FI117098B - Compsns. contg. phytate degrading enzymes - obtd. by expression of their genes in Trichoderma, used partic. for producing animal feed compsns. - Google Patents

Abstract

(A) a novel compsn. comprises a phytate degrading enzyme (PDC) produced by (a) transferring a Trichoderma host cell with a gene encoding the PDE and (b) expressing the gene encoding the PDE in the Trichoderma host cell. Also claimed are: (B) a recombinant construct comprising a genetic sequence encoding a phytase having the amine acid sequence shown or an enzymatically active; (C) a recombinant construct comprising a genetic sequence encoding a pH 2.5 AP having the amino acid sequence shown and further comprising a genetic sequence encoding a signal sequence operably linked to the first genetic sequence, where the signal sequence is selected from the signal sequence of Trichoderma CHBI, Trichoderma CBHII, Trichoderma EGO and Trichoderma EGII and the homologous phytase signal sequence; (D) a vector comprising a recombinant construct as in (B) or (C); (E) a Trichoderma host cell transferred with a genetic sequence encoding a phytase degrading enzyme.

Description

1 1

PRODUCTION OF PHYTHATIC DEGRADING ENZYMES TRICHODERMAN

The present invention relates to a process for the production of a composition comprising an excess of phytate-degrading enzyme.

Mesophilic filamentous fungus Trichoderma efficiently secretes salivase enzymes substrate Under optimum growth conditions, it has been reported to even contain 10 40 g / L extracellular cellulose {Durand et al.,

Microb-Technol. 10: 341-346 (1988); Durand et al., Biochemistry and Genetics of Cellulose Degradation, A Press, 1988, 135-151).

The development of T. reesei transformation methods enabled the use of genetic engineering techniques (Knowles et al., EP244,234; Penttilä et al., Gene 61: (1987); Berka et al., EP215,594). The genetic engineering aids in the production profiles of cellulases have been modified, for example, to produce strains that produce larger endoglucanase I enzymes. The intense cbhl promoter used to increase endoglucanase expression (Ne \ • · *; · ** et al ,, The Molecular Biology of Trichoderma and its Help * * * to the expression of both homologous and heterologous Molecular Industrial Mycology, Leong and Berka, Marcel Dekker Inc., New York, 129-148 (1991); and Harkki, V, < - > Enzyme Microb. Technol. 13: 227 -233 (1991).

. . In addition to the processing of homologous protoforms, the production coefficient of T. reesei ♦ *

M1-I ... ... 2 (Nyyssönen et al., WO92 / 01797) and fungal] ligninolytic enzymes {Saloheimo, M. and Niku-Paavo 1 Bio / Technol. 9: 987-990 (1991)). For better expression, the desired gene is inserted into 5 expression cassettes and ligated into T. reese by prot transformation (Harkki, A. et al., Bio / Technol. 7: (1989); Nyyssönen et al., WO92 / 01797; (Saloheimo,

Niku-Paavola, M.-L. Bio / Technol. 9: 987-990 (1991)).

heterologous filamentous fungal promoters such as Asp * 10 amdS, arqB and glucoamylase (GA) may function at least to some extent, (Penttilä et al., Gene 61; (1987); Knowles et al., EP244,234) requires homologous expression. use of the promoter. In addition, better yields have been obtained by producing the desired fusion protein (Harkki, A. et al. Bio / Technol. 7: (1989); Nyyssönen et al., WO92 / 01797). The yields of T. reeseiste heterologous proteins have been 10-15 <Phytate, which is a f os f ori store of plant seeds, part of the human and animal diet. Phytate Phos 20 is difficult to obtain in monogamies because it mixes with polyvalent metal ions and s m proteins. Therefore, the phytate degradation] • · * is of interest. Plant-phytin-degrading enzymes: phytase and acid phosphatase, which convert β, β 25 to inositol and inorganic phosphorus, produce bacteria (Powar, VK and Jagannathan, VJ, J. Bac: *: 15: 1102-1108 (1982); Cosgrove, DJ, Aust J. Bio!

23: 1207-1220 (1970) and Cosgrove, D.J. et al., Aust. J

1 ^ 6: 1348-1351 (1968)). Both phytase and pH 2.5 acid phosphatase are required for complete degradation of plant phytin.

Industrial applications require significantly higher yields than naturally occurring ones. The gene encoding the phytase has been isolated and characterized for a short time from A. f icuum (Van Gorco EP420 / 358 or WO91 / 05053) and the phytase production is lis niqer by inserting the gene copy number in a cassette 10 containing a strong home Aspergillus promoter e.g. The gene encoding Gorcom et al., EP420,358 or WO91 / acid phosphatase has been isolated and characterized from A. niqer (MacRae et al., Gene 71: 339-348 (198i) Knowing the better production methods for compositions containing phytase and pH 2.5 acid phosphatase. the inventors have developed a highly efficient recombinant production method for producing these compositions.

According to the invention, there is provided a process for producing a composition comprising an excess of phytate-degrading enzyme and at least 20 Trichoderma enzymes selected from β-glucan.

· * '*: Activity, CBHI, CBHII, EGI and EC

To produce the composition, a recombinant constant comprising a gene encoding a phytate digested enzyme selected from a phytase and a pH 2.5 acidophosphate * * · · · · 25 is prepared wherein said phytase has the amino acid sequence No. : 8:] and at a pH of 2.5 acidophosphase • * · amino acid sequence [SEQ ID NO: 8:]. : 2:], and function * * the signal sequence associated with said gene. Composition 4 1

Figure 1. Sequence of peptide # 816 ([SEQ ID NO: oligo PHY-31 ([SEQ ID No: 64:]), peptide # 1110 ([SEQ ID NO: oligo PHY-34 ([SEQ ID NO: 65: ]) and oligo PHY-35 (No.: 52:]) pH2.5 acid phosphatase oligonucleotide PH 5 17 mer mixture with 64-fold degeneration of inosine Peptide # 816 is derived from the degradation of native acid phospho endoproteinase Lys-C PHY-34 is 17meer with 128-fold degeneration PHY-35 is 17me not with 64-fold degeneration Both PHY-34 and PHY 10 are essential for the complete product of peptide # 1110 Peptide # 1110 is derived from native acidophos: trypsin degradation.

Figure 2. Nucleotide sequence 2.1 kb Sphl fragment with pH 2.5 acidophos f a re gene [SEQ ID NO. : 1:],; 15 derived amino acid translation [SEQ ID NO: 15

Intron donor, lariate and acceptor * as determined by cDNA sequencing are

Nucleotide sequence corresponding to peptides # 816 (

Well. : 57:]) and # 1110 ([SEQ ID No: 62:]) are below; The genomic nucleotide sequence was determined with M13-d; : * "(Sanger, F., et al., Proc. Natl. Acad., 1974) using United States Bioc * 9 -: ***! Sequencase II- putty.

| t: ": Figure 3. Phytase tryptic p <25 amino acid sequences # 792 [SEQ ID No:: 43:] and # 420 No.: 23:] and derived oligonucleotides [SEQ ID NO: 3 :, * * 5 and: 6 :] used in the 1 *** • 2 serial PCR amplification of the phytase probe.

5

Figure 5. Nucleotide sequence of the phytase gene [SEQ ID Nos. : 7: (DNA) and: 8: (amino acid)].

Figure 6. PCR primers used in the preparation of phytase fusion fragments [SEQ] 5: 9: and: 10: and: 11: and: 12: j.

Figure 7. Structure of pALK171. phytase; which had its own signal sequence, was fused to the cbl motor. Only the most relevant restriction sites are shown.

10 Figure 8. Structure of pALK172. The phytase gene was inserted into the cbhl signal sequence. Only relevant: restriction sites are shown.

Figure 9. Plasmids pALK173A and pAJ The plasmids carrying the phytase gene with the promoter and the selection label, the amdS gene, are shown. In plasmid pALK173A, the transcriptional orientation of phytase and amdSc is the same; je mid pALK173B, the transcriptome orientation of the two genes is opposite to each other. Figure 10. Western blot results of samples of native strains of Trichoderma host strains and transformant-producing transformants. Belt 50 ng purified Aspergillus ALKO 243 phytase • ·

, · *, Pulse 2: 15 ng of endoF-treated Aspergillus AL

25 phytase; Lanes 3 and 10: T. reesei ALKO 23 ^ lanes 4-5 and 11-12: T. reesei ALKO 233 transfected with ** / 171FR / A4 and A13, respectively; Zones 6 and; reesei ALKO 2221? Lanes 7-8 and 14-15: T1: ALKO 2221 transformant 171FR / A5 and A9, respectively 30 Lane 9: T. reesei ALKO 2221 transformant D2 1: ': lane 16: T. reesei ATCC56765; Zones 17, 18,

raaeai »HiPPRCTCi f yanef / NMnani · 4 I · niPD / AOl Λ 1 1 A

11 6 pH 2.5 for acid phosphatase fusion fragments [Nos. : 13: and: 14: and il5:].

Picture 12. Structure of plasmid pALK533. acid phosphatase gene having its own signal sequence inserted into the cbhl promoter.

Figure 13. Structure of plasmid pALK532, acid phosphatase gene fused to your cbh1 signal here and to the promoter.

Figure 14. Western blot of Trichoderma trans formants, 10 producing pH 2.5 acid phosphatase. Lane 1: 10 ng purified Asjus ALKO 243 pH 2.5 acid phosphatase; Zone; endoF treated Aspergillus ALKO 243 pH 2.5 ha] phytase; and Lanes 3-9: 60 µg of protein already transformed with Trichoderma reesei ALKO 2221 transformant KA-31, KA-17, KB-44, KB-18, SB-4 and KA-28 from supernatant.

Definitions 20 Many of the terms used in recombinant DNA technology are described in the description below. For the purpose of the Explanatory Note and the Claim ···, a clear and uniform understanding, * V '· 25 including the area covered by these terms, is given by the following definitions: es.

* j] ··; Gene. DNA sequence containing tern] S.: (template) for RNA polymerase. RNA, which is a copy of or does not encode a protein. RNAs <30 encoding a protein called messenger RNA in l.f: eukaryotes it copies RNA polymer! 11 7 and the so-called "antisense RNA". Further, the antisense RNAs are not readable because the antisense RNA contains end-translational codons {stop codor 5 Complementary DNA or cDNA contains banting genes that are synthesized by reverse transcription of the first mRNA and thus give rise to intermediate sequences (introns). Genomic DNA genes have or do not have introns.

10 Cloning vector. Plasmid, phage DNA t A DNA sequence capable of delivering gene information, especially DNA, to a host cell *, characterized by having y] more endonuclease recognition sites where the DI 15 sequences can be cleaved into a specific model without reducing the biological activity of the vector. to which the desired DNA can be inserted in order to clone it into a host cell. The cloning vector may also have a label suitable for the identification of the cells mapped to the cloning vector tr * 20, and an origin of replication that allows the vector to be maintained and repeats in one or more prokaryotic or eotic hosts. Labels include, for example, 4 ···, cyclin resistance or ampicillin resistance, • Γ *. Of the 25 expression vectors "is also referred to as" vel · "Plasmid" is a cloning vector, most commonly a renc * * ** / / DNA, which is maintained and replicated ** in at least one host cell.

«· V! The expression vector. The transporter or vector 30 is similar to a cloning vector but does not enhance expression of the gene cloned into it *! *. lfoon lrnn rrQön -i r-r »-I-r * ar» 4 * n rm λ -f -I-1 »{(? 4 4-9en1in If 1 /« 8 r vary depending on whether the vector is mapped to express gene in a prokaryotic eukaryotic host cell and may contain transcriptional elements such as transcriptional elements (upstream activation sequence decision sequences and / or translation initiation sites).

Host. The host is a prokaryotic or (ootic cell used as a carrier or recipient of the recombinant).

Eukaryotic host. A eukaryotic can be any cell of a eukaryotic organ, including, for example, an animal, a plant, a yeast, a host of the invention. An "inventive host" is a filamentous fungal host that is modj to produce phytase and / or pH 2.5 acid phosphate by the methods of the invention.

Functional derivative. A "functional derivative" or protein of a nucleic acid of protein 20 which is chemically or biochemically (or derived) from a protein or nucleic acid which retains its biological activity (functional or structural), which is characteristic of a * 25 protein or nucleic acid. Protein 4 * ·· A nucleic acid mutant is a biochemical or chemical derivative of a protein or nucleic acid {@ **, a "functional derivative" includes * tt f .:: "mutants", "fragments", "variants of the" "," 30 git "or" chemical derivatives "that retain the desired activity of the native molecule.

• Ί '. The present invention is directed to reducing the toxicity of the molecule, removing or removing any unwanted side of the molecule, etc. Additives that mediate such effects are described in Remington's Pharm; 5 Cal Sciences (1980). Methods for attachment to the molecule are known in the art.

Fragment. By "fragment" of a molecule, such as a nucleic acid of a protein, is meant the genetic sequence of n «amino acid or nucleotide, and in particular the functional derivatives of the invention.

Variant or analog. By "variant" or "analog" of a protein or tartaric acid is meant a molecule which is substantially similar in structure and biological activity to an i molecule, such as that encoded by a fatal allele.

The hosts according to the invention instead of producing endogenous phytase, T. reesei produces large amounts of other enzyme components, such as betaglucan degradation activator, which is important e.g. in feed applications. Here: «* The use of 25 reesees as a production organism for fungal * * reptiles and pH 2.5 phosphatase results in · * *

'··· secretion of different enzyme compositions than J

i .: Aspergillus. Also, when using Trie

; earth phytate-degrading enzyme compositions J

30 some difficult handling problems with Aspergillus compositions (e.g.

»· * * Λ Λ Λ Λ 7_ _ ^ \ L * 1 i. I-m ti ti * t— Λ *« _! _! * Λ— Λ— ti .. 11 swells Asperqilli often mucoid and thick m; case.

Larger amounts of phytase and pH 2.5 ha] phytase (as compared to * 5 theses in Asperqillus) can be produced by expressing T. reesei by inserting a DNA sequence from A. niqer coding for the phytase or pH 2.5 acid phosphatase into the T. reesei expression cassette. sii the cbhl promoter and the Aspergillus amdS gene tri10. The transfection of the resulting construct into T. reesei hosts produces stable transformants that express phytase or pH 2.5 acid phosphate in large amounts in a novel enzyme activity assay.

The mixture produced by T. reesei contains] betaglucanase activity but low glycase activity. In addition, the amount of recombinant T.sub.1 strain produced by shaking cultures is found to have a secretion level of the major cellulase, endogenous cellobiase <20 g, greater than 1 g / L. The pH2.5 amount of phosphatase by shaking cultures of the recombinant strains is less than 0.5 g / L.

·, * ·· Aspergillus niqer var. awamori ALKO 24: 38854) (IFO4033) phytase and acid phosphatase (op. * ·. · 25 2.5) were overexpressed in Trichoderma reese Tricl.

. * · *. cellobiohydrolase 1 (cbh1) promoter control

«• e • ·. In addition, the phytase gene was expressed under its own promoter i S ii «* la» ts «* Two genes containing the cbh 30 engine were made for both genes: phytase or acid phosphatase * I« ί .__ 1_______i m «.__« _ . .__. . ,. "11

sequences (and not the entire vector used to maintain the sequences) and obtain strains did not contain any "foreign" sequences such strains were suitable for industrial purposes. The three strains of Trichoderma reesei, ATCC

(RutC-30), ALKO 233 (VTT-D-79125) and the low ascoproteinase producing strain ALKO were used as hosts for phytase expression. When expressing phagease, only T. reesei ALKO 2221 10 was formed. When the phytase was expressed on the cbh1 <promoter in Trichoderma, the best transformation was devoid of any E. coli sequences yielded approximately 3,600 times more phytase: untransformed A. niqer ALKO 243 in lysis cultures. When using the 15 phytase promoter, The reesei transformant11a was about 120 µg compared to A. niqer ALKO 2 * Best acid phosphatase activities, which was about 240 times higher than the levels obtained with A. 20 ALKO 243 strain.

The molecular weights (by SDS'PAGE) of the phytase and] acid phosphatase secreted by Trichoderma are those of the corresponding enzymes secreted by Asperqillus. The difference appeared to be due to differential Λ.ϊ 25 glycosylation.

, * ·· # The rate of return of phytase obtained when · '**. gillus gene expressed in Trichoderm under Trichoderm I t a ™ motor control was surprisingly high.

The use of T. reesei as a host for fungal phosphatase 30 pH 2.5 leads to a completely different J.! ! enzyme compositions as with Aspergil i ·· »» I ry____,, _, «*. ,. ,! _ · 11 12 all Trichoderms. Trichodermat is categorized on the basis of their physical similarity. T._ was previously known as T. viride Pers. it ninqii Oudem; it was sometimes classified as a separate group of 5 T. lonqibrachiatum. The whole Trichoderma-i is characterized by fast-growing colonies, already. bushy or pustular, branched diophores with vial-shaped fialides and transparent or green conidia at those ends (Bj 10 J., Can. J. Bot. 62: 924-931 (1984)).

T. reesei fungi are clearly defined (the genus T lineage derived from strain Ql · on, a common genetic lineage derived from the simple nucleus of a particular QM6a isolate. Only these strains are called reesei.

Classification by morphological means is a single list and recently published molecular DNA fingerprinting and hybridization model of the cellobiohydrol 20 (cbh2) gene in T. Theses in the lonqibrachiatum clearly demonstrates their differentiation (Meyer, W. et al., Curr. Genet. 2J! (1992)? Morawetz, R. et al., Curr. Genet. 21 (1992).

However, there is evidence for the similarity of the different 1, ··· * derma species in molecular * cells observed in macromolecular osv I. »ΊΙ / nucleic acid and amino acid sequence conservation

* Common to different Trichoderma species. E

30 Why Cheng, C., et al., Nucl. Acids. Res. 1 ·· * · · (1990), presents the nucleotide sequence of T. viride cbhl »-« ~~ · · -

> f · -. ****** t M *. i_ »t M 1 * _ * _ * M

The 13 identified amino acid sequences had 81% homolo reesei with the phosphoglycerate gene. Thus, those classified as T. viride species and T. species must be genetically very close to 5 a.

In addition, there is a great similar

Under Trichoderma Transformation Conditions. Although virtually all industrially important hoderma species are present in the Trichodei 10 lonqibrachiatum described above, there are other Trie] species not included in this section. Such are, for example, Trichoderma harzianum, which b.

as a roller against plant pathogens. The Transfo method for this Trichoderma species also is kel 15 (Herrera-Estrella, A. et al., Molec. Microbiol.

843 (1990)) and is substantially the same as those disclosed in the applications. Thus, although Trichoderma harzianu is considered to belong to the Lonqibrachiatum section, the method used by Herre] rella for the preparation of spheroplasts; 20 before transformation is the same. Herre] rella's presentation shows that there is no such significant difference in Trichoderma spp. * Ί would suggest that the inventive trans f <··· method would not work in all Trichoderma.

** .: 25 In addition, fungal species share a common function in the transcriptional control of fungi. glycol. At least three of your A. nidulans promoters. ···! sin amdS, arqB, and qpd have been found to give time to i * »nick expression in T. reese. amdS: lie ja arqB: 1J. . One or two copies of the gene are sufficient for expression of selection phenotypes (Penttilä et al., Ill 14 1r in various databases. These institutions and titles are listed in O'Donnell, K. et al., Hemistry of Filamentous Fungi; Technology and Pr <DB Finkelstein et al., Butterworth-Heir 5 Stoneham, MA, USA, 1992, 3-39.

