CN101460612B - Phytase variants - Google Patents

Abstract

The present invention relates to a phytase which has at least 74% identity to a phytase derived from Citrobacter braakii and comprises at least one alteration as compared to this phytase. These phytase variants have amended, preferably improved, properties, such as thermostability, temperature profile, pH profile, specific activity, performance in animal feed, reduced protease sensitiliby, and/or an amended glycosylation pattern. The invention also relates to DNA encoding these phytases, methods of their production, as well as the use thereof, e.g. in animal feed and animal feed additives.

Description

Inositol six-phosphatase variants

Sequence table reference

The application comprises the sequence table existed with computer-reader form.Computer-reader form is incorporated herein by reference here.

Invention field

The present invention relates to and with the phytase being derived from Bu Shi citric acid bacillus (Citrobacter braakii) ATCC 51113, there is at least 74% identity and comprise at least one phytase changed (that is, being its variant) compared with this phytase.The invention still further relates to the DNA of these phytases of coding, their preparation method, and their purposes, such as, purposes in animal-feed and animal feedstuff additive.The maturing part of Bu Shi citric acid bacillus ATCC 51113 phytase is included in sequence table as SEQ ID NO:2.

Background of invention

Background technology

Sequence from the phyA gene of Bu Shi citric acid bacillus is submitted to EMBL/GenBank/DDBJ database by Zinin etc., and accession number is AY390262.Corresponding phytinic acid enzyme amino acid sequence is present in UniProt/TrEMBL database with accession number Q676V7.The maturing part of Q676V7 expection is included in sequence table of the present invention as SEQ ID NO:4.

WO-2004/085638 is with the aminoacid sequence of the phytase of the SEQ ID NO:7 Bu Shi citric acid bacillus YH-15 that to disclose from preservation be KCCM 10427.The maturing part of this aminoacid sequence is included in this as SEQ ID NO:3.This sequence is also present in database Geneseqp, and accession number is ADU50737.

WO 2006/037328 discloses the wild-type phytase (that is, the SEQ ID NO:2 of this paper) of Bu Shi citric acid bacillus ATCC 51113, and is included in its variant in this sequence table equally, namely as SEQ ID NO:6.

WO 2006/038062 and WO 2006/038128 discloses with the aminoacid sequence of the citrobacter freundii of accession number NCIMB 41247 preservation (Citrobacter freundii) P3-42 phytase gene.This aminoacid sequence is included in this as SEQ ID NO:9.

An object of the present invention is to provide and there is amendment, the phytase of the characteristic of advantageous embodiment.The limiting examples of these characteristics is: thermostability, temperature curve, pH curve, specific activity, the performance in animal-feed, protease sensitive and/or glycosylation pattern.

Summary of the invention

The present invention relates to phytase, it has at least 74% identity with SEQ ID NO:2 and at least one position in lower group comprises at least one changes being selected from compared with SEQ ID NO:2: 1, 2, 3, 4, 5, 31, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410 and 411, condition is this phytase is not SEQ IDNO:3, is not SEQ ID NO:4, and is not SEQ ID NO:6.

The invention still further relates to phytase, itself and SEQ ID NO:2 have at least 74% identity and comprise at least one following change: 1H, K, R, 60P, 105E, 106A, G, 155F, 157F, 173P, 175L, 188P, 205P, 215M, 231P, 254Y, 280P, 330D and/or 371P; Condition is this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, is not SEQ ID NO:6, and its variant not being SEQ ID NO:9 and listing in FIG.

The invention still further relates to the DNA of these phytases of coding, their preparation method, and their purposes, such as, purposes in animal-feed and animal feedstuff additive.

Accompanying drawing is sketched

Fig. 1 corresponds to the table 2 of WO 2006/038062 and discloses the multiple variant of citrobacter freundii NCIMB 41247 phytase with SEQ ID NO:9 aminoacid sequence;

Fig. 2 is the comparison of the phytase of SEQ ID NO:2 and 9.

Location number in Fig. 1 is with reference to the numbering of SEQ ID NO:9.Corresponding SEQ ID NO:2 position can obtain (that such as, applies the variant P229S of numbering Fig. 1 of the application is meant to variant P207S) by deducting 22.

Detailed Description Of The Invention

First aspect, the present invention relates to phytase, it has at least 74% identity with SEQ ID NO:2 and at least one position in lower group comprises at least one changes being selected from compared with SEQ ID NO:2: 1, 2, 3, 4, 5, 31, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410 and 411, condition is this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, and is not SEQ ID NO:6.

Identity per-cent is determined as described in save at " phytase polypeptides, identity per-cent " one.

