence signature:

(A) length: 26 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:15:Met Lys Phe Phe Asn Ala Lys Gly Ser Leu 10Leu Ser Ser Gly Ile Tyr Leu Ile Ala Leu 20Thr Pro Phe Val Asn Ala 26

Claims (31)

1. An enzyme obtained from Aspergillus, wherein the enzyme has the following characteristics: a.MW is 33,270D±50D b. pI value is about 3.7 c. Arabinoxylan degradation activity d. The optimum pH is from about 2.5 to about 7.0 (more preferably from about 3.3 to about 4.6, more preferably about 4) e. The optimum temperature is from about 40°C to about 60°C (more preferably from about 45°C to about 55°C, more preferably about 50°C); Among other things, this enzyme cleaves arabinose from the xylose backbone of arabinoxylan. 2. An enzyme having the sequence shown in SEQ.I.D.No. 1, variants, homologues or fragments thereof. 3. An enzyme encoded by the nucleotide sequence shown in SEQ.I.D.No.2 or its variant, homologue or fragment, or a sequence complementary thereto. 4. A nucleotide sequence encoding the enzyme of claim 1. 5. A nucleotide sequence encoding the enzyme of claim 2. 6. A nucleotide sequence having the sequence shown in SEQ.I.D.No.2, its variant, homologue or fragment, or its complementary sequence. 7. A nucleotide sequence according to any one of claims 4 to 6 operably linked to a promoter. 8. The nucleotide sequence according to claim 7, wherein the promoter is a promoter having the sequence shown in SEQ.I.D.No.3 or its variant, homologue or fragment, or its complementary sequence. 9. A promoter having the sequence shown in SEQ.I.D.No.3 or its variant, homologue or fragment, or its complementary sequence. 10. The promoter of claim 9 operably linked to a GOI. 11. The promoter of claim 10, wherein the promoter GOI is operably linked, wherein the GOI comprises the nucleotide sequence of any one of claims 4-6. 12. A terminator having the nucleotide sequence shown in SEQ.I.D.No. 13, or its variant, homologue or fragment, or its complementary sequence. 13. A signal sequence having the nucleotide sequence shown in SEQ.I.D.No. 14, or its variant, homologue or fragment, or its complementary sequence. 14. A construct of the invention comprising or expressing any one of claims 1 to 13. 15. A vector comprising or expressing the invention according to any one of claims 1 to 14. 16. A plasmid comprising or expressing the invention according to any one of claims 1 to 15. 17. A transgenic organism of the invention comprising or expressing any one of claims 1 to 16. 18. The transgenic organism of claim 17, wherein the organism is a fungus. 19. The transgenic organism of claim 18, wherein the organism is a filamentous fungus, preferably Aspergillus. 20. The transgenic organism of claim 17, wherein the organism is a plant. 21. A method of preparing an enzyme according to any one of claims 1 to 3, comprising expressing a nucleotide sequence according to any one of claims 4 to 8. 22. The method of claim 21, wherein said enzyme has a sequence shown in SEQ.I.D.No.1, or a variant thereof, a homologue or a fragment, and said nucleotide sequence has a sequence shown in SEQ.I.D.No.2 , or a variant, homologue or fragment thereof, or a complementary sequence thereof. 23. The method according to claim 21 or 22, wherein the expression (partial or total) is controlled by using a promoter according to claim 9. 24. A method for expressing GOI by using a promoter, wherein the promoter is the promoter of claim 9. 25. Use of an enzyme according to any one of claims 1 to 3 or an enzyme prepared by a method according to any one of claims 21 to 24 to degrade arabinoxylan. 26. Use according to claim 24, wherein the enzyme is used in combination with a xylanase, preferably an endoxylanase. 27. A composition of enzymes that degrade arabinoxylan, the composition comprising an enzyme according to any one of claims 1 to 3 or an enzyme prepared by a method according to any one of claims 21 to 24; and a xylanase. 28. Plasmid NCIMB 40703, or a nucleotide sequence obtained therefrom for expressing, or controlling the expression of, an enzyme capable of degrading arabinoxylan, or controlling the expression of another GOI. 29. A signal sequence having the sequence shown in SEQ.I.D.No. 15 or variants, homologues or fragments thereof. 30. Use of an enzyme according to any one of claims 1 to 3 or an enzyme prepared by a method according to any one of claims 21 to 24 to prevent indigestion and/or to increase nutrient absorption in pharmaceutical or food production. 31. An arabinofuranosidase having arabinoxylan degrading activity, which immunoreacts with an antibody against the purified arabinofuranosidase having the sequence shown in SEQ.I.D.No.1.

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