Construction of hosts according to the invention

Processing of the genes * of the hosts according to the invention according to the invention is possible by * purifying a protein encoding the enzyme or by partially or cloning the genetic sequences by polymerase reaction (PCR) technology, capable of encoding the desired protein? and by expression of the genetic sequences, starting with the term "genetic sequence" into a vi nucleic acid molecule (preferably DNA). Gene «sequences capable of encoding protein derived from various sources. These sources include, for example, 20 mi-DNA, cDNA, synthetic DNA and their combination The most preferred genorai DNA source is the fungal genome * to. The most preferred source of cDNA is a cDNA library • · made from fungal mRNA grown in c pathways known to induce expression of the desired mRNA protein.

· * "· The genomic DNA of the invention contains 1 ··· {naturally occurring introns. In addition, · $ *, genomic DNA can be obtained by means of the gene sequence! I *« promoter region and / or the 3'-transcriptional region. genomic DNA v "j / obtained with the help of genetic sequences that construct v! ·

* * * C f A 4 _ 1 A4 ^ 41 «« M DATA A 1 A «A / X« MA. A A A X

1 · 15 transcribed regions and / or mRNA 5 and / or translated regions can be maintained and used for scriptural and translational weather Genomic DNA can be extracted and purified from which 5 host cells, especially fungal host cells, naturally express the desired protein (s). technology.

Suitable DNA sequences (either genomic DNA or cDNA) for cloning the vector are randomly excised or cleaved enzymatically or into suitable vectors to generate a recombinant gene: (either genomic or cDNA) * DNA sequence encoding the desired proi or a functional derivative thereof. , can be; 15 to the DNA vector by conventional methods, end of the tel sequence for smooth or uneven ligation, digestion with a restriction enzyme to produce suitable expression, proper cohesive ends, alkaline phosphatase treatment to prevent non-integration and ligation with suitable gasses. Such methods are es: Maniatis, T., (Maniatis, T. et al., Molecular ((A Laboratory Manual), Cold Spring Harbor Laboj K.J. Second Edition, 1988)) and are well known * 25 J ankles.

· * “: Sequences encoding the desired gene s: * -t * {**. These libraries can be screened and the desired gene) · *, identified by any method that s% i * s select the sequence encoding one. , 30, or a protein, for example, by a) hybridization with a transcription protein product produced by immunoprecipitation of a s-nucleic acid probe or probes, 4 * 11 16 mRNA.

Oligonucleotide probes recognizing a particular protein, which can be used to identify clones of a protein, may be prepared when the amino acid sequence of the protein or the nucleic acid of the DNA encoding such a particular protein; sequence. Alternatively, pure antibodies to the protein can be prepared and identified to detect the presence of unique protein minants in transformants expressing the desired clone. The sequence of the residues of the peptides Amin <is now defined by 1 alpha or single alpha short: A list of such alpha-alphabetic abbreviations can be found in, for example, Biochemistry, Lehningc Worth Publishers, New York, NY (1970). When am; the posterior sequence is shown horizontally, unless defined, with the amino terminus to the left and the carboxy terminus. Similarly, unless otherwise noted or clearly apparent, the 5 'end of the nucleic acid sequence * * · is shown on the left.

Because the genetic code is degenero: • # * · * More than one codon can encode a particular ant po (Watson, JD, In: Molecular Biology of the *: *: 3rd Ed., WA Benjamin, Inc., Menlo Park, CA | et · ·

356-357). Peptide fragments are analyzed for selI

a to identify the amino acid sequences tested. The oligonucleotides with the lowest degenerates can be best performed by identifying that although all members of this group contain oligonucleotide sequences capable of the same peptide fragment and thus contain the same oligonucleotide sequence as the 5 fragment encoding gene. , only one group contains a nucleotide sequence identical to an exon encoding a nin sequence. Any member is included in a group and is capable of hybridizing DNA, even in the presence of other members of the group, to use an unfractionated oligonucleotide in the same manner as used to clone a single oligonucleotide encoding a peptide using one or more oligonucleotides of each amino acid sequence. <the desired protein. The likelihood that a particular gonucleotide actually contains the actual Iine coding sequence can be estimated by tut; abnormal base pair ratio and frequency, 20 specific codons are used (to encode a particular acid) in eukaryotic cells. Using the "1 rules of use" identifies a single olig <RTI ID = 0.0> otid </RTI> sequence or collection of nucleotide sequences: which, in theory, have the "most likely" nu] 25 sequence capable of encoding your protein.

A suitable oligonucleotide or oligonucleotide: 4 * capable of encoding a particular gene fra <... · (or which is complementary to such olig <30 otide or oligonucleotide group) may be. . tis by methods known in the art (see &quot; 4 · 4 · 6 * 18 18, et al., Molecular Cloning, A Laboratory 1, Cold Spring Harbor Laboratories, Cold Spring Harl (1982)), and Haraes, BD, et al., Acid Hybridization, A Practical Approach, IRL 5 Washington, DC (1985). The members of the above gene that have been found to be capable of hybridization are analyzed to determine the amount and quality of the coding sequences they contain.

To detect the desired DNA coding sequence, the aforementioned DNA probe is labeled with a wound group. Such a detectable group can be any material that has a physico-chemical detectable property. Such n ations have been developed for nucleic acid hybridisation and generally a label useful in those colonies is also suitable for the present invention. Particularly useful are radioactive agents such as 3ZP, 3H, 14C, 35S, 125I or the like. Whatever radioactive label having a suitable Signa 20 sufficient half-life can be used, the oligonucleotide is single-stranded, it can be 1 using a kinase reaction. Alternatively, polynucleotides are useful nucleic acid * * * ** *! as radiation probes when labeled with a non-radio-labeled fluorescent group such as biotin, enzyme] * «♦ * ··.

In summary, the determination of a partial protein sequence * · * · allows the identification of a theoretical "probable" DNA sequence or collection of DNA capable of coding. . the peptide in question. By constructing the oligonucleotide

III

a «A ^ - 1 -. 1 1 ^ a - - i_i_ ^ Ί 1 a. ^ 1 1, 11 si.

In an alternative gene cloning procedure, the library is prepared using the expression velocity by cloning into the expression vector DNA or mi * 5 cDNA prepared from a cell expressing the protein. The library then monitors for members expressing the expressed protein, for example, by screening the library for antibodies to the protein.

The methods set forth above are capable of identifying genetic sequences that encode a protein or a biological or antigenic fragment of a protein. Such characterization of the aesthetic sequences to produce the recombinant protein above, the desire for expression of the proteins encoded by these sequences reveals those clones that find proteins having the c protein of the desired protein. These properties may include the ability to specifically bind to an antibody, including the ability to produce an antibody capable of binding to a non-recombinant protein. . enzymatic activity in a cell that is a prot · · · · · / part, and the ability to produce a non-enzymatic (but sp * * * ··· * 25) function in the receiving cell.

• i *! The DNA sequence can be truncated by methods known in the art to isolate the desired gene! a mosomal area containing more »» »f. * · *; countries as needed to utilize the gene in a k m 30 compliant host. For example, restriction op; can be used to cleave full length m 4 · *** * β ΐ U a 1 e a 4 α1% 4- a j ^ «t« 1 11 20

Exonuclease III and Bal31. Other nucleases are well known.

If the coding sequence and the functionally linked promoter are inserted into the receiving 5 ootic cell as a non-replicating DNA (or R as a non-integrating molecule), the encoded pro-expression may take the form of a linked (non-stable) expression.

Preferably, the coding sequence is linked to a 10 (or RNA) molecule, for example, a closed cytosolic annular molecule that is incapable of native replication, or preferably a lineage molecule linked to the host chromosome. Gene-stable transformants can be obtained by Konsi 15 vector methods or the transformation method: wherein the desired DNA is inserted into the host chromosome, such as by insertion into a de novo or by transformation of a vector that incorporates itself into a functional chromosome, for example transposon or DNA elements. DNA sequence incorporation into chromosomes. Use is made of one that is capable of linking the desired gene set to the ♦ + *! / Fungal host cell chromosome.

* '**, 25 Genes encoding phytase or * * (> *: acid phosphatase under the control of a suitable promoter) are linked in a plasmid construct and inserted into the m' cell by transformation. Vector type i from V host. A practical embodiment of the invention was used as a filamentous fungus Trichoderma.

: Trichoderma, in particular T. reesei, the association of Sue 21 n with the locus relevant to this locus.

Cells that stably link the linked DNA to their chromosomes are selected from one or more tags that allow the host to select which expression vector the chromosome label can confer, for example, biocide resistance <e.g. . The marker gene of choice 10 can either be directly linked to the DNA gene sequences to be introduced or be co-transfected into the same cell. Keksinnönmu! the genetic tag of the hosts is amds, which encodes what is involved and allows Trichoderma to grow as <15 the only nitrogen source.

In order to express the desired protein and / or its Akti: derivatives, transcriptional signals that are suitable for my father to transcribe are required. The cloned coding sequences obtained by the above methods; read in double stranded form, can be functionally linked to sequences that control trai. thionic expression in the expression vector, * »« can be linked to a host cell, either prokaryotic * * 4 ** ·· [25 eukaryotic, to produce a recombinant protein • # 'a functional derivative thereof. Depending on this, the strand of the coding sequence is functionally linked to the transcriptional expression coding gene, it is also possible to express antisense 30 or a functional derivative thereof.

; · '. Expression of a protein in various hosts "capable of expressing" a polypeptide, containing expression control sequences containing transcriptional regulatory information and sequences operably linked to the nucleotide sequence encoding the pol 5.

A functional linkage is a linkage in which it: is linked to a regulatory sequence (sequence; such that expression of the sequence occurs by or under the control of the weather sequence. A Kahc 10 sequence (e.g., coding sequence and 5 'linked promoter control sequence) is said to be operably linked the desired protein was transcribed by the mRNA and if the nature of the linkage between the two DNA secs1 does not (1) result in a junction, (2) does not interfere with the ability of regulatory expression to direct the protein, antisense RNA; Thus, the promoter region is functionalized into a DNA sequence if the promoter was able to use this DNA sequence for transcription.

The regulator needed for gene expression; the exact nature of the regions may vary with different 1 <* »* and cell types, but generally have 5'-ρέ • * *; ·· '25 transcriptional and 5 * terminal non-translational (non-cooc * · sequences containing transcription) or * initialization eg TATA code, mask sequence {, · · CAAT sequence or similar.

: T: set 5'-non-transcriptional control sequence sJ

<30 regions containing the promoter functional, as applied to the transcriptional control of the gene in A - 11/23, and preferably fungal regulatory systems. 1 uses a variety of transcriptional and Tramal regulatory sequences, depending on the host cell t1. Preferably, these regulate signals in the li: 5 native state to a specific gene that is abundantly expressed in the host cell.

In eukaryotes with transcriptional translation, such controls contain or do not contain an initiator-meth (10-AUG) codon, depending on whether such a methionine is contained in the cloning gene. Such regions generally contain a promoter region sufficient to initiate RNA synthesis in the host cell. Promoters of the Fis tis fungal genes encoding a trait-capable mRNA product are preferred and, in particular, strong promoters can be used provided they also function as promoters in the cell. Preferred strong promoters for Ί dermal include the T. reesei cbhl gene promoter or its cellulase gene promoter, e.g., cbh2, eq eq! 2. Using Trichoderma regulatory elements, protein expression can take place in Asy. lus nidulans regulatory elements I. / (e.g., arqB gene promoter 1a amdS qee • a * ··· * 25 motor), Aspergillus niqer regulatory elements • · * 5 (e.g., phytase promoter or · · ♦ amylase promoter ori). The expression of such non-lipid controlling elements by s: T: is very low compared to the use of Trich 30 elements and in particular to the use of T. Rees: elements.

• · * · 11 24 intervening codons encoding thionine. The full presence of codons results in either a fusion protein (if the AUG codon is in the same reading frame as the DNA coding for dreams) or a point mutation (5 AUG codons are not in the same reading frame as the p n coding sequence).

It may be desirable to construct a fuu, a tein containing a partial coding sequence of a protein (usually at the amino terminus of the 10 coding sequences (partial or assembly of the phytase-degrading enzyme of the invention) which may not function as a signaling sequence for secretion of Hal. For example, a fusion protein sequence may or may not be designed for subsequent deletion of the desired peptide sequence, and may be designed to allow secretion of the protein from the t-host or to localize the protein therein. a signal sequence or a derivative of a function * of this sequence that retains the ability to direct this 1 ««

Secretion of 25 peptides. AspergiJ

* "· Wire / secretion signal sequences also work for Trie \ .. 5 mass.

* ·

J Transcriptional aJ can be selected

ΓΓ: sensory signals that allow for repi or activation so that functionally the expression of the appendix: genes can be modulated. Adjust t «* * 25

If desired, the 3'-end of the non-transcribed and / or unwanted protein coding sequence can be obtained by cloning the 3'-end untranslated region for either the scriptural termination control element or those elements that are polyadenylated in eukaryotic cells. When the vit expression control sequences do not function properly in the host cell, the functional sequences in the host cell can be replaced.

The vectors of the invention may consist of other functionally linked regulators, such as DNA elements, that receive antibiotic resistance or replication to maintain the Started vector in one or more plaque cells.

In another embodiment, in particular; In order to maintain the appropriate vectors in procary cells or S. cerevisiae cells, the I20 sequence is included in a plasmid or virecek capable of independent replication in the host host. Any vector can be used; purpose. In Bacillus hosts, the desired DNA may be necessary.

*; · ** 25 The amount of a particular plasmid or virus vector; Important factors in this context are: the ease with which the receiving cells containing the vi * «··“ can be identified by selecting one of the receiving cells without the vector; desired vector instrument in 30 specific hosts; and whether there is a desired "sukkuloidc: ·" between different host cell types.

t «· · 26

Cycle and Inheritance, Cold Spring Harbor Labo Cold Spring Harbor, NY, pages 445-470 (1981); J.R., Cell 281203-204 (1982); Bollon, D.P., et__ Clin. Hematol. Oncol. 10: 39-48 (1980)? Maniati in 5: Cell Biology A Comprehensive Tr

Vol. 3, Gene Expression, Academic Press, NY (1980)) and are commercially available.

Once the vector or DNA sequence of the constructs or constructs has been prepared for the purpose, the DNA construct (s) will be introduced into a suitable host cell by any suitable means, for example by transformation. Following vector digestion, the receiving cells are grown on a lective medium that favors the growth of vector s 15 cells. Expression of the sequences of the cloned gene sequence results in production of the desired pro or fragment of this protein. This i. E. Can take place continuously in transformo cells or controlled, for example, through expression.

Fungal transformation is also carried out according to known methods, for example, k · β · β · for example homologous recombination with the lithium * * m *. / Gene stably into a fungal host and / or the ability of a thousand cells to express a particular protein.

¥ · * * **

Production of Antibodies «» * · «• *? * .. ·: Γ: The following description refers to the various] 30 methods known in the art of immunology «j, * · General Principles of Immunology • f * · 11 27

Plenum Press, New York (1980)); Campbell, A. ("Antibody Technology," in Laboratories in Biochemistry and Molecular Biology, Vo (Burdon, R., et al., Eds.), Elsevier, Amsterdam 5)); and Eisen, H.N., in Microbiology, (Davis, B.D., et al., Harper & Row, Philadelphi 0)).

An antibody is said to be "capable of nuclear" if it is able to react with the molecule and thereby bind to the antibody. By "epitope" is meant the portion of the hapten that can be recognized and the antibody can bind. An antigen may have γ more epitopes. An "antigen" is capable of producing an epitope that binds to an antigen only in an animal. By the aforementioned specific thio is meant that the antigen reacts very closely with the corresponding antibody and not with antibodies raised by other anti.

The term "antibody" (Ab) or "monoclonal <antibody" (Mab) is used herein to mean any molecule as well as fragments thereof, for example Fab and F (ab ') fragments) which * 1 25 antigen binding. Fab and F (ab ') 2-f]: ·· .1 you lack the • 1 fragment in the whole antibody, they are eliminated for faster circulation • · 1 2. ,,! they may be less nonspecifically bound to tissue than whole antibody (Wahl et al. 30 Nucl. Med. 24: 316-325 (1983)).

The antibodies of the present invention are capable of binding phytase or pH 2.5 ha phytase.

Cells expressing phytase or acid phosphatase, or a fragment or mixture thereof containing phytase or pH 2.5 ha phagease or fragments, can also be elicited to induce serum production of polyclonal antibodies capable of s phytase or pH 2.5 happofosfataasiproteiinin. At pH 10, the phytase or pH 2.5 acid phosphatase can be purified from other polyclonal media by standard protein purification methods, in particular by affinity chromatography using phytase or pH 2.5 acid phosphatase or fragments.

The phytase or pH 2.5 acid phosphatase protease may also be synthesized by chemical purification by HPLC for free contamination. Such a preparation is administered to an animal to produce polyclonal substances of high physical activity.

Monoclonal antibodies can be 1 using hybridoma technology (Kohler et al., 256: 495 (1975); Kohler et al., Eur. J. Immunol.

...

25 (1976); Kohler et al., Eur. J. Immunol. 6: 292 * * ** · * Hammerling et al. , in: Monoclonal Ant: '':: and T-Cell Hybridomas, Elsevier, N.Y., pages! • · * (1981)). Usually in such methods: phytase or pH 2.5 acid phosphatase: 30 is immunized with an antigen. Spleen cells from such an animal are extracted and ligated into a suitable I 11 29 bitter HAT medium and cloned with restriction dose as described by Wands, J.R., et al. roenteroloqy 80; 225-232 (1981), the disclosure of which is hereby incorporated by reference. A variety of hybridoma cells are analyzed to clone those that secrete antibodies capable of binding the phytase or pH 2.5 happ re-protein antigen.

Further cell lines capable of producing antibodies recognizing the epitope of the phytase-2.5 acid phosphatase protein may be obtained by applying the above methods.

Antibodies against phytase or pH 2.5 hap, on both the highly conserved and weak regions, are useful in studying the biosynthesis and catabolism of phytase and acid phosphatase, and in studies requiring the protein to be antigenized or quantified in the composition.

20

Production of phytase and acid phosphatase

The highest levels of phytase yield were obtained by Tri ». , countries transformed with linear DNA] * · * 11 with the cbhl promoter (about 3,800 PNU / ml, Kati lock 8). About 3,500 and 3,600 PNU / ml were grown * **: best T. reesei ALK02221 and ALK0233 were obtained · * »

formants that did not contain E. coli. Both phytase and cbh1 signaling] appeared to work equally well and similar levels of sin production were obtained using T. reesei AI

30 than any hitherto reported amount of hygene protein expressed Trichodermi The range of enzymes which precede co-phytase with the strains of Trie 5 according to the invention is different and less expensive than the Asperqillus broth preparations. Both endoglucose and cellobiohydrolase activities are substantially higher when using the derma host of the invention. Phytase Glycosylation Model o: 10 Different When Phytase Expressed Trichodermall produces a phytase protein which passes as a band in Western analysis.

Best pH 2.5 for the production of acid phosphatase or Trichoderma-trans formant sa c 15 APNU / ml broth in shake culture on a toose-based medium. As with the phytase, the signal sequence of phosphatase and cbh1 is as well.

The compositions of the invention which contain phytase may be used to remove phytic acid or ii-hexaphosphoric acid from a raw material, i.e. a phytin-containing raw material, and in batches; plant material. Phytase removes phosphate. . phytinic acid and destroys its ability to interfere with mini «« · 25 absorption. When used as an animal feed additive, the phytase compositions of the invention release · · * *; phosphorus bound to cereals and thus we: »* * significantly reduce the need for additional phosphorus in feed rations and reduce the environmental load.

• · * * · '**; 30 The phytase and m acid phosphatase produced according to the invention can be purified using known pi 31 EXAMPLE 1

Method for measuring phthase activity

Principle Phytase acts on phytate (5 olhexaphosphate) to liberate inorganic phytate. The amount of inorganic phosphate liberates becomes dye, which is formed when the phosphomolybdate is reduced.

Unit of activity One unit of phytase is the amount of enzyme which liberates 1 nmol of inorganic phosphate sodium phytate per minute in a standard solution.