The Position Number mode of location number reference SEQ ID NO:2, as described in save in " Position Number mode ".Position corresponding with these SEQ ID NO:2 location numbers in other phytase as " determining corresponding position number " save described in determine.

Phytase of the present invention is the variant of the phytase of SEQ ID NO:2, and namely they are different from SEQ ID NO:2, changes because it comprises at least one compared with SEQ ID NO:2.

In an embodiment, phytase of the present invention comprises at least one compared with SEQ ID NO:2 at least one position being selected from lower group to be changed: 1, 2, 3, 4, 5, 31, 46, 52, 53, 55, 57, 59, 76, 82, 99, 100, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 137, 141, 161, 162, 164, 167, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 218, 223, 241, 273, 276, 285, 286, 299, 314, 331, 339, 362, 379, 385, 406, 410 and 411.

In another embodiment, phytase of the present invention is not SEQ ID NO:9.

In further embodiment, phytase of the present invention is not the variant of the SEQ ID NO:9 that Fig. 1 lists.

In a preferred embodiment, phytase of the present invention comprises at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, T, 41P, 46C, D, E, 52C, E, 53V, Q, 55D, I, 57Y, 59C, 74A, 76G, 82E, 84Y, 91C, P, 99C, 100C, 104A, 105F, 107D, E, G, 109A, G, 111P, 114H, N, T, 115Q, 116A, E, P, T, Q, 117D, E, K, 118I, L, M, T, 119G, K, R, S, 120K, S, T, Q, 121A, D, M, P, T, V, 122D, 123P, S, 124L, T, V, 136P, 137P, 141C, 154P, 161P, 162C, 164D, E, 167Q, 171T, 176C, 177C, 179G, I, K, N, Q, 180A, E, G, T, 181D, G, I, K, 182H, K, S, Q, 183A, L, P, S, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K, R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L, Q, 276K, R, 281H, 282P, 283P, 284P, 285G, N, R, 286K, Q, 289P, 294T, 299L, 308A, 314G, N, 316D, 324N, 331K, 339D, 351Y, 355P, 362K, R, 379K, R, 385D, 406A, 409D, E, 410D, E and/or 411R, K.

" changing for changing the nomenclature used, as replaced, lacking, inserting herein." one joint in describe in detail.

Phytase of the present invention preferably comprises at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, 46E, 52C, E, 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D, E, G, 109A, 111P, 114T, 115Q, 116AT, 117D, 118T, 119K, R, S, 120S, 121D, P, T, 122D, 123P, 124L, 137P, 141C, 161P, 162C, 164E, 167Q, 179K, 180E, T, 181D, K, 182H, K, Q, 183L, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K, 202N, 218Q, 223E, 241Q, 273L, 276K, R, 285G, R, 286Q, 299L, 314G, N, 331K, 339D, 362K, R, 379K, R, 385D, 406A, 410D, E and/or 411R, K; And/or the amino acid of wherein position 179,180,181,182,183,184,185 and 186 is replaced by KEKHQ, KEKQQ, KEKKV or KTDKL.

In another preferred embodiment, the amino acid of position 179,180,181,182,183,184,185 and 186 is replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL.

The invention still further relates to the identity the phytase comprising at least one following change with SEQ ID NO:2 with at least 74%: 1H, K, R, 60P, 105E, 106A, G, 155F, 157F, 173P, 175L, 188P, 205P, 215M, 231P, 254Y, 280P, 330D and/or 371P; Condition is this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, is not SEQ ID NO:6, and its variant not being SEQ ID NO:9 and listing in FIG.One preferred embodiment mysoinositol six-phosphatase comprise change 1K.In other preferred implementation, phytase comprises following change combination: 280P/282P/283P, 155F/254Y and/or 155F/157F/254Y.

The preferred phytase of the present invention comprises and is selected from a following change: 52C, 141C, 162C, 31C, 52C, 99C, 59C, 100C, 141C/199C, 4P, 5P, 111P, 137P, 161P, 52E, 57Y, 76G, 107D, 107G, 109A, 1*, 1*/2*, 1 ^*/ 2 ^*/ 3 ^*, 121T, 273L, 285G, 286Q, 299L, 362K, 331K/55D, 107E, 46E, 82E, 119R, 119K, 164E, 223E, 276R, 276K, 362R, 379R, 379K, 385D, 410D, 410E, 411R, 411K, 53V, 121D, 167Q, 196Q, 200K, 202N, 218Q, 241Q, 285N, 314N, 314G, 406A, 179K/180E/181K/182H/183Q/184 ^*/ 185 ^*/ 186 ^*,

179K/180E/181K/182Q/183Q/184 ^*/185 ^*/186 ^*，

179K/180E/181K/182K/183V/184 ^*/185 ^*/186 ^*，

179K/180T/181D/182K/183L/184 ^*/185 ^*/186 ^*，111P/241Q，1K，

114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L and

114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L。

Phytase of the present invention can be the variant of any wild-type or variant phytase.In a particular embodiment, it is SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, the variant of the phytase of SEQ ID NO:6, SEQ ID NO:9, or the variant of any one inositol six-phosphatase variants also in FIG listed relevant to SEQ ID NO:9.