15 Measuring conditions

Substrate Sodium phyta * pH 5.0

incubation temperature 37 ° C ± 0.5 ° C

incubation time 15 minutes 20

installations

water bath 37 ° C

water bath at 50 ° C

. # Spectrophotometer t φ «25 Test tube mixer (vortex) 5 ... # Phosphate-free glassware • * • *

Reagents All solutions are prepared in: deionized water, Milli-Q or similar water.

1. Citrate buffer (0.2 M. pH 5.0). Va! 0.2 M solutions in water and sodium tr <11 32 2. Substrate. Dissolve 1.00 g of natate (Sigma P-3168) in 70 ml of citrate; Adjust the pH to 5 with 0.2 M citric acid and make up to 100 ml with citrate buffer.

5 substrate solutions should be prepared daily.

3. 15% (w / v) TCA solution. It is prepared from acetic acid (Merck 807).

4. 10% (w / v) ascorbic acid solution. Va; ascorbic acid (Merck 127). Store in 10 loaves. The solution remains stable for seven days at 5. 2.5% (w / v) Ammonium molybdate solution, made up to 2.5 g (NH4) 6Mo7024 * 4H2O, Merck 1182) in water to 100 ml.

6.1 M sulfuric acid. Add 55.6 mL k <15 of H 2 SO 2 (Merck 731) to about 800 mL water and make up. Allow to cool and make up to one ml with water.

7. Reagent C. Mix 3 parts 1 M acid and 1 part 2.5% ammonium molybdate, add 20 parts 10% ascorbic acid and mix well, reagent C must be prepared daily.

Dilution of the sample Dilute the samples with the buffer. Weigh duplicate samples of each dilution. Weigh out 9 999 mg of the enzyme powder in a sample of approximately 250 mg, dissolve in buffer and make up to 25 ml in a volumetric flask, diluting as necessary.

999 • »• 99 9 9 9 999 9 9 0 9 Ψ · 9 9 9 9 33

Laimennustaulukko_

Estimated Active Recommended Dilution Volume PU / ml__dilution__ja_ 2000 1 + 19 20 5 20000 1 + 199 200 40000 1 + 399 400 100000 1 + 999 1000 500000 1 + 4999 5000 10

Analysis

Hydrolysis Pipette 1.0 ml sample containing 20-190 PU into two test tubes. Li 15 2.0 ml of 15% TCA solution in another tube (blind and mix. Tubes without TCA in a water bath at 37 ° C and equilibrated for 5 minutes. Using a stopwatch, hydrolyze by successive addition of 1.0 ml of substrate (equilibrated 10 at 37 ° C) into each tube and mix, After incubation for exactly 15 minutes, r is stopped by adding 2.0 mL of TCA to each tube Mix and cool to room temperature *: 25 1.0 mL of substrate is also added to the blind test p has been kept at room temperature) and stirred m · '1 · If a precipitate forms, it must be separated by a centipede for 10 minutes at 2000xq.

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

3.6 ml of water for each test tube. Inkub warm-up lacca 9 imi minimum na n na na h v 34 34 34 11 v 34 34 (Merck 4873, dried in a desiccator to a volume of 1 ml. Make the following dilute with water from a stock solution and use as a standard.

5

Dilution Phosphorus concentration Phytase activity in nmol / ml PU / m 1: 100 90 240 1: 200 45 120 1: 400 22.5 60 10 * The corresponding phytase activity (PU / ml) is calculated by dividing the phosphorus concentration (nmol / ml) by the hydrolysis time. (15 minutes) and multiplying by four (total volume after hydrolysis reaction / tet volume) and 10 (dilution before inorganic 15 analysis of its phosphorus)

Pipette 4.0 ml of each dilution into a test tube. Pipette also 4.0 ml of water into a test tube (reagent blank test). Add 4.0 π of 20 g C and mix. Incubate at 50 ° C for 20 minutes and cool at room temperature

· *** · laan. Measure the absorbances at wavelength I

«· · \ J reagent-blind test. Let's make a standard

···, plotting absorbance vs. phytase activity (E

* * 25 A new st disassembly must be made for each test set.

Calculation The blank absorbance of the test is reduced by the absorbance of the sample (the difference should be 0.

35

Preparation of feed and other insoluble matter for phytase analysis Weigh t.

2.5 g aliquot in two 50 ml beakers Add 20.0 ml citrate buffer. Stir] with a magnetic stirrer for 30 minutes at hi. Transfer about 10 ml of each! and centrifuge the solids for 10 minutes at 2000 x g. Place 2.5 ml supernal PD-10 gel filtration columns (Sephadex G-25M, 1010 and 17-0851-01) equilibrated in 25 citrate buffers. Remove the eluate. This; add 3.5 ml of citrate buffer to the column and k <eluate to a graduated cylinder, make up to 5.0 ml with citrate buffer (Law 15 Factor 2) and determine the phytase activity. A) Oral PU / g is obtained by multiplying the measured activity by ml) 40 (dilution factor x volume of extract buffer divided by the exact weight of the sample (g). Reference et al. Anal. Chem. 28: 1756-1758 (1956).

20 EXAMPLE 2

Determination of acid phosphatase activity, Principle Acid phosphatase acts on p-4 * * 25 phenylphosphate by releasing inorganic fc * ··· *. The specific amount of liberated inorganic phosphate turns to a color formed by the reduction of phospho-molybdate.

| Activity unit One acid phosphatase? i * · ': 30 (HFU) is the amount of enzyme which liberates 1 nmol of inorganic phosphorus p-nitrc 36 incubation temperature 37 ° C ± 0.5 ° <incubation time 15 minutes · 5 Equipment

water bath 37 ° C

water bath at 50 ° C

Spectrophotometer Vortex Mixer 10 Centrifuge (Hereaus Biofuge 17S. 3090 1 equivalent)

Phosphate-free glassware

Reagents All solutions are prepared in deionized, Milli-Q or similar 1: 1. Glycine buffer (0.2 M, pH 2.5) Dissolve 15.014 g of glycine (Merck 4 'in about 800 ml of water. Adjust pH to 1 M with hydrochloric acid. 80 ml) and dilute to 1000 ml with hydrogen 2. Substrate (30 mM)

Dissolve 1.114 g of p-nitrophenylphos (Boehringer, 738 352) glycine buffer in a volume of 100 ml buffer] f f · '· 25 A new buffer must be prepared daily with 3.15% (w / v) TCA solution • * * ·; Prepared from trichloroacetic acid (Me 807) J; * <4. 10% (w / v) Ascorbic acid solution 1 '* * t * r, 30 Prepared from ascorbic acid (Merck) Keep cool. The solution is stored in approximately 7,731 ml of water. Allow to cool and adjust the volume to ml with water.

7. Reagent C

Mix 3 parts of 1 M sulfuric acid and 2.5% ammonium molybdate, add 1% ascorbic acid and mix well with reagent C. Daily dilution of specimens Dilute the specimens in a sine buffer. Each dilution was spiked. Weigh accurately 250 mg of the enzyme powder into the sample, dissolve in buffer and; 15 in a 25 ml volumetric flask;

Dilution Table: 20 Estimated Ac- Recommended Dilution Dilution HFU / ml Factor 20000 1 + 19 20 V ·: 200000 1 + 199 200 O 25 400000 1 + 399 400 - \ t 1000000 1 + 999 1000: * ··; 5000000 1 + 4999 5000 ·· * t I-30 t Analyze The 38-second clock is started by hydrolysis by adding 0.1 ml of enzyme dilution into the test tube at appropriate intervals and mixing. After exactly 15 m, the reaction is stopped by adding 2.0 ml 5 to each test tube. Mix and let; to room temperature. Also add 0.1 ml of sample to test tubes (kept at room temperature and mixed. If precipitate forms, centrifuge at 2000 x g for 10 minutes.

10 Liberated orthophosphorus: Pipette 1 sample after hydrolysis into test tubes. Add ml of water to each test tube. Add 4.0 ml of C and mix. Incubate at 50 ° C; and cool to room temperature for 15 minutes against absorbance reagent blind assay above) at 820 nm.

Standard Prepare a 9.0 mM phosphate-toluene solution. Dissolve and dilute 612.4 mg (Merck 4873, dried in a desiccator, silica gel in a 20 ml graduated flask. Make the following Laimer in water from the stock solution and use as a stock.

: Dilution Phosphorus Concentrate- Happophosphate I. / thio Activity • ft nmol / ml HFU / ml * ft ft ^ ^ mb · __ ft! 25 1: 100 90 2400 • «1: 200 45 1200 1: 400 22.5 600 * · * ♦ Corresponding acid phosphatase activity (H obtained by inoculating phosphorus concentrate inmol / ιηΐΐ 39 test tubes. Also pipette 4.0 ml water into a single tube (reagent blank test) Add 4.0 ml of C and mix, Incubate at 50 ° C for 1 minute and cool to room temperature M 5 absorbances at 820 nm against the reagent Assay Plot the standard curve of absorbance vs. phosphatase activity (HFU / ml) and make a new standard for the Ji series. the absorbance of the assay was reduced from the absorbance of 10 samples (difference should be 0 to 1000) Phosphatase activity (HFU / ml) is plotted versus dilution factor To calculate the activity of 1 powder (HFU / g), the result (HFU / ml) must be multiplied by 25 (ml) and divide by <15 tea with exact weight (g).

Preparation of feed and other insoluble matter for phosphatase analysis Weigh ti

2.5 g of stock sample into two 50 ml beakers: Add 20.0 ml of glycine buffer. Stir] with 20 magnetic stirrers for 30 minutes at hi. Transfer about 10 ml into each of the t fugue tubes and centrifuge the solids for 10 minutes at 200 x g. Place 2.5 ml supernal PD-10 gel filtration columns (Sephadex G-25M, I

• *: .v: 25 cia 17-0851-01) equilibrated with 25 glycine buffers. Remove the eluate. This; 3.5 ml of glycine buffer is added to the column and the eluate is added to a graduated cylinder.

** *:. *. to 5.0 ml with glycine buffer (Law, ···] 30 factor 2) and assay for phytase activity. The HFU / g Al is obtained by multiplying the measured activity ptt. i τ a / 1 -,.: au .. i .: _ i. j i 40 EXAMPLE 3

Phytase and pH 2? 5 acid phosphatase pool

To indicate how one of ordinary skill in the art would purify f 5 and pH 2.5 acid phosphatase, the following is provided, 1. Phytase

Enzyme purification. Operations were performed at 10 ° C at 4-8 ° C unless otherwise noted. The cell-free Aspergillus niqer var. awamori ALK (Concentrated broth concentrate.

Ammonium sulfate precipitation. The culture medium concentrate (990 ml) was kept in an ice bath j. 15 g of ammonium sulphate per ml were added (70% saturated). After 30 minutes, the precipitate was separated from the centrifuge for 15 minutes at 10,000 x g and discarded

Hydrophobic interaction chromatography. The sieve was placed on an Octyl-Sepharose CL-4B (Pha: 20 column (5 cm x 17 cm) equilibrated with 0.436 g (NH4) 2 SO4 per ml in 20 L Tris / HCl, pH 6.2). The column was washed with 500 ml of stock solution and developed with 500 ml of a linear gradient of 70-0.0% ammonium sulfate in 20 of 25 Tris / HCl (pH 6.2).

10 ml fractions were collected and collected. Phytase and acid phosphatase activity was detected. Mouth: Phytase activity eluted with gradients <• Fractions eluted with the following procedure i * · * * 30 eluted fractions after the phytase activity fraction and containing most of the acid phosphate on 41 1 'filtration columns equilibrated with bis-Tris / HCl solution (pH 6.2). Sample 24.5 was applied to a DEAE-Sepharose (Pharmacia) column (7 cm) equilibrated with 50 mM bis-Tris / L 5 (pH 6.2). The column was washed with equilibration (100 ml) and developed with 200 ml line * gradient containing 0 * 0.5 M NaCl in equilibrium.

Gel filtration. The combined active fi 10 was concentrated using a Centricon -30 microcontroller to a total volume of 600 μΐ, 100 μ1: η ar run at about 23 ° C and 0.3 Superose 12 HR 10/30 HPLC column (Phai equilibrated with 50 mM bis-Tris / HCl-luc 15 (pH 6.2).

Cation Exchange The combined active fraction was retained in 50 mM sodium formate (pH 3.8) using a Centricon -30 microconcentrator. The sample was applied in two 2 ml portions of Mono S HR 5/5 FPLC CoJ (Pharmacia) equilibrated with 50 mM ns formate (pH 3.8) at about 23 ° C. The washes were washed with equilibration buffer (10 ml) and the bound protein was eluted at a flow rate of 60 ml / h for 20 linear gradients containing 0 * 430 mJ *. **: 25 in equilibration buffer.

»M ♦ ·» 9 2. Acid phosphatase * «« * M ·: Gel filtration. Hydrophobic Interaction »M * /:. Fractions from 30 tographs containing * · · most of the acid phosphatase activity were loaded with DEAE-Sepharose (Pharmacia) (5 ml) in Konse chin n 1 -h racim Hat ulcol la Irfirma (42 ml). cm x 7 cm) equilibrated in 50 mM hi / HCl (pH 6.2). The column was washed with 100 mL equilibrium discipline and developed with 200 mL linear gradient <5 containing 0 ^ 0.5 M NaCl in equilibration buffer

Anionivaihtokromatoqrafia. Combined a! The fractions were concentrated and transferred to 20 r Tris / HCl (pH 6.0) by ultrafiltration using an Am 10 membrane. The sample was run on four 3.5 mL 10 Mono Q HR 5/5 HPLO columns (Pharmacia) equilibrated with 20 mM bis-Tris / HCl (pH 6.0) at about 23 ° C and 60 mL / h washed with 10 mL equilibration buffer and the bound inin was eluted with 20 ml linear gradient containing 0 to 350 mM NaCl in equilibration buffer.

Gel filtration. The active fractions were pooled, concentrated and transferred to 20 mM bis-T] (pH 6.0) containing 150 mM NaCl, with a Cei -30 microconcentrator to a total volume of <20 100 μ 100, the portions were run at 23 ° C and v: 18 ml. / h Superose 12 HR 10/30 via HPLC (Pharmacia) equilibrated on a screen.

Anionivaihtokromatoqrafia. The combined α] 25 fractions were transferred to 20 mM histidine / HCl (pH 5 on a 10 gel gel column. Samples were run in 1 ml portions of Mono Q HR 5/5 HPLC column on Mac) equilibrated with sample buffer, noii: and at a flow rate of 60 ml / h. ϊ 30 was washed with 5 mL of sample buffer and bound protein with 20 mL of linear gradient containing

43 H

Table 1. Summary of purification of Aspergillus niger phytase

Step Size - Size - Specific - Available - Pure: Female Window Act (Ken Ti Protein (%)) Visibility Visibility __ (PU) (mg) (PU / mg) ___

Education- 44866S0 2119 2117 100 1 filtrate______

Ammonium 3771750 1263 2986 84.1 1.4 Sulphate Supernatant_________

Octyl Sep- 1765881 32.3 54671 39.4 26 harose___ _____ DEAE-Sepha- 1453470 8.4 173032 32.4 82 - rose___

Superose 12 1010888 5.7__177349 22.5 84

Mono S__827566 3.0__275885 18.4 130

Table 2. Summary of purification of Aspergillus niger acid phosphatase

Step Size- Koko- Species- Pure: Female Acne Prophylaxis (]: Sorbitin Active (¾) Oral (mg) Volume O __ (ΗΓ0) ___ (PU / mg) ___ • e

Education- 11652-2119 54990 100 1; filtrate 3000 ** * —— - ™

Ammonium sul- 88275000 1263 69893 75.8 1.3, *! *. fate su- • S f supernatant_____ 44 EXAMPLE 4

Characterization of Purified Phytase 1a pH 2.5 Acid Phase Peptide Digestion 5 Native Purified Phytase (70 pg)

In Tris-HCl, pH 7.9 was digested with 2% (w / w) cin (TPCK treated, Sigma) for 2 hours at 37 ° C, followed by a further 2% (w / w) trypsinization for 21 hours. One batch of native purified 10 volumes in 100 mM Tris-HCl pH 8.0 was treated with 2% (w / w) trypsin for 20 hours at a temperature followed by 2% (w / w) trypsin for 6 hours. The peptides have been purified as described above

Another batch of purified native phosphate was alkylated using 4-vinylpyridine as follows: To lyophilized phosphatase (75 μg) was added 0.5 M Tris-HCl pH 7.5 containing 6 M guanidine hydrochloride, 2 mM EDTA and 34 mM DTT. After addition of 1 µl of 4-pyridine (Sigma), the reaction mixture was detected at room temperature (22 ° C) for 1 hour. F

was stopped by the addition of 10 μΐ 1.4 M DTT. Al * phosphatase was purified by HPLC on a C-1 reverse column (TSK TMS 250; 0.46 x 4 cm) using a 70% ACN / 0.06% TFA gradient (80% to 30% 0.1% TF).

The fractions that absorbed the wave at 218 nm were pooled and evaporated in a Spe * ψ vacuum centrifuge. The dried sample was suspended in s * 'p 70 mM Tris-HCl pH 9.1 and digested with 2% lysyl endopeptidase C. (Wako Chemicals) for 2 hours at 37 [deg.] C. After another 2% (w / w) addition of lendopeptidase C, I incubated ISmnnfi broad T7 niHannaff in OC fnnf Tie in Tie 45

was performed on a C-18 reverse phase HPLC column (Vydac B5; 0.46 x 25 cm) for 90 minutes with a gradient C

ACN / 0.06% TFA (100-40% with 0.1% TFA). A wavelength of 218 nm was used for P 1 detection.

5 Amino-terminal sequencing of the purified peptides as well as the nai proteins was performed by cleavage of the ne gas-pulsed-liquid sequencer (Calcium J * mann, Journal of Protein Chemistry 7: 242-243 (: PTH amino acids released were analyzed online using a narrow channel reverse phase). -HPl tetta.

Sequencing of the carboxyl terminus of phytase

One batch of purified phytase (53 μg) was added with carboxypeptidase Y (Sigma, 0.6 U) in sodium acetate pH 5.6 containing 10% ur 0.05% SDS at room temperature (22 ° C). Samples of the seizure were taken at different times. Samples were plated in a Speed ^ Vac vacuum centrifuge and derivatized with a phenyl isothiocyanate (PITC) Pj (Waters association) amino acid analysis kit. The amino acid derivatives were analyzed by a reverse HPLC apparatus with a Pico-Tag C-18 well; sample volumes were similarly analyzed with some * 25 standard amino acids.

ti »•» »·» »·

Results and Their Review «« «i» • «» ««:, \ The sequences could be extracted from the peptides, 30 was the "double sequence" (for both phytase and phos t * », Tables 3 and 4) because they quantitatively p a 'f λϊ a ΐ cfaan -ia / or f λϊ nan ΒΔίττιαηββΐ 1 ifn 117 46

Amino-end sequences derived from phytase # 1081; [SEQ. ID NO.:50:]) were similar not identical to the A. ficuum phytase am sequences (LAVPASRNQSSGDT) [SEQ ID No.:17:], y 5 presented in Ullah, A.H.J. Prep. B-18: 459-471 (1988). The dit obtained by trypsin digestion is shown in Table 3. One peptide] phy) [SEQ ID NO: 23:] was identical to the peptides within the sequence ficuum phytase (M110 QEPLVRVLVNDR); Ullah, A.H.J. Prep. Biochem. 18: (1988). Sequencing of the carboxyl terminus of the phytase yielded the sequence XSA-OH.