Phytase of the present invention can comprise the combination of a change (replacement) or multiple change (replacement) further, and they are selected from the combination of change (replacement) listed in each row of Fig. 1 or change (replacement).

The variant of the phytase of especially preferred SEQ ID NO:2 is following: R339D, N4P, G5P, Q111P, E1 ^*, E1 ^*/ E2 ^*, E1 ^*/ E2 ^*/ Q3 ^*, M273L and N286K; And their arbitrary combination; And SEQ ID NO:3, the variation of 4 and 6.

The especially preferred phytase of the present invention comprises at least one following change: 339D, 4P, 5P, 111P, 1 ^*, 1 ^*/ 2 ^*, 1 ^*/ 2 ^*/ 3 ^*, 273L and/or 286K.

The invention still further relates to and with SEQ ID NO:2, there is at least 74% identity and comprise the phytase of at least one following change:

(i) 141C/199C, 91C/46C, 52C/99C, 31C/176C, 31C/177C, 59C/100C and/or 162C/247C;

(ii) 41P, 91P, 136P, 137P, 154P, 161P, 355P, 111P, 240P, 282P, 283P, 284P, 289P, 4P and/or 5P;

(iii) 52E, 55I, 57Y, 104A/105F, 107D, G, 109A, G, 76G, 84Y, 121T, 362K, 273L, Q, 285G, R, 286K, Q, 294T, 299L, 331K/55D and/or 351Y;

(iv) 1 ^*, 1 ^*/ 2 ^*or 1 ^*/ 2 ^*/ 3 ^*;

V () be K179 wherein, T180, T181, E182, K183, S184, T185 and K186 are replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL;

(vi) 119R, K and/or 411R, K;

(vii) 107E and/or 164E, D;

(viii) 362R, K, 276R, K, 379R, K, 409D, E, 223E, 385D, 46D, E, 410D, E and/or 82E;

(ix) 218Q, 324N, 200R, K, 121D, 196Q, 202N, 406A, 167Q, 53V, Q, 241Q, 314N, G, 239Q and/or 285N;

(x)114H/115Q/116E/117K/118M/119G/120T/121M/122D/123P/124T，

114H/115Q/116Q/117D/118I/119K/120Q/121V/122D/123S/124L，

114H/115Q/116P/117E/118I/119G/120K/121M/122D/123P/124V，

114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L，

114H/115Q/116Q/117D/118I/119K/120Q/121A/122D/123P/124L，

114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L or

114N/115Q/116A/117D/118L/119K/120K/121T/122D/123P/124L；

(xi) 31T, 74A, 171T, 203T, 281H, 316D and/or 308A; And/or

(xii)339D。

Prepare the strategy of variant

By homology modeling (modeling), utilize the structure of intestinal bacteria (E.coli) AppA phytase as template (Protein Data Bank id.:1DKO; Lim etc., Nat.Struct.Biol. (2000), vol.2, pp.108-113 (volume Two, 108-113 page)) build the structure of Bu Shi citric acid bacillus ATCC51113 phytase.

Molecular dynamics (MD) simulation and electrostatic calculations are carried out to this structure.Also identify the disulfide linkage (disulfide bridges) of presumption and the position of proline(Pro), and other sites of the potential importance relevant with various desirable enzyme characteristic.Finally, the glycosylation site (stretching, extension (stretches) of NXT or NXS) of presumption is identified.

Within the framework of modeled structure and analog result, these all proposals are assessed, especially focus on temperature end assessment thermostability.

Corresponding inositol six-phosphatase variants by method preparation known in the art and as tested described in experimental section.

Phytase polypeptides, identity per-cent

Phytase is the polypeptide with phytase activity in this article, namely the hydrolysis of catalysis phytinic acid (salt) (phytase) (phytate (myo-inositol hexakisphosphate)) generates (1) inositol (myo-inositol) and/or (2) its list, two, three, four and/or pentaphosphate and (3) inorganic phosphate.

Term phytinic acid enzyme substrates includes, but are not limited to (i.a.) in this article, phytinic acid and any phytate (salt of phytinic acid), and phosphoric acid salt listed in superincumbent (2).