No results were obtained from the sequencing of native and alkylated phosphatase am (Table 4) The peptide (7Lpho, # 817 [SEQ. ID NO.:53;]) was identical to the amino acid sequence of amino acid sequence of A. ficuum acid phosphatase (pH optic FSYGAAIPQSTQEKQFSQEFRDG). , AHJ and Cummings, Prep, Biochem. 17: 397-422 (1987) and another (1C

20 # 941 [SEQ. ID NO.:54:]) seems to be this

Peptide 3Tpho (peptide # 1106 in Table 4; SEQ I] 61 :) could also be a peptide ULpho extension (] # 943-2 in Table 4; SEQ ID No.:60 :) because of its four overlapping amino acids: FSE "25 The peptide ILpho (peptide # 816 in Table f ** ID No.: 57 :) contains phytases and phosphatases

'·· I set forth the RHGXRXP [SEQ

: 18:] # reported in Ullah, * ** · et al., Biochem. Biophys. Res. Comun. 178: 45-53 i ··· · j ·: ·. The peptide was very homologous but not idenl

One phytase peptide (# 675, [SEQ. ID NO.:37:], 47,117

The results show that A. niqer fyt is homologous to A. ficuum phytase but not. The same result is observed with acid phosphatase (pH optimum 2.5).

5

Table 4. Peptide sequences of pH 2.5 acid phosphatase obtained by digestion of either trypsin (T) or endoproteinase Ic (L) with the isolated enzyme. The corresponding nucleotide positions are also read in __tU .__

Peptide-Peptide Sequence Nucleotide Numeric_N-Terminal FSYGAAlPQSTQEK 193 ... 234 # 8I7; 7Lpho

fSEO ID No.:53:I

QFSQEFRDGY 235 ... 264 # 941; lOLpbo [SEQ ID No.: 54; | YGGNGPY 280 ... 300 # 938: 2Lpbo {SEQ ID No.: 55: 1 VSYGIA 310 ... 327 # 1111: 4Tpho {SEQ ID No.:56:l RHGERYPSPSAGK 376 ... 414 # 816: ILpho l ' \ l | SEQ ID No.:57:j DIEEALAK 415 ... 438 # 847: 5Lpbo%: {SEQ ID No.:58:1 '*: ARYGHLWNGET 595 ... 627 # 943-1: ilLpbo [SEQ ID No. 59: |

; VVPFFSSG 628 ... 65I

# 943-2: IlLpbo (SEQ ID No.:60 :) FSSGYGR 640 ... 660 11 '48

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Cloning and Sequencing of Aspergillus niger pH 2.5 Optimal Happc 5 Gene 1. Summary of pH 2.5 optimal acid phosphatase gene chlorine and sequenced in Aspergillus niger fungus. Translated nucleotide sequence c 10 amino acids of acid phosphatase. The gene for this protein was isolated by a set of oligonucleotide probes based on the peptide sequence of the isolated protein.

A. Design of oligonucleotide probes pH 2.5 coding for acid phosphatase (AF) was done by hybridizing the degenerate oligonucleotides obtained from the peptide sequence.

A. niger var. awamori strain ALKO 243 (ATCC 388 20 2.5 AP internal peptide fragments have been previously identified and sequenced as described previously in the patent.

17 mer degenerate oligonucleotide, F

* was made from acid phosphatase peptide # 816 (ILpho tau * * ·! .. * 25? 4 [SEQ. NO.:57;]). Containing neutral inosine gives one perfectly compatible $ 6 combination in the PHY-31. Figure 1 shows the nucleotide sequence 9 * of the oligonucleotide PHY-31; corresponding peptide sequence.

30 B. Hybridization of Oligonucleotide Probes! ·. specificity 117 55 was probed as whole genomic DNA was examined from ALKO 243. Genomic DNA was isolated by a neutral method. Finely powdered, freeze-dried was digested with 4% SDS-TE buffer. The residual cells were p 5 and the supernatant was removed and extracted with the same amount of Tris-saturated phenol: chloroform solution. The genomic DNA was precipitated with NH4OAC and EtOH. The pelleted DNA was purified with ultracentrifuge CsCl solution and treated as described in Maniatis et al. Molecular Cloning / A La] ry Manual, Cold Spring Harbor Laboratory, Cold Harbor, NY, USA (1982)). Hybridization with genomic-1 [γ32P] ATP-tagged degenerate oligons] Maniatis et al. Molecular Cloning, A Lab <15 Manual, Cold Spring Harbor Laboratory, Cold Harbor, NY, USA (1982) was performed at 42 ° C over filters on oligonucleotide hybridization (6X SSPE, 0.5% SDS, 3X Denhardts, 100 μq / ml the binders were washed twice: for 30 minutes twice at room temperature with 2XSSC, 0.1% SDS, and once for 5 minutes at fresh temperature overnight on Kodak X-Omat Ar film with a reinforcement shade.

25 A. niqer ALKO 243 genomic DNA was tested by probing pH 2.5 oligonucleotide 1 * * * * Hybridization was performed as genomic hybridization.

Of the AP oligonucleotides, only PHY-31 showed specificity for its hybridization to genomic DNA.

: T: 30 dissection into one prevailing and one smaller, but showed sufficient specificity for screening the strings.

56 23kb fragments. The fragment was ligated with BamHI-cleaved dephosphorylated Dash II vector (Stratagene). Attached DNA pa in vitro using Gigapack Gold kit 5 (Stratagene). The packaged phage was used to infect E. coli P2392. The lambda library screened] with the gonucleotide PHY-31 pH 2.5, found the AP gene under the conditions indicated in the genomic hybridization (B) section. Twelve hybridizing spots for additional characterization. Bacterial phage DNA isolated from each condition was digested with restri donuclease and assayed with either PHY-31 and PHY-31 and PHY-35 derived from the unrelated pH 2.5 AP peptide (Figure 1). One of the 15, AP99, contained a 2.1 kb Sphl fragment and a previously identified Southern analysis of the genome that strongly hybridized to both assays. Strong hybridization to the two oligonucleotides from different peptide sequences gives the assumption that 20 AP99 contains the pH 2.5 AP encoding sequence at 2.1 kb. therefore to M1 and M13mp19 for sequencing. Translation of this: nucleotide sequence revealed a peptide. sequences with the N-terminated peptide (Table] 25 Immediately upstream of the N-terminal peptide · '· «** typical fungal secretion signal sequence, river **: from the methionine start codon at position 136. All t ·· sequences were present in simple 01 except # 1108 (peptide) 9Tpho in Table 4 [SEQ. I] 30 63:]) starting at nucleotide position 1384. Lobmids were identified by all three readings 57 using pH 2.5 AP specific alli A. niqer var. Awamori ALKO 243 were grown on Rl medium containing liter 2.0% maize (Sigma), 1.0% protease peptone (Difco), 5 glucose, 5.0g NH4NO3, 0.5g MgSO4-7H2O, O.SgKCl, FeSO4 · 7H2O Total RNA was isolated by McAda and Doi by LiCl precipitation (McAda PC e Meth. Enzymol. 97: 337-344 (1983). Polyadenylod hetti RNA affinity purification was performed on a total of 10 using an oligonucleotide (dT) cellulose probe: (Pharmacia) as specified by the manufacturer for gonucleotide PCR primers. UPPHOS (5 'GAATTCCGAGTi TCATGGGCGCG-3') [SEQ ID No.:66:] and DOWNPHOS (5 'TCCCGGGACCTACCCCTCTGCAT-3') [SEQ ID No.:16:] were sequenced according to the genome sequences to which the EcoRI restriction site was located. UPPHOS and DOWNPHC are located in reverse and are separated by 97S in the genomic clone. First Strand Synthesis with 1 BMB cDNA Kit by Manufacturer's Recommendations for Mug 20 1.0 Mg mRNA and DOWNPHOS. PCR amplification of cDNA mRNA Komi with oligonucleotide primers UPI DOWNFOS gave a 850 bp specific product, PCR amplification of plasmid DNA with the same sequence). produced the expected 1006bp product. The gel purified 25 cDNA PCR product was digested with EcoRI and cloned; 9 * *** · '18 for double strand sequencing using * ·: States Biochemical Sequencase II kit. The primer * * * lifted the 850 bp fragment of cDNA and waited for the jj *: bp fragment from the cloned genomic DNA. Amp]: T: Sequencing of a 30 dun cDNA fragment showed the presence of three ♦ introns, each with a cons; * mushroom donor. lariate and accentor sequence. Wed 58 11 in the nucleotide sequence, was calculated for Mr! The 2.1 kb Sph1 fragment in pUC18 (pAP-1) contained a pH of 2.5 above the AP sequence.

EXAMPLE

Production of Aspergillus niqer phytase by Tricl reesei

Experimental Arrangements 10 1. Bacterial strains, phages and plasmids

The coli strains DH5α used in cloning and sequencing (Hanahan, D., "Techniques for 15 Formation of E. coli," in DNA Cloning, v. Glover, DM, ed., IRL Press, Oxford, 109-135 (Bethesda Research Laboratories, Gaithersburg) , ME and XL-1-Blue (Bullock, WO, et al., BioTecli 5: 376-378 (1987); Stratagene, La Jolla, CA.

20 E. coli Y1090 (r ~) (Huynh, DS, et al., "Constrain and screening cDNA libraries in gtl and gtll cloning, vol. 1, Glover, DM, ed., IRL Press, C 49-57 (1985). ); Promega Biotec Protoclone GT S

; Madison, WI, USA) as a host for the cultivation of the gtll phage Aspergillus niger var. awamori ALKO 243 38854) was used as a donor of the phytase gene. The strains ATCC56765 (RutC-30), ALKO 233 (VTT-E 56, Bailey and Nevalainen, Enzyme Microb. TeS: 3: 153-157 (1981)) and ALKO 2221 were used as the phytase (): 30 recipients. ALKO 2221 is a mutant with low aspartyl assay activity that is already 1 by UV mutagenesis from the strain ALKO 233.

11, 59, 1 USA (1984).

The vectors used for cloning were (Boehringer, Mannheim, FRG) and pALK307, pIBI76 (IBI, New Haven, Conn., USA). To form pALK307 5, about 940 bp of NaI-PvuI frac (941 bp actually) has been deleted from pBI76. T only change in pIB176. Plasmid pAMHHO (Neva! H., et al., "The Molecular Biology of Trichoder Application to the Expression of Both Homes and Heterologous Genes," in Molecular Indu Mycology, Leong and Berka, eds., Marcel Dekker New York, 129 -148 (1991)) contains the Trichoderma cbhl promoter and decision regions. The plasmid (Kelly and Hynes, EMBO J. 4: 475-479 (1985)) contains the acetamidase gene of Aspergillus nidulans. The p3SR2 raid was kindly provided by Dr. M. Hynes versity of Melbourne, Australia).

2. Culture medium and growth conditions 20 cultures of E. coli were performed at 37 ° C overnight and filamentous fungi were grown at 5 to 7 days for enzyme production, and for 2 days when mycelium was grown for 4 µl of DNA. .

E. coli was grown on L medium (Man

..! * T., et al., Molecular Cloning, A Laboratory M

Cold Spring Harbor Laboratory, Cold Spring Harbo *%:, :: USA (1982)) supplemented as needed with ampicillin! 30 (50 - 100 μς / τιιΐ). Inclined PD agar (Potato De Broth, Difco, Detroit, MI, USA) was used to grow Asperg in Trirhoderma strains. Asnar 11 '60 disclosed in Penttilä et al. (Pentti et al., Gene 61; 155-164 (1987)). Transformants were plated on a selective acetamide-CsCl medium, M., et al., Gene 61: 155-164 (1987)) on PD inclined surfaces.

For plate-screening high-phytase-producing products, cbh1 ptor / phytase transformed T. reesei} were grown for 3 to 5 days in 10 Trichoderma minin-10 non-glucose plates supplemented with 1% sodium phytate (Sigma, St. MO, USA), 1% Solka with Floc and 1% protease peptide (Difco). When the phytase promoter-containing correction was used for transformation, the producers of phytase rur 15 were screened on plates containing 1% sodium phytate and 1% proteose pef but no sodium phosphate.

For 5 days for growth of phytase by A. niqer ALKO 243 in soybean meal medium containing q 20 and mineral salts (all Merck * East, Darn FRG): pH was adjusted to 5.0. From the phytase expression cbh1 promoter, T. reesei transformants were plated for 7 days (250 rpm) on lactose-based # * f medium. With a phytase promoter containing * * · l, ' Twenty-five transformed T. reesees were grown on * 1 in 1 hoderma medium supplemented! ψ e ^ soybean meal, but no sodium *. ·. * phate was added.

»*! J: Soya flower medium (soya flower medium)» »:: 30 dye is as follows (per liter): 50g soybean glucose, 5.0g NH4NO3, 0.5g MgSO4 * 7H2O, 0.5 <: 0.183 σ FeSO * 7HO. dH 5.0. LactoseDohiininen 11 61

Plasmid DNA from E. coli (large scaled using Qiagen columns (Diagei Dusseldorf, FRG) according to the manufacturer's instructions, screened using Holmes and Quigley Men 5 (Anal. Biochem. 114: 193-197 (1981)). Chromosomal DNA from Asperqillus was isolated from a lyophilized list as described in Clements and] (Curr. Genet. 9: 293-298 (1986)) and Raeder and (Lett. Appl. Microbiol. 1: 17-20 (1985)).

4. Cloning Methods The standard DNAs described in Maniatis et al. 15 Lecular Cloning, A Laboratory Manual, Cold Spray Laboratory, Cold Spring Harbor, NY, USA (Restriction enzymes, T4 DNA) were mainly used. ligase, DNA polymerase Klenow fragment, T4 DNA polymerase, polynucleotide kinase, and EcoR1 methylase used in DNJ 20 rations were from Boehringer (Mannheim, New York Biolabs (Beverly, MA, USA). The other nuclease was from BRL (Gaithersburg, MD). , USA) and Pharmacia (Uppsala, Sweden), each enzyme was tested according to the manufacturer's instructions.

* · ♦ ***: To prepare the gene bank, chromosomal DNA was partially digested with Haell1. EcoRl met ^ • * ·; processing, size fractionation, and packing are carried out in accordance with Paloheimo et al. (Palc: M., et al., Appl. Microbiol. Biotechnol. 36 :: m · ——— - - - - 30 (1992)). 2-8 kb fragments were used in the construction of gene m.

• · Tf 1 o on ^ ami πρπ o Ί a rvti i H i volr f · πτί ί ti fohtin 62 n (FMC Bioproducts, Rockland, ME, USA) by the frozen phenol method (Benson, SA, Bio / Tec 2: 66-68 (1984)) or by using the GeneClean® tc maid ™ kit (BIO 101 Inc., La Jolla, CA, USA) according to the teacher's instructions.

Sequencing was performed directly by the plasmid Sanger method (Sanger, F., et al., Proc Acad. Sci. USA 74: 5463-5467 (1977)) using T7, pUC / M13 primers and amplification primers and] Sequenase sequencing kit (United States) Bioc] Corporation, Cleveland, OH, USA), cbhl promoted phytase gene fusions were sequenced on an automated sequencer (Applied Biosystems 373A, Foste: CA, USA) using a Taq DyeDeoxy ™ Terminator 15 Sequencing Kit (Applied Biosystems). The oligonucleotides used were synthesized on an Applied Biosyste Synthesizer except for the pUC aliquoted from the United States Biochemical Corporation.

The DNA probes were labeled using the Bc ger non-radioactive DIG DNA Labeling and Di on kit according to the manufacturer's instructions.

Hybridizations were performed at 68 ° C. ·,: Manufacturer's instructions (Boehringer). Spot Screening: * Amei Hybond N nylon filters were used for 25 Southern blot hybridizations.

After screening the spots with antiserum, phytase-specific polyclonal Antis * * KH1236 was prepared. KH1236 was made against purified and deglycc * * ·: 30 phytase preparation (M. Turunen, Alkc.

At the National Public Health Institute (Helsinki, Finland).

5. Transformations E. coli strains were transformed according to Hanahan's method ("Techniques for Transformation of 5 li /" in DNA Cloning, Vol. 1, Glover, DM IRL Press, Oxford, 109-135 (1985)), and T. reesei as in Penttila et al. (Gene 61: (1987)). When the ligated fragments were transformed by the transformant in Table 7), the ligation mixture was not transformed but used as such in the transfections. Prior to sporulation, potato dextrose agar (for PD surfaces, T. reesei transformants were transferred to a dense medium and purified for conidia. 15 6. Enzyme and protein measurements

For enzyme analysis, T. reesei; was separated from the culture medium by centrifugation] for 5,000 to 10,000 rpm (Sorvali 20 Dupont Company, Wilmington, Delaware, USA). A. The cultures were centrifuged for 40 minutes

10,000 rpm (Sorval SS-34). Phytase activity ti from culture supernatant in an amount of inorganic <, ·. ; phosphate released by sodium phytate subunits • ··

I., * 25 in enzymatic hydrolysis at 37 ° C

f · * · „previously described. One phytase normalized; i «*" 5 (PNU) is defined as the amount of phytase activity produced by the A. niqer strain ALKO 243 under the conditions of use.

30 Phytase production sodium phytate analyte;

the joila was visualized by pouring reagent C

64

Amyloglucosidase activity (AGU) mi using 1% Zulkowsky starch (Merck) as a tin and riveting glucose units released after boiling with DNA reagent (see

5) after 10 minutes at 60 ° C

at pH 4.8. Proteases (HUT) were measured 4.7 as in the Food Chemicals Codex (1981).

2% hemoglobin (Sigma) as substrate. E

canase (ECU) and cellobiohydrolase (FPU) Act 10 were measured as in IUPAC * in Measure

Cellulase Activities (IUPAC Commission on Biote gy, Measurement of Cellulase Activities, Bioc

Engineering Research Center, Indian Institute ofology, Delhi, India, pages 5-7 and 10-11 (1981; 15 hydroxyethylcellulose (Fluka AG) in 50 mM sodium buffer, pH4.8) and Whatman No. 1 was printed as substrates. differed from that described in the method and a solution of 50.0 g of 2-hydroxy-3,5-dinitrobenzoic acid (Mei in 20 L of deionized water) was prepared. Then 80.0 was added slowly using a magnetic stirrer and sodium potassium tartrate (Merck) was added and the solution was heated to C): The female volume was adjusted to 5 Isa, the solution was filtered through 1 What was No. 1 and protected from light.

CBHI protein was determined from culture supernatants by running on SDS-polyacrylamide gel and 1 [···; * CBHI protein band (T. reesei ALKO 233: 2221 strains) or by dot blotting with V Σ 30 Schleicher & Schuell's (Dassel, FRG) Minifold. ™ Sample Filtration Manifold (T. reesei ATCC56765) 65 7. SDS-PAGE and Western Blot Analysis

Sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) and Western blot were performed as described by Laenunli (Laemmli, U.K., Nature 2885 (1970)) and as described by Towbin et al. (Towbin, H., Proc. Natl. Acad. Sci. USA 76: 4350-4354 (1979). Western blot visualization of the re-protein was done using polyclonal rabbit Antis <10 KH1236. Immunocomplexes were visualized by spot screening.

8. Polymerase Chain Reaction (PCR) 15 PCR reactions were performed on a Techne Thermal PHC-2 (Techne Ltd., Cambridge, UK) in a 1 'volume. The reaction mixtures contained 0.2 mM I dNTP (3'-deoxynucloside-5'-triphosphate, Pha] in 10 mM Tris buffer (pH 8.3), 50 mM KCl, 1.5-20 MgCl 2, and 0.01% (w / v) gelatin. as follows: 95 ° C (plasmid) or 100 ° C mosomal DNA) / 5 minutes before addition of Tag DNA pole (1-2 units, Cetus Corp., Emeri, CA, USA) and 100 μ1: η paraffin oil coating, c 25 cycles of 95 ° C / 1 min, heat treatment of 60 ° C / 1 min • ft #! ...: 52 ° C on a self-PCR, amplification 72 ° C / 2 min (reverse * '·· min) for 30 cycles. The final amplification time was 9 mil to ensure complete strand synthesis: the plasmid or DNA fragment used is temple * * · 30 the amount of template used is 5 to 10 ng and 20 to 50 pmol of primer is added. When chromosomally hfl to homnl λλΗ · i n λ trj »ef aav · 66 tions, Innis, M.A., et al., Eds., Academic Pre; Diego, pp. 219-227 (1990)). The PCR fragments were purified with the GeneClean® or Mermaid® kit (agarose as needed) or with Qiagen tips. Fragment! 5 was filled using DNA polymerase I Clenolent.

SCORE

10 A. Molecular Cloning of Aspergillus niger Phytase 1. Production of Phasease Probe by Sequential Amplification 15 oligonucleotides used in PCR reactions and corresponding ALKO 243 phytase pe] # 792 (15 phy) and # 420 (10 phy) amino acid sequences (Table 3) previously) is not 20 in Figure 3 (nucleotides 1409-1480 and 1727-171 in the restart sequence, see Figure 5). Two PCR reactions were performed. In reaction A, sens gonucleotide 1 (# 792) was used and antisense oligonucleotide (# 420) was used; reaction B used sense oligonucleotide 25 3 (# 420) and antisense oligonucleotide 2 (# 792) • · * ··· [PCR reaction A generated a single band of ♦ * bp, whereas the product formed in the PCR amplification reaction was concluded. that region <j encodes peptide # 792 was located in the phytase sequence: T: 30 moiety compared to the region encoding peptide ·

The primary PCR fragment (A) was used as a template.

_ * 4- A «M A AA n ^ O« M A A Ta 4. i A A M AA A n -M. The DNA sequence derived from the DNA sequence also contained the known acid acids from peptide # 792 and # 420 which were not present in the primer sequences. The 350 bp PCR fragment serves as a probe for DNAbank.

5 2 · Screening of DNA Bank

The gene bank contained about 1.9 x 10 6 pfu forming units) / ml, with about 99.5% being an insert. From 80,000 spots, screened, two positive clones 6 and Hael-5 were found. Clones were isolated, purified inserts (5.6 and 5.2 kb) were cloned into EcoRI-introduced pALK307. The clones also reacted with the phytase 15 serum KH1236. Inserts were restricted and a BamHI-Sphl restriction fragment of about 1 kb of the hybrid clones was detected (see Figure 4 Hybridization of the Phytase Sequence Clone Hae2-6, which contained a large portion of the 20 sequences encoding 15 internal tryptic peptides found at Napp terminus. The N-terminal and promoter region containing giclones were screened from a gene bank using Ink reverse PCR (Ochma), "Amplification of flanking sequences by PCR," in PCR Protocols, A Guide to Meth <♦ <RTI ID = 0.0> * * </RTI> Applications, Innis, MA, et al., Eds., Academic ss, San Diego, pp. 219-227 (1990).

• «* • i ·» o * 30 3.5 'end amplification and phytase gel promoter sequence reverse PCR: 68 r

the primers used were bases from the phytase regions 1243-1257 (antisense primer) and 1304-132 that primer) (see Figure 5). In reverse PCR, S

the digested ALKO 243 DNA was a 1.2 kb PCR 5,350 bp PCR fragment hybridized to the reverse fragment and the cloned sequence was found to contain the upper parts of the phytase gene as well as the N-terminal peptide sequence.

4. Isolator of the whole phytase gene 1.2 kb PCR fr obtained by reverse PCR. thi was used as a probe for screening 80,000 l of which seven positives were identified. The complete phytase gene was isolated with a 6 kb phage] insert.

The 2.4 kb Sph1 fragment, which: the phytase gene and promoter region, was cloned into 307 (digested with Sph1), giving mu <20 pALK169 (Figure 4). The restriction map of f] containing the Sph1 fragment and the position of the phytase gene in the fragment are shown in the plasmid map.

The phytase sequence is shown in Figure 5. the gene encoding the phytase protein corresponds to «« ·

E 25 gillus ficuum phytase sequence, which is juJ

• *

Gist brocades (Delft, The Netherlands) in PCT Patent Publication No. EP420,358 or WO91 / 05053 which has 1 · · · derived amino acids. Each difference in amino acids results from a nucleotide change and s: T: 30 results from a difference in strains. Also, 33 nucleotide differences (69 TAG) between the two sequences in the structural gene 1 sequence were found to be 96.3% different between the two phytase sequences; Gist-brocad Alkon, is shown in Table 5.

• * «4« • ft · • ft ft ·· • ft • »• ft * • ft ft ft« »ftftft« «ftftft ft ft ft ftftft ft ftft» tft * ft ft ft. 70

Table 5. Sequence and amino acid differences between Gist and Alko phytase sequences »: regionΛ;:. nt; no ah no: i Giist'n nt \ Alkon = nt i. Gistrn: -: --— =. — aa--, aa-a ^ = ^^ = SSSS3 = S ^^ = 5 signa- 39 ( -7) CT G CT A Leu lis- 40 (-6) T CT GCT Ser vein _________ presumed 59 (-) AG (-) intron 61 (-) AT (-) 65 (-) AT (-) 72 (-) AG (-) 85 (-) CT (-) 86 (-) CT (-) 88 <-) TG (-) __136 (-) T__A __ (") 10 construct- 191 11 AGT ACT Ser gene 210 17 CAG CAA Gin 258 33 GCA GCG Ala 287 43 GTC GCC Vai 300 47 GAG GAT Glu 312 51 GGA GGT Gly 369 70 GAC GAG Asp 419 87 GCG GTG Ala 369 91 GAC GAT Asp 501 114 G AA GAG Glu 549 127 CGG CGA Arg 565 136 GTT ATT Vai 570 137 CCA CCG Pro 613 152 AAG GAG Lys 624 155 ATC ATT Ile 669 170 CCC CCG Pro f \: 756 199 TTC TTT Phe 809 217 GTC GCC Vai:: 846 229 TCC TCT Ser 849 230 GGT GGC Gly 976 273 AAC CAC Asn. * ·% 997 280 TTG CTG Leu • · «« 1! I Λ i II - 1 * «* • t IM * * M ·.

And a 71 a construct- 1005 282 AAG AAA Lys gene 1008 283 TAT TAC Tyr (continued) 1020 287 GGT GGC Gly 1083 308 CTG CTC Leu 1113 318 AGT AGC Ser 1125 322 ACT ACC Thr 5 1136 326 AGC AAC Ser 1140 327 CCG CCA Pro 1149 330 TTT TTC Phe 1182 341 TCG TCC Ser 1185 342 CAT CAC His 1188 343 GAC GAT Asp 1203 348 TCC TCT Ser 1206 349 ATT ATC Ile 10 1218 353 TTA TTG Leu 1245 362 CTA CTG Leu 1284 375 GGA GGG Gly 1321 388 TTG CTG Leu 1344 395 TGT TGC Cys 1350 397 GCG GCC Ala 1413 418 CCG CCA Pro, _ 1414 419 GTT ATT Vai 15 | _ 1499 I 447 TTT | TCT I Phe

Table 5. Nucleotide and amino acid differences between Gist and Alko phytases. Nucleotide numbers are calculated from the first methionine Met (AUG) signal sequence: amino acid numbers from the N-terminal leucine Leu (see Different amino acids in sequences written in dark 20 text).

♦ «V · 9 • ♦ * • * 25:» ·· * B. Construction of plasmids by phytase yl • · **. to fulfill the Trichoderma Theses ♦ · »'• t •» «·« j ϊ *; 1. PCR fragments it for exact cbhl-fyte t *! *! 30 fusions * 4 72 et al., Bio / Technology 1: 691-696 (1983)). Figure shows primers for construction of PCR fragments 171 (phytase signal sequence) was constructed at SacII site in the cbh1 promoter region in the 5 'PC 5 lane. The XhoI site (14 nucleotides from the phytase gene) was used in the 3 'primer. The 5 'primer <mer containing 19 nucleotides of the "tail" cb motor sequence (preceded by a signal sequence from the phytase signal sequence. 3'-sub102-mer. PALK172 fcbhl signal sequence) was constructed by using the Sfil-5b cis sh1 cis in the primer and the Sali site of the phytase (7 leukides at the N-terminus) in the 3 'primer. In this case, the primer was 46-mer, which contained 28 nucleotides 15 and 18 nucleotides of the phytase N-terminal sequence, the primer was 24-mer. For all primers, three to five additional nucleotides at the ends of the PCR fragments after restriction site sequences to provide proper cleavage. pALK169 was used as temp 20 na in PCR reactions.

The PCR reactions yielded the expected β fragments: the fragment containing the desired fusion had 71 bp and 800 bp for pALK172.

*. **: 25 2. Feeding of the plasmid with the cbhl promoter ···: pALK171 (phytase signal sequence and pALK172 (cbhl signal sequence)) * * * * · Plasmid pALKl70L was made by cleavage of 30 recombinant pALK169 genes in Sph. As an XhoI fragment by working with SphI-XhoI-cleaved pALK307 73, no phytase fragment containing the fusion Eris SphI (treated with T4 DNA polymerase) was present in the PCR amplification - Sac11 and the cbhl promoter and Plasmid were added: 5,101, previously cleaved between NdeI (the Klenow fragment of DNA polymerase I) and the Sac terminus, The resulting plasmid (pALK177 7) contains a phytase gene fused to the promoter, to construct pALK171, an selectable label was isolated. p3;

As a SphI-Kpn1 fragment, the ends were treated with polymerase, followed by amdS ligation with a SphI k <17 7.5 (treated with T4 DNA polymerase) of a linear fragment containing no bacterial sequences, isolated with pALK.

cleavage with Xbal and it was used for transitions.

pALK172 was constructed essentially! like pALK171 (see Figure 8). The 800 bp PCI 20 ment was cleaved with SalI and ligated with X1 (supplemented with DNA polymerase I Klenow fragmentmenl SalI cleaved pALK170L. Also, fusions and PCR fragment sequence were dipped by sequencing. Phytase PCR fragment 25 was isolated from pALK170. Sph1 (treated with T4 DNA j: * '* Rase) -SfiI fragment and ligated with pAMH; * ·; which had been cleaved with NdeI (supplemented with DNA dust / Klenow fragment) -SfiI. ] sex fragment containing the amdS gene, liqat f # * * r | / 30 to pALK172S in the same way as constructing '171. In this case too, Xbal's L · a ί eama an τγλ1γ + · λτ ϊ e + af t'anp f Anna af ί λι osa lra.mf f> 74

3. Construction of plaques containing the phytase promoter: pALK173A and pALK173B

The phytase gene with its own promoter was inserted as a Sph1 fragment from pALK169 and the Kpn1 site of Liga p3SR2 (in both cases, the fti ends were complemented with T4 polymerase), the approximately 11.2 kb plasmids pALK173A and pALK173B16 in Figure 4. and Figures 7 and 8 in pALS172A of p3SR2, the two genes, phytase and amdS, are in the upstream position, in pALK173B they are in the opposite position (Figure 9). EcoRI was used to unpolarize pALK173A and pALK173B when using unpolarized plasmids in transformations.

15 C. Transformation of Trichoderma reesei Screening for Transformants T. reesei ATCC56765-? The circular plasmic Xba I fragments from pALK171 and pALK172 promoter were transformed into strains ALKO 233 and ALK 20. The linearized pALK171 and j plasmids as well as the circular and linear% * 12573A and pALK173B (phytase promoter) plasmids were also transformed with T. reesei ALKO 233 and 2221.

«« I

·, “· Format frequency (transformantity ^ g DNA) v <

* '· 3 to 60 when using pALK171 or PALI

isolated transformants. When transformants were used, the frequencies of pALK171 or pALK172 annular plasmas were about 50 / μg T. reesei ALKO 233 for ALKO 2221 and about 100 / μq T. reesei ATCC567 * 75 from 4.5% to 13.2%. T. reesei for ALKO 233 and AL strains and 1-2% of T. reesei ATCC56765 kan: Table 6 shows the number of transfections screened for the transformants from the phytase plate. The clones that surrounded the blue halo ring colony defined as positive clones.

• »• m · • · 1 ♦ · • 1 0 · · ♦ · m ·« 0 · • ··· • 1 • 1 ·· # • m • 1 0 «·» «• 1« • 1 · • 00 0 76

Table 6. Positive transformants and total number of k-ions tested in plate analysis

SBSBBSBSSaSBSSSBSBBqsaSSSS ^ ^ BSSBBaBBySBBaB BBSSIBSBdSBBBaeBBaBB

PALK171 fragment 49% (47/96) 46% (33/71) 23% (15/6 annular plasmid 35% (6/17) 23% (5/22) 32% (22/6 linear)

plasmid 41% (29/71) 49% (27/55) __ ND

PALK172 fragment 47% (48/103) 30% (34/113) 11% (8/7 annular plasma

di 17% (4/23) 13% (2/15) 12% (12 / 1C

linear

plasmid 37% (22/59) 24% (11/45) __ ND

PALK173A

annular plasmid 75% (9/12) 70% (14/20) ND

linear

plasmid 67% (10/15) 64% (14/22) __ ND

pALK173B annular plasmid 40% (4/10) 63% (15/24) ND

,. linear

**. * ·: Plasmid 63% (10/16) 67% (8/12) ND

Table 6. Positive, \ s transformants and total number of clones tested in plate analysis

The table shows the number of positive transformants in the plate assay and the total number of transformants tested for f reactivation: · β in the plate assay. Only those transformants: T: shown, which grew well in choice studies and plate analysis. Positive phytase producers were considered to be strains that showed clear phytase activity in Malia analogues.

77

The transformants that looked the best on the producer dish were grown in shakes. Inoculations were taken either directly from the inclined surfaces or from the inclined PD surfaces after conidia. Strains of T. reesei ATCC5676! 233 and ALKO 2221 transformed with pALK and fragments derived from pALK172, 7 clones from each transformation set were tested for purity and phytase production using a shake culture 10 When transformed using the circular dots pALK171 or pALK172, 13 and 12 purified esses ATCC56765 and Rees <233 and ALKO 2221 transformant strains were grown using linearized pALK171 or pALK172 p15 and about 20 transformant strains each in growth shake dishes. T. reesei ALKO 233 strain; transformed with linear pALK? 73A /? mids, seven of the pALK173A and two pALK173A circular plasmids, four pALK173A and 20 pALK173B) transformants, which showed phytin activity on the plates, were purified and tes1 shake cultures. T. reesei Tested in ALKO 2221-tr piston shaker cultures; the quantities were as follows:

4M

! .. * 25 three pALK173A and five pALK173B and ring chip *; · ** plasmids, six pALK173A and six pALK173B tr <pistons.

«· ♦ · ··· • ·; D. Trichoderma Transformant Phytase t * Yl 30 Yield * * m 4 aC ^^ rc at νΛ «* at Tr, Λ 78 S {sD-Qo-QQQQQ oo ° Q Ξ SD o ς ^ <η2Γ · ΐ2 <· η - 22222 ntNM-ZdZlZ ^ ZfN ö V v 2 2 VV 2 ö

iS

B - f I § finOQ ^ Q ^^ Q ^ QQQ OOOOQOQQOQO o «nmo OOO ^ [ϋ'ώ ^ ηΖίΛα ^ ν 222 r-rfTtm2rf22 <^) 2 ^ <n - - - Ό - - - <-λ § U0 .s 3 ° Q® 9SI2ÖQQQQ ^ <n ^^ 9 <n95ovQ - μγοο ^ νμ9- 31 - 2 - 2 - (N22222 V 1t v V2niZZN2m ^ 10 ^ 22 ^^ 2 ^ ™ _ '5Γ "c.

en G

ga ovQ ^ floo ^ PS000 ScsSSDSQQt.QS - SSSSSSS ^ ^ <^ 2os2oooo2-222 VS v v2vZZnZv -vvvvvvv ^ o> {Λ <jj g 5 JS £ 3 ££££££££ 3 ££££ 3 £ £££££££££££££! S jd jd .2 i

«I: OOOOOOOOOO OOOOOOOOOO OOOOOOO

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• · · w ··· s «f 4 Ϊ-Η · · ΓΠ Λ Λ _.

79

The best transformant 11a gave no: PNU / ml. Using T. reesei strains ALKO 233 and ALI yielded approximately the same degree of production, the phytase-producing T. reesei ATCC56765 transfo 5 produced approximately 1,800 PNU / ml. Both the phytase and the cb female sequence appeared to work equally well; phytase yield was obtained using T. Rees 2221 or ATCC56765 as host. The activity of the phytase obtained with T. reesei AL1 was higher at 10 in the phytase signal sequence.

Some of the transformants do not produce any detectable CBHI protein, which is most likely to indicate the transformation of the DNA to be transformed into the cbh1 locus. CBHI deficiency: The influenced screened transformants produced, for example, and among the normally CBHI producing non-CBHI negative transformants, there are good producers.

The highest levels of phytase production using the circular and line plasmids of pALK171 and pALK172 are shown in Table 8. The best yields were obtained with T. reesei ALKO 2221, which has been formed with the linear plasmid pALK171 (phytase: additional sequence).

• ·· 25:: ··· • · · ·

M

• · · »· • · ···« · ♦ · · • ♦ ♦ II · ·

Ml I · · • I I • 80

Table 8. Phytase production of T. reesej strains transformed with TtengaRmaigos or with the linear plasmid pALK171 or pALK172______ f PlaBatid Transform • '·: \ flf ·': W8m -: Bittcytoi ·. pine. '.' i f · '·' · ^ 116 ALCO 233 pALK171 annular maize- C13 650 nen C23 530 C4 240 linear A22 1,610 A21 1,270 AI 7 1,180 pALK172 annular maize- C13 1,360 nen C21 540

Cl 500 linear A20 1,420 A27 1,330 ____A32__980 ALKO 2221 pALK171 annular maize- C2 1,630 nen C8 1,290 C32 810 linear A14 3,800 A8 3,660 B14 3,610 pALK172 annular maize- C3,480 nen C4 190 C6 170 * · ♦ · · \ *60 linear · 1,790 B9 1,390 1 TU '* ··] ATCC5- pALK171 ringasmai- A74 2,030 6765 nen A75 1,980: Bll 1,870 t * «* it! pALK172 ringasmai- B3 2,250 nen B23 1,970 B1 1,030 81

The A. niqer phytase promoter can also express the gene in Trlchoderma. However, the enzyme yields are much lower than those of the cb engine homologous to T. reesei: Transformants from the culture supernatants having the promoter having their own promoter had from about 1 to about 14 PNU / ml with T. Rees 233 transformants and ca. From 6 to about 120 T. reesei with ALKO 2221 transformants (Table 10a · a 1 aa 1a • a:: • a »f · aa • aa •: aa • 1 # aaaaa a1 a · a · · A · aaaaaa 82

Table 9. Re-production of the phy -__ of T. reesei strains transformed with the circular or linear plasmid pALK173A or pALK173B

Kanta - ^ lasmidi Plasmidi- Twuasfox ^ II; ί :: · v · Ϊtilli f littp m • '•' ^^ iiiiuoto y ^^ ll 1P; pine; = "· | i ί jllljl ALKO 233 pALK- annular bag- C25 14.2 173A nen C29 6.6 C23 1.2 linear D4 8.8 D18 5.8 D6 3.9 pALK- annular bag- C8 3.5 173B nen C6 <E10 <linear D13 5.5 ____ D31 __ <ALKO 2221 - annular- E3 32.5 173A nen E5 31.7 C22 25.0 linear D24 37.5 D36 8.3 D17 5.8. ·.: pALK- annular- Cl3 115.8 "· 173B nen C28 65.0 · / **: C27 50.8 *« 4 # e linear D9 36.7 D26 22.5 * ··· * D21 18.3. f% ----- I »*« «· · '··. Table 9. Phasease production of reesei transformants transformed with annular or linear plasmid * '* pALK173A or pALK1738 (phytase promoter). The table contains a brochure. . phytase activity of transformant broth supernatants 83 enzyme composition of phytase preparations produced by E. T. reesei

Phytase is expressed in 5 volumes in T. reesei strains and the composition of enzyme activities in the supernatants of T. transformants is different in Aspergillus supernatants (Table 7). Both glucanase and cellobiohydrolase activity were substantially higher when used as T. reesei 10 as a production host compared to the A? Niqer strain. Useful reesei strains also produced relatively less amylase activity than A. phytase protein produced by A. niqer ALKO 243-cat F. Trichoderma trans formants.