ENZYME website (http://www.expasy.ch/enzyme/) on network is the information storage that the nomenclature of enzyme is relevant.The suggestion of its Main Basis international bio chemistry and NK of molecular biology association (IUB-MB) (Nomenclature Committee of the International Union of Biochemistryand Molecular Biology), it is described that all kinds (the Bairoch A.The ENZYME database of the enzyme through characterizing providing EC (the enzyme council) number, 2000, Nucleic Acids Res28:304-305).Also can name handbook see the enzyme of NC-IUBMB, 1992.

According to ENZYME website, there will be a known three kinds of dissimilar phytases: so-called 3-Phytase (another name 1-phytase; Phytate 3-phosphohydrolase, EC3.1.3.8), so-called 4-phytase (another name 6-phytase, based on the title that 1L numbering system instead of 1D are numbered, EC 3.1.3.26), and so-called 5-phytase (EC 3.1.3.72).For the present invention, whole three kinds are included in the definition of phytase.

In an embodiment, phytase of the present invention belongs to acid histidine phosphatases family, comprises the phytase of intestinal bacteria pH 2.5 acid phosphatase (gene appA) and fungi as Aspergillus awamori (Aspergillus awamorii) phytase A and B (EC:3.1.3.8) (gene phyA and phyB).Each Histidine acid phosphatase has the sequence similarity in Liang Ge district jointly, and each district is round a conservative histidine residues center.These two Histidines look like the catalyst mechanism that take part in enzyme.First Histidine is positioned at N-terminal portions and forms phosphorus-histidine intermediate, and second is positioned at C-terminal portions and may play a role as protophobe.

In embodiment more specifically, phytase of the present invention has conservative active site motif, i.e. R-H-G-X-R-X-P, wherein X represents any amino acid (see SEQ ID NOs:2, the amino acid/11 6 to 22 of 3,4,6 and the amino acid 38-44 of SEQ ID NO:9).In a preferred embodiment, conservative active site motif is R-H-G-V-R-A-P, and namely amino acid/11 6-22 (with reference to SEQ ID NO:2) is RHGVRAP.

For the present invention, the activity of phytase is determined with FYT unit, and a FYT is that per minute discharges the ortho-phosphoric enzyme amount of 1 micromole inorganic under the following conditions: pH 5.5; Temperature 37 DEG C; Substrate: concentration is the sodium phytate (C of 0.0050mol/l ₆h ₆o ₂₄p ₆na ₁₂).Suitable phytinic acid enzyme assay is FYT and the FTU assay method described in the embodiment a kind of WO00/20569.FTU is for measuring the phytase activity in feed and premixture.Assay method in embodiment 1 (" mensuration of phosphatase activity " or " mensuration of phytase activity ") can also be utilized to determine the activity of phytase.

In an embodiment, phytase of the present invention is separated.Term used herein " separation " refers to that, as determined by SDS-PAGE, at least 20% is pure, and preferably at least 40% is pure, more preferably at least 60% is pure, also more preferably at least 80% pure, most preferably at least 90% is pure, even most preferably at least 95% pure polypeptide.Concrete, preferred polypeptide is " form that (essentially) is pure substantially ", that is, described polypeptide preparation thing is substantially free of other polypeptide raw materials of (essentially free of) combination natural with it (nativelyassociated).Such as, this can by preparing polypeptide to realize with the purification process of the recombination method known or classics.

Dependency between two aminoacid sequences is described by parameter " identity ".For the present invention, the comparison of two aminoacid sequences is determined from the Needle program of EMBOSS software package (http://emboss.org) version 2 .8.0 by utilizing.Needle program performs the overall comparison algorithm (globalalignment algorithm) described in Needleman S.B. and Wunsch, C.D. (1970) J.Mol.Biol.48,443-453.Substitution matrix used is BLOSUM62, and it is 10 that breach opens point penalty (gapopening penalty), and gap extension penalty (gap extension penalty) is 0.5.

Identity degree between the aminoacid sequence (SEQID NO:2) related in aminoacid sequence of the present invention (" invention sequence ") and claim, be the number by exact matching in two sequence alignments, calculate divided by one the shortest in the length of " invention sequence " or the length of SEQ ID NO:2.Result represents with percentage identities.

When exact matching (representing this situation with " | " in comparison example hereafter) occurs when the same position of overlap has identical amino-acid residue for " invention sequence " and SEQ ID NO:2.The length of sequence is the total number of atnino acid (such as the length of the amino acid/11-411 of SEQ ID NO:2 is 411) in sequence.