Media samples of transformed T. reesei strains ATCC56765, A and ALKO 2221 of transformants (ALK171 fragment) were analyzed by i blot analysis (Figure 10). The following sample was lysed: Zone 1: 50 ng purified Aspe: ALKO 243 phytase; Zone 2: 15 ng endoF hand

Aspergillus ALKO 243 phytase; Zones 3 and; reesei ALKO 233; Lanes 4-5 and 11-12: T: 25 ALKO 233 transformant 171FR / A4 and A13, cont.

Zones 6 and 13: T. reesei ALKO 2221; Lane 8 and 14-15: T. reesei ALKO 2221 transformant 1.5 'and A9, respectively; Zone 9: T. reesei ALCO 2; ! #! · Ansformant D2; Zone 16: T. reesei ATCC5676! * T: 30 lanes 17, 18, 19: T. reesei ATCC56765 transfected 171FR / A21, Ali, and A23, respectively. Each 1 ft M A Mmm Λ. . 1 1. A M · _ _ i- * * M 1 Jaaaa-MMaa m. M ^. —A X. «3 _ 1. 1 Λ 1 ^ 1 m. M a 84 at cosylation levels. The t-phytase of T. reesei ALKO 233 strain was visible by Western blot and the 6 to 9 end lines secreted by T. reesei ALKO 2221 strain of about 45-65 kDal, the lowest being the deglycosylated Aspe phytase (45-48 kDal SDS). -PAGE C.). The phytase secreted by the T. reesei ATi strain was 65-80k composed of three to five protein bands. N? Aspergillus Phytase Molecular Weight SDS-PAGE: 10 about 80-85 kDal. The protein produced by the transformant transfected with the PALK172 fragment or pALK173A / B had similar banding.

Conclusions

The level of production of Aspergillus phytase utilized T. reesei as the host for the production was astonished high. Using T. reesees, the phytase is produced in a novel composition different from that produced by Aspergi ^ 20 and contains enzymes found in feed applications. Aspergillus phytase prot produced in Trichoderma, a molecule different from that produced by Aspergillus »This] appeared to be due to a different glycosylation, mi. * ·«. 25 enzyme activity.

* «• · * • · • 9 EXAMPLE 7.

• 9

Aspergillus niger pH 2.5 acid phosphatase 9 9 9 ··· · production in Trichoderma reesei · «* -: 30

Experimental Arrangements ♦ 85 (1990); Stratagene, La Jolla, CA, USA) hosts the construction of the constructs pALK601 (Figures 12 and 13) and pAP-1 (Figures 12 and 5) The plasmid pALK610 contains the T. reesei cbhl-promoto 5 and the decision sequences and the Aspergillus nldulans -a plasmid. pAP-1 contains the Asperqillu; var. awamori ALKO 243 (ATCC 38854) pH 2.5 happ again gene.

Trichoderma reesei strain ALKO 2221,;

10 low aspartyl protease mutant from T. reesei strain ALKO 233 (VTT-D-79125) U

genesis, pH 2.5 was used as the receptor for the acid phosphatase.

2. Culture medium and culture conditions E. coli strains were grown on L-medium Niatis, T., et al., Molecular Cloning, A Lab <Manual, Cold Spring Harbor Laboratory, Cold 20 Harbor, NY, USA (1982)). if necessary, had been treated with ampicillin (50 μ9 / αι1). The E. coli plant was made at 37 ° C overnight.

PD Agarino Studies (Potato Dextrose- »

Difco, Detroit, Michigan, USA) was used Trich <. ***; 25 stocking. Bowls and trays, joi1 ** * *. were transformed by T. reesei, were reported in Penttilä et al. (Penttil et al., Gene 61: 155-164 (1987)). Transformants * were transfected with selective acetamide-CsCl ali: 30 (Penttilä, M. et al., Gene 61: 155-164 (1987)) onto sloping PD surfaces. T. reesei transf * * 86 3. DNA treatment DNA treatment was performed as above for phytase, mainly standard 5 (Maniatis, T., et al., Molecular Cloning, A Lab Manual, Cold Spring Harbor Laboratory, Cold

Harbor, NY, USA (1982)). Plasmid DNA E was isolated using Qiagen columns (Diagei Dusseldorf, FRG) according to the manufacturer's instructions. H and Quigley's method was used for rapid screening of E110 plasmid DNA (Holmes and Quigley, Biochem. 114: 193-197 (1981)).

DNA ligase, a Klenow fragment of DNA polymerase I, used for DNA purification from Boehringer (Mannheim, FRG) and New Biolabs (Beverly, MA, USA). Each one was not used according to the manufacturer's recommendations. DNA flasks used for digestion or transformation were isolated from low melting point rose gels (FMC Bioproducts, Rockland, ME by freeze-thaw phenol method (Benson, S.A. Techniques 2: 66-68 (1984)) or

Kit (BIO 101 Inc., La Jolla, CA, USA) as directed by the person skilled in the art.

, ···. Sequencing of the fusion of the 25 cbhl promoter and the pH 2.5 hap again gene was performed using pUC / Ml «·· and amplification primers using / ·· * DyeDeoxy ™ Terminator Cycle Sequencing Kit: • ♦ 9

Lt: (Applied Biosystems) and Automatic Sequence <* ««: 30 (Applied Biosystems 373A, Foster City, CA, USA) The oligonucleotides used were synthesized; • «. . __ _ _. ___ 87 4. Transformations

Transformations were performed as shown on the phytase. Transformation of E. coli strains XL1-Blue t5 was performed according to the manufacturer's (Stratagene La Jolla, CA, USA), T. strain transformation was performed essentially according to P et al. * (Penttilä, M-, et al., 61: 155-164 (1987)). Novozym 234 used in 10 niprotoplastics was Novo Industri AS penhagen, Denmark). Prior to sporulation, transformants of PD-vinopi T. reesei were purified on cone selective acetamide medium.

15 5. Enzyme activity assay

For enzyme analysis, mycelia differed from the culture medium by centrifugation for 15 minutes at 3,000 rpm (Sorvali SS-34, Dupont C 20 Wilmington, Delaware, USA). The pH 2.5 acid phospho enzyme activity was measured from a culture supernatant using paranitrophenyl phosphate (St. Louis, USA) as substrate as before: One pH 2.5 acid phosphatase activity unit '♦ «* · ·] 25 µl 1 nmol inorganic phosphate per minute Γ *. of rophenyl phosphate substrate pH 2.5 warms • # 37 ° C. One unit normalized to acid phosphatase • * * ·· * is defined as the amount of acid phosphatase active: «9!:: Produced by the A. niger strain ALKO 243 to be used» «« V: 30 under nutrition conditions (see Example 6, Section 2)

Amyloglucosidase activity (AGU) decay • · j - j - i_ ** -λ. j.ams: i 1 u ία, i_ __ i. m - - Ί. · * .... |.

88

Chemicals Codex, National Academy Press, Wash DC, USA, p. 496-497 (1981)) using 2% heme (Sigma) as substrate. Endoglucanase (I cellobiohydrolase (FPU) activities were measured by 5 IUPAC guideline Measurement of cellulase act, IUPAC Commission on Biotechnology, Measurem Cellulase Activities, Indian Institute of Technology, Delhi, pp. 5-7 and 10- 11 (1981)). 1% hydroxyethyl 10 lose (Fluka AG) in 50 mM Na citrate buffer (and Whatman no. 1 paper was used as substrate. The DNA used differed from the IUPAC Commission on Biotechnology, Ment. Of Cellulase Activities, Biochemical Engi 15 Research Center , Indian Institute of Technology, India, pp. 5-7 and 10-11 (1984)) and was made by first dissolving 50 g of 2-hy 3,5-dinitrobenzoic acid (Merck) in 4 liters of Deion water followed by 80 g of Ogon. NaOH was added over h 2 using a magnetic stirrer and 1.500 g of K-Na (Merck) was added and dissolved in a hot solution (maximum temperature 45 ° C). The entire volume was made up to 5 liters, filtered through Whatman, protected from light.

6. SDS-PAGE and Western Blot Analysis * Sodium dodecylsulfate-polyacryl · · · · · · gel electrophoresis (SDS-PAGE) and Western blot : 30 were performed according to Laemmli methods (Li UK, Nature 227: 680-685 (1970)) and Towbin et al., 89 provided by Public Health Laboratory (Helsinki, CBHI visualization of pH 2.5 acid phosphate transformants by Western blot analysis of mouse monoclonal antibody). an anti-rabbit IgG and an anti-mouse IgG alkaline phono-conjugate using agent CI-261 (Ah <5 al., Eur, J. Biochem 200: 643-649 (1991)) and the toBlot ™ Immune Transfer System (Promega, M USA); color-forming substrates were provided for the detection of immune complexes.

10

7. PCR

PCR reactions were performed on a Techne thermal PHC-2 (Techne Ltd., Cambridge, UK) in a volume of 15 volumes. Reaction mixtures contained 0.2 mM i dNTP (Pharmacia pH 8.3), 20-50 pmol each of a, and 10 ng of plasmid template in 10 mM Tris buffer:

8.3), 50 mM KCl, 1.5 mM MgCl 2 and 100 µg / ml Gelai The following arrangement was used: 96 ° C / 10 mir 20 Taq DNA polymerase addition (2 units, Boe] Mannheim, FRG) and 100 μΐ paraffin oil, denat 95 ° C / 1 min, heating at 60 ° C / 1 min, amplification 'min for 30 cycles. Final amplification time in minutes to confirm lane synthesis. PCI

«·. ·· *, 25 blades were cleaned with Mermaid ™ Kit.

The ends of the \: ment were filled in using the DNA polymer · ·. / Klenow fragment.

ft m • Ψ «· t

· '· ": RESULTS

A. Vector constructs pH 2.5 acidol 30 e 90 var. awamori ALKO 243 pH2.5 linked to its acid phosphatase gene signal sequence in a Trie reesei expression cassette containing the cbl ·: engine and decision sequences. pALK533 contains 5 transformants of the AmdS gene of Aspergillus nidulans as a mammalian tag.

A fusion between the cbh1 promoter and the pH 2.5 acid phospho signal sequence was made. The primers used for the PCR fragments are shown in Figure 11. The SacII site of the cbh1 promoter region was used in the primer, and the N pha N1 of the acid phosphatase gene was downlinked. The 5 'primer was 39-mer: contained the cbh1 promoter sequence signal sequence of the preceding 19 nucleotides of the tail, linked to the first 20 nucleotides of this acid phosphatase signal sequence. The 3 'primer was pH 2.5 acid phosphatase and 30 mer. pAP-1 (Figure 12) was used in templ <PCR reactions. The PCR reaction yielded the expected 206 bp 466 bp fragment, the 466 bp PCR fragment containing the acid phosphatase signal sequence, and ligated into pAF-1 digested with DNA, Klenow fι 25 DNA and plasmid digested with MluI to give p102 * (Figure 15). Fusion and PCR fragment sequences to ensure no PCR amplification. * * ··· * errors occurred.

: Plasmid for construction of pALK533 (Km V · 30 pH 2.5 acid phosphatase gene containing fi; isolated from plasmid pl02 Sphl (amplified * rtrt 1 * »n A Vt 3 n β -ί T VI A« A .. * m mm am 4 µl of 1 µm C of TT polymerase Klenow fragment The linear fragment used for the transform was separated with Vek Xbal.

5 2. Construction of plasmid pALK532

Plasmid pALK532 consists of Aspergillus var, awamori ALKO 243 pH 2.5 acid phosphatase ligated into a Trichoderma reesei CBHI expression 10 cassette containing the cbh1 promoter and additional sequence and decision sequences. pALK532 s also serves as a selection marker for the transduction of the Aspergillus nidulans amdS gene.

The fusion of the cbh1 signal sequence and the pH 2.5 happ 15 gene was performed by PCR. The primers to be used for the PCR fragment are shown in Figure 11. The 5f primer used in the Sf ta cbh1 signal sequence was a 46-mer containing 28 nucleotides of the tail fused precisely to the acid phosphatase N-; 20 to the first nucleotide. The 3 'primer was the same as in the pALK533 construct. pAP-1 was used as a template in the PCR reaction. The PCR reaction yielded a 418 bp fragment of length.

; A 418 bp PCR fragment containing * * 25 signal sequence was digested with MluI and Liga pAP-1 digested with HindIII; DNA polymerase I with Klenow fragment and digested with * «· * to obtain plasmid p51 (Fig. 13), Fui *: PCR fragment was sequenced to confirm ϊ 30 vi amplifications during PCR amplification;

Plasmid for construction of pALK532 (Figure 13) with 92 fragments and digested with SfiI. pALK532 was made by restriction with Xba I a 7.8 kb fragment which did not contain bacterial sequences, which fragment was used for transfoins.

B. Transformation of Trichoderma reesei Screening for transformants Trichoderma reesei transforms ALKO 2221 separately with the Xba I fragments of plasmids pALK532 and pALK533. The transformation frequency (formants / μ <$ DNA) ranged from 2 to 30.

44 transformants of T. reesei ALKO 2221 / pALK532 transfon and 103 pALK533 were purified and grown in shake flasks.

C. Production of pH 2.5 Acid Phosphatase by Tricl Transformants 20

The best pH 2.5 acid phosphatase transformants are shown in Table 10. The best level of activity was 240 APNU in shake cultures on lactose-based medium.

«·« «* F · 9 999 • 9 9 | 9 · f 999 I · V 9 999 9 9 9 9 9 9 9 999 9 M · 9 9 9 9 9 9 9 9 9 93 ö I e O M P-t ^ S ai CO Jd Ή

M ^ M

tn - ω ------ o Q> έ ^ + - 1 ^ Ä '9.2 - 1¾ «^ Oi-jpQQQQQQ O g g + l.g« «whZ222222 o E-» ^ w .¾. »2 S So fi Cö ϋ O OOOOqOqqOQ ^ g S * 3

25 O OOffiCOÖJCOldSNS m o ^ .S

ti to n ^ c * ^ Zio2Zu) Z £ ί -S

m 1 5 S

fi s p,: JS ^ r ~. :: ¾ 00 {NCDWCOQ ^ QQhQ r-1 J-g '9 »Jf: ¾ S« g: 0 «

· * · :: * 5J f ± M CJ

m 9 «, (J ^ li t -s £ + 2" oo MNTTNflinflQofl CD Φ S - rt · w «^ co z« 2 2 ^ 2 ^ S ^ Et 8 2i§ ^ <T3:.> "* j_» rt Ä 3 o 3

:: ¾4 S M

::: ¾ ++ ++ I + C ++ ++ · ä «S - £ pu,: S -_ <WW ^ rtW ^ 'rtW'rtWW 6 m 2« i3 ___ λ> ϋ rsrr: fl td ;: B S Λ

-¾ w OOOOOOOOOO rt .2 O

. · * ..; P:. TfTiMrtCJrtOJOioro i-t m bO

ΐ ψ. : 1¾.¾ o {N <N {NMWW rt rt rtrt «MF ^ i '"' i li _ 1¾ ·.: Ft ® ^ * * ·; w wcoeorteowMcoMN 2 ö 3 ... Ή Mrtcortnnwnnn ^ 'Sw • · · «A · in ιο mm tn in mmm tn ^ wj-ι: ... S φ ►JhdhdrtJj.Jrti.djjj φ« 55

:: # «Jcccccc ^ cc tccSS

* ;;. *: ft aftftftftftftftftft αm 9 .:. .0 1 · * · rt <m ¢ 0 *: --- :: - ':'. '. V ______ {β O rt cd ft a - _ d ft? 3 = t 0 fa g 5 94

Both acid phosphatase and cbhl-Signa acted equally well and approximately the same level of acid phosphatase activity was achieved. ] derma transformant SC-9 produced the best 5 levels of acid phosphatase activity, which was no more than that of the native Aspergillus Nige awamori strain ALKO 243 in the corresponding strains.

Two of the nine top 10 producers responded by Western blot analysis of CBHI monoclonal antibody, suggesting that the expression cassette was associated with the cbhl locus of these two trp mins (Table 10).

15 pH 2.5 acid phosphatase preparations produced by D. T. reesei pH 2.5 acid phosphatase is expressed in high concentrations in T. transformants and T.

The activity of some other enzymes in the supernatants differs from that of Aspergillus stants (Table 10). Both canase and cellobiohydrolase activities were significantly higher when used! «F 25 T. reesei'ta. T. reesei-trans formant it yields .....

\; relative to less glucoamylase activity kv • * · e ... * niger ALKO 243 strain.

• Ψ • · »•« «« t ··: E. Trichoderma-trans formant ti en support • it - V * 30 pH2.5 identification of acid phosphatase ψ · • · ♦ 2 4 «m ^ j ÄTt / Λ 95

Trichoderma reesei was grown from the supernatant of transformants SC 31, KA-17, KB-44, KB-18, SB-4 and KA-28 (Figure 14).

5 acid phosphatase secreted by T. reesei transformants was detected as four 50-66 kD lines. This is probably due to the different degree of glycation of the secreted 2.5 acid phosphatase protein. Compared to Aspergillus niqer var. At pH 2.5 produced by the ALKO 243 (66 kD) strain, most of the acid phosphatase proteins produced by T. reesei are smaller than those produced by gillus.

All references are included with this vi. Once the invention has been fully described, it will be apparent to one skilled in the art that the scope of protection may be broad and corresponding to a range of concentrations, values, and the like without affecting the inventive concept of the invention or embodiments.

«T • * · • *» M 9 · 9 · • * • · * 9 9 99 • · ·· »9 9 9 9 * 9 9 * 99 · * 9 99 9 · 9 9 9 9 9 9 96

Sequence Listing

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(C) CITY: Espoo (D) STATE OR COUNTRY: (E) COUNTRY: Finland {F) POSTAL NUMBER: FI-02260 (ii) NAME OF THE INVENTION: Production of Phytase-Degrading Enzyme by Trichoderma ΐiii) NUMBER OF SEQUENCES: :

{A) TO: Borenius & Co Oy Ab <B) STREET: Tallberginkatu 2 A

(C) CITY: Helsinki (D) STATE: (E) COUNTRY: Finland (F) ZIP Code: FI-00180 (v) MACHINE CODE FORMAT: (A) TOOL TYPE: Diskette (B) COMPUTER: IBM PC compatible

V ·: (C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patent Release # 1.0, Version # * · * · · * · t. ··· [(vi) CURRENT APPLICATION DETAILS:! * ·; '(A) APPLICATION NUMBER: (B) SUBMISSION DATE: 0: ( (C) CLASSIFICATION:; (vii) PRIORITY APPLICATION INFORMATION: (A) APPLICATION NUMBER: US 07 / 923,724 99 (ix) TELECOMMUNICATION CONTACT INFORMATION: ^ (A) PHONE: (B) TELEFAX: (2) SEQUENCE ID NO: 1 DETAILS: (i) SEQUENCE ) LENGTH: 2071 base pairs (B) TYPE: Nucleic Acid (C) STRENGTH: Single strand (D) TOPOLOGY: Both (ix) FEATURES:

(A) NAME / EXPLANATION: CDS

(B) LOCATION: Field (136..916, 971..1088, 11 1305..1737) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GCATGCTGGA CCGCAATCTC CGATCGCCGG GTATAAAAGG TCCTCCAAAC C 60 .... CGATATGTAC CCCGCTCGTC ATCTCCAATC CTCTCGAGAG CACCTTCTCC A i2o * I I ♦ • »· * i

AATTGTACCT TCGCA ATG CCT CGC ACC TCT CTC CTC ACC CTG GC

· «· 171« *

!. ·: Met Pro Arg Thr Ser Leu Leu Thr Leu AI

»M

I · · * ♦ «15 1 ft · lift loo Ί - TCA ACC CAG GAG AAG CAG TTC TCT CAG GAG TTC CGC GAT GGC 267

Ser Thr Gin Glu Lys Gin Phe Ser Gin Glu Phe Arg Asp Gly 30 35 40 ATC CTC AAG CAC TAC GGT GGT AAC GGA CCC TAC TCC GAG CG *) 315 lie Leu Lys His Tyr Gly Gly Asn Gly Pro Tyr Ser Glu Arg 45 50 55 TAC GGT ATC GCT CGC GAT CCC CCG ACC AGC TGC GAG GTC GA1 363

Tyr Gly lie Ala Arg Asp Pro Pro Thr Ser Cys Glu Val Asp 65 70 ATC ATG GTC AAG CGT CAC GGA GAG CGC TAC CCG TCC CCT TCi 411 lie Met Val Lys Arg His Gly Glu Arg Tyr Pro Ser Pro Ser 80 85 90 AAG GAC ATC GAA GAG GCC CTG GCC AAG GTC TAC AGC ATC AAC 459

Lys Asp lie Glu Glu Ala Leu Ala Lys Val Tyr Ser lie Asn 95 100 105,, GAA TAC AAG GGC GAC CTG GCC TTC CTG AAC GAC TGG ACC TAC • * # * · 507 • I »'... · Glu Tyr Lys Gly Asp Leu Ala Phe Leu Asn Asp Trp Thr Tyr 110 115 120 «* · * ·« ·

Icct aat gag tgc tac tac aac gcc gag acc acc agc ggc ccc: 7: 555

Pro Asn Glu Cys Tyr Tyr Asn Ala Glu Thr Thr Ser Gly Pro. . 125 130 135 ιοί * | ^ GGC CAC CTC TGG AAC GGT GAG ACG GTC GTG CCC TTC TTT TC '] 651

Gly His Leu Trp Asn Gly Glu Thr Vai Val Pro Phe Phe Ser 160 165 170 TAC GGA CGT GTC ATC GAG ACG GCC CGC AAG TTC GGT GAG GGI 699

Tyr Gly Arg Val lie Glu Thr Ala Arg Lys Phe Gly Glu Gly 175 180 185 GGC TAC AAC TAC TCC ACC AAC GCT GCC CTC AAC ATC ATC TCC 747

Gly Tyr Asn Tyr Ser Thr Asn Ala Ala Leu Asn Ile He Ser 190 195 200 GAG GTC ATG GGC GCG GAC AGC CTC ACG CCC ACC TGT GAC ACC 795

Glu Val Met Gly Ala Asp Ser Leu Thr Pro Thr Cys Asp Thr 205 210 215 GAC CAG ACC ACC TGC GAC AAC CTG ACT TAC CAG CTG CCC CAG 843

Asp Gin Thr Thr Cys Asp Asn Leu Thr Tyr Gin Leu Pro Gin 225 230. . GTC GCT GCT GCC CGC CTA AAC TCC CAG AAC CCC GGC ATG AAC »» «• · 891 • • a

Val Ala Ala Ala Arg Leu Asn Ser Gin Asn Pro Gly Met Asn * ·! 240 245 250 «* a · •« · i! *: GCA TCT GAT GTC TAC AAC CTG ATG G GTATGTGAT TACGGTACAA Ί * «· * 945 * a ♦

Ala Ser Asp Val Tyr Asn Leu Met ». 255 260 i 4 102> | Ί CGT CCC TTC TCC AAC TGG ATC AAC GCC TTT ACC CAG GAC GAi 1044

Arg Pro Phe Ser Asn Trp Ile Asn Ala Phe Thr Gin Asp Glu 270 275 280 AGC TTC GGT TAC GTT GAG GAT TTG AAC TAC TAC TAC TGC GCl 1089

Ser Phe Gly Tyr Vai Glu Asp Leu Asn Tyr Tyr Tyr Cys Ala 290 295 TGAGTTTACC ATTTGATCCA TTATTGTCTT GGATCAGCTA ACGATCGATA 1144 GGT GAC AAG AAC ATG GCT GCT GTG GGT GCC GTC TAC GCC AAC 1192

Gly Asp Lys Asn Met Ala Ala Vai Gly Ala Vai Tyr Ala Asn 305 310 315 CTC ACC CTC CTG AAC CAG GGA CCC AAG GAA GCC GGC TCC TTG 1240

Leu Thr Leu Leu Asn Gin Gly Pro Lys Glu Lower Gly Ser Leu 320 325 330

, AAC TT GTACGTTCTCG GCAGAATCAG AGTCTCACAA AAAGAAACTC TTC

1296 ♦ · * ··· * Asn Phe * 1 335 «« «f f •« • «e

ί TATAGTAG T GCC CAC GAC ACC AAC ATC ACC CCC ATC CTC GCC

Ala His Asp Thr Asn Ile Thr Pro Ile Leu Ala 1 i ♦ 340 345 103 11 GGC AAC CCC TAC TCG ATC GGC AAC ATC GTG CCC ATG GGT GGC 1440

Gly Asn Pro Tyr Ser Ile Gly Asn Ile Val Pro Met Gly Gly 365 370 375 ACC ATC GAG CGT CTC AGC TGC CAG GCC ACC GCC CTC TCG GAC 1488

Thr lie Glu Arg Leu Ser Cys Gin Ala Thr Ala Leu Ser Asp 385 390 ACC TAC GTG CGT CTG GTG CTG AAC GAG GCT GTA CTC CCC TTC 1536

Thr Tyr Val Arg Leu Val Leu Asn Glu Ala Val Leu Pro Phe 400 405 410 TGC ACC TCC GGA CCG GGC TAC TCC TGC CCT CTG GCC AAC TAC 1584

Cys Thr Ser Gly Pro Gly Tyr Ser Cys Pro Leu Ala Asn Tyr 415 420 425 ATC CTG AAC AAG AAT CTG CCA GAC TAC ACG ACC ACC TGC AAT 1632 lie Leu Asn Lys Asn Leu Pro Asp Tyr Thr Thr Cys Asn 430 435 440

,, GCG TCC TAC CCG CAG TAT CTG AGC TTC TGG TGG AAC TAC AAC

it · * · 1680 '... · Ala Ser Tyr Pro Gin Tyr Leu Ser Phe Trp Trp Asn Tyr Asn' 1 ·! 445 450 455 «··» I I · ···

• ACG GAG CTG AAC TAC CGC TCT AGC CCT ATT GCC TGC CAG GAG

• II 1 T. 1728 ► · ·

Thr Glu Leu Asn Tyr Arg Ser Ser Pro lie Ala Cys Gin Glu. 1,465,470

104 H

TTGATATTCA AGTTTGGTGG TGACGATCAC CTTGTTAATA GTCTTGTACA C 1837 GAATGTAAAT AATGATAATA GCAATGATAC ATGTTGGAAT CTCGTTTTGT Ί 1897 CATAGGCGCT TTGGGGGTGT ATTTTTAGGC GTTAGACTTA TTTTCAATTC C 1957 CGGTCAGTAA ATGAATCATC AATTATTCAA ATGCAATGCT GTATACGTGA fl 2017 TTAAGACGCA GCTACTAGCT, GACTGCTTGG TTACTTTCTG TGTACACCGC 2071 (2) SEQ ID NO: 2 INFORMATION: (i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 479 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECYL TYPE: protein • 1 V ·: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: «« t • f • 4 4 4! / ·· Met Pro Arg Thr Ser Leu Leu Thr Leu Ala Cys Ala Leu Ala i 5 10 • 4 «I · • 4 4 ϊ

Ala Ser Ala Phe Ser Tyr Gly Ala Ala Ile Pro Gin Ser Thr • · · 20 25 30 · · lii Lys Gin Phe Ser Gin Glu Phe Arg Asp Gly Tyr Ser Ile Leu * · ... ... ς Art Art Ac.

105 1

Arg His Gly Glu Arg Tyr Pro Ser Pro Ser Ala Gly Lys As 85 90

Glu Ala Leu Ala Lys Val Tyr Ser lie Asn Thr Thr Glu Ty 100 105 11

Asp Leu Area Phe Leu Asn Asp Trp Thr Tyr Tyr Val Pro As 115 120 125 '

Tyr Tyr Asn Ala Glu Thr Thr Ser Gly Pro Tyr Ala Gly Le 130 135 140

Ala Tyr Asn His Gly Asn Asp Tyr Lys Ala Arg Tyr Gly Hi 145 150 155

Asn Gly Glu Thr Val Val Pro Phe Phe Ser Ser Gly Tyr G] 165 170

Ile Glu Thr Ala Arg Lys Phe Gly Glu Gly Phe Phe Gly T} 180 185 1 «

Ser Thr Asn Ala Ala Leu Asn lie lie Ser Glu Ser Glu V <195 200 205 • ♦ · 4 1 • ·. · 2. Ala Asp Ser Leu Thr Pro Thr Cys Asp Thr Asp Asn Asp G] · 1 ». ·. : 210 215 220 • it • 1 • ·

/ 2 Cys Asp Asn Leu Thr Tyr Gin Leu Pro Gin Phe Lys Vai AJ

: - · 2 225 230 235 • · 1 • · t «

Arg Leu Asn Ser Gin Asn Pro Gly Met Asn Leu Thr Ala Se 2,245,250

Ml 41 2 • «106 1 '

Tyr Vai Glu Asp Leu Asn Tyr Tyr Tyr Cys Ala Gly Pro Gly 290 295 300

Asn Met Ala Ala Vai Gly Ala Val Tyr Ala Asn Ala Ser Leu 305 310 315

Leu Asn Gin Gly Pro Lys Glu Lower Gly Ser Leu Phe Phe Asn 325 330

His Asp Thr Asn lie Thr Pro lie Leu Ala Ala Leu Gly Val 340 345 350

Pro Asn Glu Asp Leu Pro Leu Asp Arg Val Ala Phe Gly Asn 355 360 365

Ser lie Gly Asn lie Val Pro Met Gly Gly His Leu Thr lie 370 375 380

Leu Ser Cys Gin Ala Thr Ala Leu Ser Asp Glu Gly Thr Tyr 385 390 395

Leu Val Leu Asn Glu Ala Val Leu Pro Phe Asn Asp Cys Thr 405 410 • · «« «• · · •. ·" · Pro Gly Tyr Ser Cys Pro Leu Ala Asn Tyr Thr Ser lie Leu 420 425 430 • · 9 • · * *. ··· * Asn Leu Pro Asp Tyr Thr Thr Thr Cys Asn Val Ser Ala Ser ··· * 435 440 445 · «· ♦ * · * * ·

Gin Tyr Leu Ser Phe Trp Trp Asn Tyr Asn Thr Thr Thr Glu: 450 455 460 * «· 1 • · •» 107 1 (A) LENGTH: 17 base pairs (B) TYPE: Nucleic Acid (C) STRENGTH: Single strand (D) ) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TAYTAYGGNC AYGGNGC 17 (2) SEQ ID NO: 4 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 17 bp (B) ) STRENGTH: Simple strand (D) TOPOLOGY: Both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: • · 4 • »4 •»

: “CARGGNGTNG GNTAYGC

• · * · '. : 1 7 • · ♦ 1 9 • · M * * * (2) SEQ ID NO: 5 DETAILS: • 4 «

· «T S

4 4 4 • * · '**' (i) SEQUENCE FEATURES: (A) LENGTH: 20 base pairs: .i .: (B) TYPE: nucleic acid ··· * fC) SPECTACY: Simple 108 (2) SEQ ID NO: : 6 DATA: (i) SEQUENCE FEATURES: (A) LENGTH: 23 base pairs (B) TYPE: Nucleic Acid (C) JURITY: Simple (D) TOPOLOGY: Both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 23 (2) SEQ ID NO: 7 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 2363 base pairs (B) TYPE: Nucleic Acid (C) JURITY: Simple (D) TOPOLOGY: Both (ix) FEATURES:

(A) NAME / EXPLANATION: CDS

: (B) LOCATION: space (404 .447, 550..1906) • aa • a aa • a "*! (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: a a • ·

| ·· ;: CATCCAGGCA CCCTTTCCCA ACGGGGGAAC TTCCGTTGTC CACGTGCCCI

60 * a · • a a · • ATCAAAGCGT CCCACGGCAA TGCTGGATCA ACGATCAACT TGAATGCAAI 120 • A A • · · • a • a a 109 1 CCATTGCACG TGGGCCACCT TTGTGAGCTT CTAACCTGAA CTGGTAGAGT 300 ATGCGAAAGT GGGATGAAGG GGTTATATGA GGACCGTCCG GTCCGGCGCG 360 CTGCCAATCG CTGCTGTGCA AGAAATTTCT TCTCATAGGC ATC ATG G (415

Met Gl GCT GTT CTA CTT CCT TTG TAT CTC CTA GCT GG GTATGCTAJ 457

Ala Val Leu Leu Pro Leu Tyr Leu Leu Ala Gly 5 10 15 CACCGGTATC TAAGTCTGAT AAGGACCCTC TTTGCCGAGG GCCCCTGAAG 517 GTGGGACTAC TGATCGCTGA CAATCTGTGC AG A GTC ACC TCC GGA 568

Val Thr Ser Gly no ^ GGT TGC AGA GTC ACT TTC GCT CAG GTC CTC TCC CGT CAT GC 760

Gly Cys Arg Vai Thr Phe Ala Gin Val Leu Ser Arg His GL · 70 75 80 TAT CCG ACC GAG TCC AAG GGC AAG AAA TAC TCC GCT CTC ΑΊ 808

Tyr Pro Thr Glu Ser Lys Gly Lys Lys Tyr Ser Ala Leu 11 <90 95 ATC CAG CAG AAC GTG ACC ACC TTT GAT GGA AAA TAT GCC ΤΊ 856

He Gin Gin Asn Val Thr Thr Phe Asp Gly Lys Tyr Ala Phi 105 no 11: ACA TAC AAC TAC AGC TTG GGT GCA GAT GAC CTG ACT CCC Tr 904

Thr Tyr Asn Tyr Ser Leu Gly Ala Asp Asp Leu Thr Pro Phi 120 125 130 CAG GAG CTA GTC AAC TCC GGC ATC AAG TTC TAC CAG CGA T1 952: Gin Glu Leu Val Asn Ser Gly lie Lys Phe Tyr Gin Arg Ty: λ / 135 140 145 • «>» · • t • · · '· CTC ACA AGG AAC ATC ATT CCG TTC ATC CGA TCC TCT GGC T <• # · 1000:.:: Leu Thr Arg Asn Ile He Pro Phe Ile Arg Ser Ser Gly Se:: T: 150 155 160. . ·. GTG ATC GCC TCC GGC GAG AAA TTC ATT GAG GGC TTC CAG A <. ···. 1048 • ♦

Λ A

m V1 GTG GTC ATT TCC GAG GCC AGO TCA TCC AAC AAC ACT CTC Gl 1144

Or Vai Ile Ser Glu Ala Ser Ser Ser Asn Asn Thr Leu Asj 200 205 210 ACC TGC ACT GTC TTT GAA GAC AGC GAA TTG GCC GAT ACC G1 1192

Thr Cys Thr Vai Phe Glu Asp Ser Glu Leu Ala Asp Thr Va! 215 220 225 AAT TTC ACC GCC ACG TTC GCC CCC TCC ATT CGT CAA CGT Cl 1240

Asn Phe Thr Ala Thr Phe Ala Pro Ser Ile Arg Gin Arg Lei 230 235 240 GAC CTG TCT GGC GTG ACT CTC ACA GAC ACA GAA GTG ACC Tl 1288

Asp Leu Ser Gly Vai Thr Leu Thr Asp Thr Glu Vai Thr Tyi 250 255 GAC ATG TGC TCC TTC GAC ACC ATC TCC ACC AGC ACC GTC Gl 1336

Asp Met Cys Ser Phe Asp Thr Ile Ser Thr Ser Thr Vai As]. . 265 270 27J

• t · * ·· * * • · # CTG TCC CCC TTC TGT GAC CTG TTC ACC CAT GAC GAA TGG A ^ 1384 * ·· · ... · Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp Glu Trp Il < I .: ';' 280 285 290 • · · * · · «» · GAC TAC CTC CAG TCC CTG AAA AAA TAC TAC GGC CAT GGC GC 1432 • · ·. ···, Asp Tyr Leu Gin Ser Leu Lys Lys Tyr Tyr Gly His Gly Al < ·· »ΛΛ - * A _ _ _ _ 112 ^ CGT CTC ACC CAC TCG CCT GTC CAC GAT GAC ACC AGC TCC Aj 1528

Arg Leu Thr His Ser Pro Val His Asp Asp Thr Ser Ser As 330 335 TTG GAC TCG AAC CCA GCT ACC TTC CCG CTC AAC TCT ACT Cf 1576

Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn Ser Thr Le 345 350 35 GAC TTT TCC CAC GAT AAC GGC ATC ATC TCT ATC CTC TTT G <1624

Asp Phe Ser His Asp Asn Gly He lie Ser He Leu Phe Al 360 365 370 CTG TAC AAC GGC ACT AAG CCG CTG TCT ACC ACG ACC GTG G 1672

Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr Thr Vai G1 375 380 385 ACC CAG ACA GAT GGG TTC TCG TCT GCT TGG ACG GTT CCG Τ '1720 • *

Thr Gin Thr Asp Gly Phe Ser Ser Ala Trp Thr Val Pro Ph O 390 395 400 • »* ·» * »»

. ···. CGT CTG TAC GTC GAG ATG ATG CAG TGC CAG GCC GAG CAG G

7. 1768 ♦ · *

Arg Leu Tyr Val Glu Met Met Gin Cys Gin Ala Glu Gin G1 *** * 410 415

GTC CGT GTC TTG GTT AAT GAT CGC GTT GTC CCG CTG CAT G

t ·· 1816 113 AGC TTT GCT AGA TCT GGG GGT GAT TGG GCG GAG TGT TCT GC 1906

Ser-Phe Ala Arg Ser-Gly-Gly-Asp-Trp Ala Glu Cys Ser Ale 455 460 465 TAGCTGAACT ACCTTGATGG ATGGTATGTA TCAATCAGAG TACATATCAT 1966 ATGTATTTAC GAAGATGTAC ATATCGAAAT ATCGATGATG ACTACTCCGG 2026 GTCCCCTTCT ATCCTTCGTT CCACAACCAT CGCACTCGAC GTACAGCATA 2086 AGCATTAACA AACGAACAAA TAATATTATA CACTCCTCCC CAATGCAATA 2146 TTCATACCTC ATATAGATAC AATACAATAC ATCCATCCCT ACCCTCAAGT 2206 CATAATCAAA TCCCTACTTA CTCCTCCCCC TTCCCAGAAC CCACCCCCGA 2266: \ j GTAGTAGTAG AAGAAGCAGA CGACCTCTCC ACCAACCTCT TCGGCCTCTT 2326 • · • f · • »f

'··· [GCTATACACA CACGAACACA CCAAATAGTC AGCATGC

/ • f 2363 • # · «· · * · · · · · (2) SEQ ID NO: 8 DETAILS: • · · · φ · · · (i) SEQUENCE FEATURES: 114

Met Gly Vai Ser Ala Vai Leu Leu Pro Leu Tyr Leu Leu AJ 1 5 10

Thr Ser Gly Leu Ala Val Pro Ala Ser Arg Asn Gin Ser Tt 20 25:

Thr Vai Asp Gin Gly Tyr Gin Cys Phe Ser Glu Thr Ser H: 35 40 45

Gly Gin Tyr Ala Pro Phe Phe Ser Leu Ala Asn Glu Ser AJ 50 55 60

Pro Asp Val Pro Ala Gly Cys Arg Val Thr Phe Ala Gin V <65 70 75

Arg His Gly Ala Arg Tyr Pro Thr Glu Ser Lys Gly Lys L ^ 85 90

Ala Leu Ile Glu Glu Ile Gin Gin Asn Vai Thr Thr Phe Ai 100 105 r

Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Ser Leu Gly Ala Ai 115 120 125 ft ·· ♦ »• ·

Thr Pro Phe Gly Glu Gin Glu Leu Vai Asn Ser Gly Ile L} I. / 130 135 140 • · ft # • · · • ft ft i

• * j · Gin Arg Tyr Glu Ser Leu Thr Arg Asn lie lie Pro Phe IJ

\ s: 145 1 50 155

Ser Gly Ser Ser Arg Val lie Ala Ser Gly Glu Lys Phe IJ ***** 165 170 115

Thr Leu Asp Pro Gly Thr Cys Thr Vai Phe Glu Asp Ser G1 210 21 5 220

Asp Thr Val Glu Ala Asn Phe Thr Ala Thr Phe Ala Pro Se 225 230 235

Gin Arg Leu Glu Asn Asp Leu Ser Gly Val Thr Leu Thr As 245 250

Val Thr Tyr Leu Met Asp Met Cys Ser Phe Asp Thr lie Se 260 265 27

Thr Val Asp Thr Lys Leu Ser Pro Phe Cys Asp Leu Phe Th 275 280 285

Glu Trp lie His Tyr Asp Tyr Leu Gin Ser Leu Lys Lys Ty 290 295 300

His Gly Ala Gly Asn Pro Leu Gly Pro Thr Gin Gly Vai Gl 305 310 315

Asn Glu Leu lie Ala Arg Leu Thr His Ser Pro Val His As 325 330 «« »· · •» · *

Ser Ser Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pi 340 345 35 ♦ · ♦ ·

. **; Ser Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Gly Ile II

:; | j 355 360 365 • · · # · ·

Leu Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu S «370 375 380 ··· 4 · • · * *» 4 116

Glu Gin Glu Pro Leu Vai Arg Val Leu Val Asn Asp Arg V <420,425 4;

Leu His Gly Cys Pro lie Asp Ala Leu Gly Arg Cys Thr A: 435 440 445

Phe Val Arg Gly Leu Ser Phe Val Arg Gly Gly Asp T: 450 455 460

Cys Ser Ala 465 (2) SEQ ID NO: 9 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) SENSITIVITY: simple (D) TOPOLOGY: both (xi) SEQ ID NO: 9 : SEQ ID NO: 9: • * • * · • m «

, ··% CAACCGCGGA CTGCGCATCA TGGGCGTCTC TGCTGTTCT

39 (*) SEQ ID NO: 10 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid: ***; (C) SEQUENCE: Simple 117 ^ (2) SEQ ID NO: 11 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 46 base pairs (B) TYPE: Nucleic acid (C) SEQUENCE: Simple (D) TOPOLOGY: xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CTCGGCCTTC TTGGCGAGAG CTCGTGCTCT GGCAGTCCCC GCCTCG 46 (2) SEQUENCE ID NO: 12 DETAILS: (i) SEQ ID NO: 12 (c) T : simple (D) TOPOLOGY: both • «* · 9 • • 9 9 9 9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: · · 9 99 9 9 9

/ *; ' TTGGTGTCGA CGGTGCTGGT GGAG

(2) SEQ ID NO: 1 3 DETAILS: 9 9 9 9 9 9 9 999 999 (i) SEQUENCE FEATURES: 118 1 (xi) SEQ ID NO: 1 DESCRIPTION: SEQ ID NO: 13: CTCGGCCTTC TTGGCCACAG CTCGTGCTTT CTCCTACGGC GCTGCC 46 (2) SEQ ID NO: 14 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 30 bases (B) D) TOPOLOGY: Both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GCCATGGTTG TACGCGTCCA GCAAACCGGC 30 (2) SEQ ID NO: 15 DETAILS: (i) SEQ ID NO: 15 Length: (b) , ···] (C) DRAINAGE: simple (D) TOPOLOGY: both * · * • «· ♦ · m · * • • 999 • 9: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: • · · tt ft ft ft · 9

CAACCGCGGA CTGCGCATCA TGCCTCGCAC CTCTCTCCT

• · * <39 • «ft ^^

119 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GAATTCCCGG GACCTACCCC TCTGCAT 27 (2) SEQ ID NO: 17 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH (B): : amino acid (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Leu Ala Vai Pro Ala Ser Arg Asn Gin Ser Ser Gly 7 1 5 10 (2) SEQ ID NO: 18 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 7 amino acids (B) TYPE: amino acid • *. ·· ·. (D) TOPOLOGY: Both «4 ♦ ♦ · 9 9 9 9 9 9 99 9 9 994 4 · / • f (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: • · · 4 9 9 999 9 4 99 '· * * Arg His Gly Xaa Arg Xaa Pro 1 5 • * · i · · ···: **: (2) SEQ ID NO: 19 DETAILS: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 1 20

Lys Asp Pro Arg Ala 1 5 (2) SEQ ID NO: 20 DATA: (i) SEQUENCE FEATURES: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQ ID NO: 20 NO: 20:

Tyr Tyr Gly His Leu Gly Ala Gly Asn Pro Leu Gly P 1 5 10 (2) SEQ ID NO: 21 DETAILS: • ft »ftft ft ft ft (i) SEQUENCE FEATURES: • · · (A) LENGTH: 11 amino acids mmml (B) TYPE: amino acid • # »(D) TOPOLOGY: both • * · • ft · * • · ft • t · ··· • # • · · · 121 (2) SEQ ID NO: 22 DETAILS: ii) SEQUENCE FEATURES: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (ix) FEATURES: (A) NAME / DESCRIPTION: Peptide (B) LOCATION: 6..7 (D) OTHER INFORMATION: / Stamp = Peptide / Note = "When deduced from the DNA sequence at positions 6 and 7, serine was found." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Ala Gin Pro Gly Gin Ala Pro Lys 1 5 (2) SEQ ID NO: 23 DETAILS:. . (i) SEQUENCE FEATURES: »» · "(A) LENGTH: 16 amino acids (B) TYPE: amino acid ♦ φ:. * ·· (D) TOPOLOGY: both • • t * ♦ • · •« • · · • ♦ (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: ♦ • ♦ ·

Leu Tyr Vai Glu Met Met Gin Asn Gin Ala Glu Gin TY

* · * · * Vai (2) SEQ ID NO: 24 DATA: 122 (i) SEQUENCE FEATURES: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24 NO: 24:

Leu Tyr Vai Glu Met Met Gin Cys Gin Ala Glu Gin G

Vai 1 5 10 (2) SEQ ID NO: 25 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both • «• ·« (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: * »• · · •« · · * · Phe Ile Glu Gly Phe Gin Ser Asp Lys 1 5 • * ♦ * · • ie • · e ·: T: (2) SEQ ID NO: 25 : 26 DETAILS:. (i) SEQUENCE FEATURES: • * *. * · *. (A) LENGTH: 9 amino acids • m • ««,. , 123 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

Phe Ile Glu Gly Phe Gin Ser Asp Lys 1 5 (2) SEQ ID NO: 27 DATA: (i) SEQUENCE FEATURES: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

Tyr Ala Phe Leu Lys 1 5 (2) SEQ ID NO: 28 DATA: (i) SEQUENCE FEATURES: (A) LENGTH: 6 amino acids (B) TYPE: amino acid * # (D) TOPOLOGY: both • «« * «* ψ · 9 9 9 9 9 9 * 9 9 9 * · 9 • 9 9 9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: • · · ··· * ··· 9 9 9 9 9 9

Gly Leu Ser Phe Ala Arg 1 5 ♦ • 9 9 9 9 9 999 «<· ·. * / 91 ςρκν ^ κς.ςτΜ τη νπ · 5q φττγπλφ * 124 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

Vai Ile Ala Ser Gly Glu Lys 1 5 (2) SEQ ID NO: 30 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

Phe Tyr Gin Arg (2) SEQ ID NO: 31 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 9 amino acids (B) TYPE: amino acid v ·: (D) TOPOLOGY: both • i «« · ♦ »• · T V · • t · • »* 1 · • ·« · 9 · ♦: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: • · «· • · • ♦« • 1 ·

Phe Tyr Gin Arg Asp Ser Phe Vai Arg 1 5 • · «• 1 · • · · ··· • · ___ · / 1> ΐ cuirucMCCTM τη μπ · n φτγπλπι.

125 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

Asp Ser Phe Vai Arg 1 5 (2) SEQ ID NO: 33 DATA: (i) SEQUENCE FEATURES: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33 NO: 33:

Vai Leu Vai Asn Asp 1 5 (2) SEQ ID NO: 34 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 5 amino acids β · β; (B) TYPE: amino acid *; ./ (D) TOPOLOGY: both • «•» »··· ♦ (2) SEQ ID NO: 35 DETAILS: 126 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

Tyr Glu Ser Leu Thr Arg 1 5 (2) SEQ ID NO: 36 DATA: (i) SEQUENCE FEATURES: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: ID NO: 36:

Ser Ala Ala Ser Leu Asn Ser 1 5 (2) SEQ ID NO: 37 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 5 amino acids. (B) TYPE: amino acid (D) TOPOLOGY: both • »• 1 • 1« • · • · · · ♦ •

»M

• ♦ * ··: (xi) Sequence Description: Sequence ID NO: 37: * ·· I «·»

• · I

• i · •

Leu Lys Asp Pro Arg 1 5 • · · • · ♦ ♦ ·· * ··· *: 2) SEQUENCE ID Noise DETAILS: »(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: 127

Vai Ile Ala Ser Gly Glu Lys 1 '5 (2) SEQ ID NO: 39 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQ ID NO: 39 : SEQ ID NO: 39:

Tyr Pro Thr Glu Ser Lys 1 5 (2) SEQ ID NO: 40 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both • · • ♦ »• i · I · • «•« · · · · · · (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: ** i • · • · ♦ # · 9 ** · ·

Tyr Phe Asn Xaa Gly # · · * 1 5 ^ * i «« • · • f (2) SEQ ID NO: 41 DETAILS: 128 (ix) FEATURES: (A) NAME / DESCRIPTION: Peptide (B) LOCATION: 3 ..8 (D) OTHER INFORMATION: / Stamp = Peptide / Note = "The following are the free amino acids at position 4: Proline as phenylalanine at position 4, Serine at position 6, Leucine at position 7, and Wallin at position 8." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

Leu Glu Asn Asp Leu Asp Gly Phe Thr Leu 1 5 10 (2) SEQ ID NO: 42 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: Π amino acid (B) TYPE: amino acid (D) TOPOLOGY: both ** * ·· \ v (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: * f • · * · * 5 Leu Glu Asn Asp Leu Ser Gly Vai Thr Leu Thr * ·· 1 5 10: (2) sequence id no: 43 Information:. (i) SEQUENCE FEATURES: ♦ · ·, ··· β (A) LENGTH: 20 amino acids • * ··· fry \ mvvnnT. ___ 129 (D) OTHER INFORMATION: / Stamp »Peptide / Note =" The amino acid at position 15 may be tyrosine. " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

Tyr Tyr Gly His Gly Ala Gly Asn Pro Leu Gly Pro Ti

Or 1 5 10

Gly Ala Asn Glu 20 (2) SEQ ID NO: 44 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 3 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both a * * · (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: aa · • a

• Q.

• aa

Leu Ile Ala • aa • * 1

• a I

• aa • a • * a • a * «a · · • aa • · a • aa (2) SEQ ID NO: 45 DETAILS: aae (i) SEQUENCE FEATURES: aa *** (Δ \ DTTnriC R ami τ- ιar \ r \ r \ z> (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: 130

Vai Thr Phe Ala Gin Vai Leu Ser 1 5 (2) SEQ ID NO: 46 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQ ID NO: 46 : SEQ ID NO: 46:

Phe Ile Glu Gly Phe Gin Ser Thr 1 5 (2) SEQ ID NO: 47 DETAILS: (i) SEQUENCE FEATURES: • · (A) LENGTH: 7 amino acids «« · (B) TYPE: amino acid (D) TOPOLOGY: both

• • I

• f • * • · I ** *. ··% (ix) FEATURES: • * «(A) NAME / DESCRIPTION: Peptide. (B) LOCATION: 1 • · · (D) OTHER INFORMATION: / Stamp Peptide * · / Vmnina a— aoomacoa 1 n 4- -5 131 (2) SEQ ID NO: 48 DETAILS: (i) SEQUENCE FEATURES: ( A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

Asn Ile Glu Pro Phe Gin Vai Asn 1 5 (2) SEQ ID NO: 49 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE : SEQ ID NO: 49: * · · • • ♦. ·· *. Vai Leu Vai Asn Asp Arg • - · 1 5 • · · • i · * »•» t (2) SEQ ID NO: 50 DETAILS: «* ·« «4« 44 4 V: (i) SEQUENCE FEATURES: ( A) LENGTH: 14 amino acids: (B) TYPE: amino acid 4 «ft. · * ·. (D) TOPOLOGY: both * & * 132 (2) SEQ ID NO: 51 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 3 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (ix) FEATURES: (A) NAME / DESCRIPTION: Peptide (B) LOCATION: 1 (D) OTHER INFORMATION: / Stamp = Peptide / Note = "Cysteine was found at position 1 of the DNA sequence." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

Arg Ser Ala 1 (2) SEQ ID NO: 52 DETAILS:. . (i) SEQUENCE CHARACTERISTICS: • «* * ·" / (A) LENGTH: 17 base pairs * ··. * (B) TYPE: nucleic acid • · \ ··: (C) DURABILITY: simple: ***: (D ) TOPOLOGY: Both • »· * # •« · ft · ft ft · · · ft · ft · · (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: ft · ·

CARTTRCCNC ARTTYAA

ft · **. i * 1 * 7 133 (i) SEQUENCE FEATURES: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:

Phe Ser Tyr Gly Bottom Ile Pro Gin Ser Thr Gin GJ 1 5 10 (2) SEQ ID NO: 54 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (DJ TOPOLOGY: both) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

Gin Phe Ser Gin Glu Phe Arg Asp Gly Tyr: 1 5 10 • «i • ·· • ♦ V„! (2) SEQUENCE ID NO: 55 DETAILS: * · · • · • * »: ···: (i ) SEQUENCE FEATURES: • »: (A) LENGTH: 7 amino acids \ · '* (B) TYPE: amino acid (D) TOPOLOGY: both * · · ♦ · * * · · * · •> * * 134 (2) SEQ ID NO: 56 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:

Vai Ser Tyr Gly Ile Ala 1 5 (2) SEQ ID NO: 57 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both. . (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: * * »• · * * m '· * · * Arg His Gly Glu Arg Tyr Pro Ser Pro Ser Ala Gly: • * 1 5 10 • · • · • · · : (2) SEQ ID NO: 58 DETAILS: ·· * * • i (*) (*) (i) SEQUENCE FEATURES:. (A) LENGTH: 8 amino acids • · »

Jill (B) TYPE: amino acid t a (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: 135

Asp Ile Glu Glu Ala Leu Ala Lys 1 5 (2) SEQ ID NO: 59 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE : SEQ ID NO: 59:

Ala Arg Tyr Gly His Leu Trp Asn Gly Glu Thr 1 5 10 (2) SEQ ID NO: 60 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 8 amino acids (B) TYPE: amino acid V *: (D) TOPOLOGY : both · * · • · * 9 • · · «9 9 9« 4 · «• 9 • 44 9 · • 9« 9 «: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: 9 · # 9 9 9 9 9 9 * 4 9 9 4 9 9

Vai Vai Pro Phe Phe Ser Ser Gly 1 5 4 9 9 4 9 9 949 4 9 4 (2) SEQ ID NO: 61 DETAILS: 136 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:

Phe Ser Ser Gly Tyr Gly Arg 1 5 (2) SEQ ID NO: 62 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:

Gin Leu Pro Gin Phe Lys 1 5 (2) SEQ ID NO: 63 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both 9 9 • 99 • 99 • · • · • * 999 9 9 • 99 9 9 9! ·;! (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: «· ·

Vai A Phe Gly Asn Pro Tyr 9 9 9 9 9 9 ··· 1 5 «M • · • ♦ 999 (2) SEQ ID NO: 64 DETAILS: 137 (i) SEQUENCE FEATURES: (A) LENGTH: 17 base pairs ( B) TYPE: Nucleic Acid (C) DRAINAGE: Simple (D) TOPOLOGY: Both (ix) FEATURES: (A) NAME / DESCRIPTION: Modified Base (B) STATUS: 12 (D) OTHER INFORMATION: / mod Base = i (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 64: GTRCCNCTYK CNATRGG 17 (2) SEQ ID NO: 65 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 17 base pairs (B) TYPE: Nucleic acid (D) TOPOLOGY: Both • i • * »• ** • * • · 9 9,999: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:« * «* 99 * 9 9 9 9 9 9 CARCTNCCNC ARTTYAA 17 • i «• · · ··· 999 9 9 *** · '{7) SRfn / FNCQTM ΤΓΊ ΜΠ * - (xx) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 66: 138 GAATTCCGAG TCCGAGGTCA TGGGCGCG 28 • a •« «

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Claims (3)

A process for preparing a composition comprising a phytate digestive enzyme, and at least one Trichoderma enzyme selected from the residue of a β-glucone degrading activity, CBHI, CBHII, EGI and EGII, characterized in that it comprises the following steps wherein a. preparing a recombinant construct containing a gene, a phytate digesting enzyme selected from phytase and pH 2.5 suit phosphatase, phytase having the amino acid sequence SEQ ID NO: 8, and wherein said pH 2.5 s has the amino acid sequence SEQ ID NO; 2, and a signal sequence associated with said gene; b. transforms a Trichoderma reesei host with said DNA cone c. cultivates the transformed Trichoderma / * ease * values below 1 Trichoderma reesei host and for expression of said phytate cleavage favorable conditions; • · · · 9 • «I • · * * · d. Removes the mycelium from the culture medium. »Ψ • 9 9 • * * • 9 • a # 1. menetelmä koostumuksen tuottamiseksi, joka koostumus käsittää yl fytaattia hajottavan entsyymin yes Vähintään ylidene Trichoderma enXsyymin glukaania hajottavasta aktiivisuudesta, CBHIistä, CBHII: STA, EGIrsta yes EGII: siitä, että menetelmä käsittää seuraavat vaiheet text. Valmistetaan yhdistelmäkonstruktio, joka sisältää geenin, yok fytaattia hajottavaa entsyymiä valittuna phytaasista ja happofosfataasista, jolloin mainitulla phytaasilla on aminohapposel ID NO: S yes mainitulla pH 2,5- happofosfataasilla on aminoha]: SEQ ID NO: 2, yes toiminnallisesti main signal no sequence; b. transformoid Trichoderma reesei-isäntä mainitulla DNA consi c. viljellään transformoitu Trichoderma reesei-isanlä olosuhteissa sopivia mainitulle Trichoderma reesei-ismälle ja fytaattia entsyymin ilmentämiselle; m * • «t a !. * ·: d. poistetaan kasvualustasta sienirihmasto. • »· • $ · * *: 2. Patenttivaatimuksen 1 mukainen menetelmä, tunnettu siitä, etä mai Vs constructio saatetaan mainittuun isäntään linearessa muodossa. * *: 3. Patenttivaatimuksen 1 mukainen menetelmä, tunnettu siitä, one May • ψ · «· ♦ · 1 in 1. *. . - * ,,. « Method according to claim 1, characterized in that it is introduced when the structure in linear form in said host. • ♦ ♦ • · · 3. A method according to claim 1, characterized in that it is introduced when the structure as an annular plasmid in said host. • · • *