Embodiment 13 is comparison examples of the phytase of SEQ ID NO:2 and the phytase of SEQ ID NO:9, this example illustrates the identity per-cent how calculating these two main chains.

In the comparison example of another pure supposition below, overlap is the aminoacid sequence " HTWGER-NL " of sequence 1; Or the aminoacid sequence of sequence 2 " HGWGEDANL ".Breach represents with "-" in this example.

Assuming that comparison example:

Sequence 1:ACMSHTWGER-NL

| ||| ||

Sequence 2:HGWGEDANLAMNPS

In an embodiment, the aminoacid sequence of polypeptide with or measure as follows relative to the identity per-cent of SEQ ID NO:2: i) use Needle program, use BLOSUM62 substitution matrix, breach opens point penalty 10 and gap extension penalty 0.5, comparison two aminoacid sequences; Ii) count ratio centering exact matching number; Iii) by the length of the most short data records in two aminoacid sequences except exact matching number, iv) by iii) results conversion that removes becomes per-cent.

In superincumbent supposition example, exact matching number is 6, and in two aminoacid sequences, the length of most short data records is 12; Therefore identity per-cent is 50%.

In the embodiment of phytase of the present invention, be at least 75%, 76%, 77%, 78% with the identity degree of SEQ ID NO:2,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.In embodiment more specifically, identity degree is at least 98.0%, 98.2%, 98.4%, 98.6%, 98.8%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9%.In alternate embodiments, identity degree is at least 70%, 71%, 72% or at least 73%.

In further embodiment, phytase of the present invention is no more than 1,2,3,4,5,6,7,8 as having compared with SEQ IDNO:2, and 9 or be no more than 10 changes; 11 are no more than, 12,13,14,15,16,17,18,19 or be no more than 20 changes as having compared with SEQ ID NO:2; 21 are no more than, 22,23,24,25,26,27,28,29 or be no more than 30 changes as having compared with SEQ ID NO:2; 31 are no more than, 32,33,34,35,36,37,38,39 or be no more than 40 changes as having compared with SEQ ID NO:2; 41 are no more than, 42,43,44,45,46,47,48,49 or be no more than 50 changes as having compared with SEQ ID NO:2; 51 are no more than, 52,53,54,55,56,57,58,59 or be no more than 60 changes as having compared with SEQ ID NO:2; 61 are no more than, 62,63,64,65,66,67,68,69 or be no more than 70 changes as having compared with SEQ ID NO:2; 71 are no more than, 72,73,74,75,76,77,78,79 or be no more than 80 changes as having compared with SEQ ID NO:2; 81 are no more than, 82,83,84,85,86,87,88,89 or be no more than 90 changes as having compared with SEQ IDNO:2; 91 are no more than, 92,93,94,95,96,97,98,99 or be no more than 100 changes as having compared with SEQ ID NO:2; 101 are no more than, 102,103,104,105,106,107,108,109 or be no more than 110 changes as having compared with SEQ ID NO:2; 111 are no more than, 112,113,114,115,116,117,118,119 or be no more than 120 changes as having compared with SEQ ID NOP:2; Be no more than 121,122,123 or 124 as having compared with SEQ ID NO:2 to change.

Position Number

The nomenclature of definition amino acid position used herein is the phytinic acid enzyme amino acid sequence based on being derived from Bu Shi citric acid bacillus ATCC51113, and its mature sequence provides (amino acid/11-411 of SEQ ID NO:2) as SEQ IDNO:2 in sequence table.Therefore, in this article, the basis for numbered positions is SEQ ID NO:2, stops from E1 to E411.

When used herein term " maturation " partly (or sequence) refer to by the polypeptide portion of the polynucleotide containing this polypeptide of coding as the emiocytosis of its part heredity parts (genetic equipment).In other words, mature polypeptide part refers to is cutting away signal peptide part, and the polypeptide portion that propeptide parts (if there is any propeptide parts) is later remaining.Signal peptide part can be undertaken predicting (such as SignalP) by program software known in the art.Be included in sequence table of the present invention using the SEQ ID NO:2 signal peptide part of expection as SEQ ID NO:8, SEQ ID NO:8 is encoded by SEQ ID NO:7.SEQ ID NO:2 is the maturing part of expection.Normally, first amino acid of the maturing part of enzyme can by determining the N-end sequencing of purifying enzyme.The inevitable existence owing to propetide of any difference then between signal peptide part and maturing part.

Change, as replaced, disappearance, inserts

The variant of phytase can comprise various types of change relative to template (namely with reference to or the aminoacid sequence that compares as SEQ IDNO:2): amino acid can with another amino acid replace; An amino acid can be lacked; An amino acid can be inserted; And the arbitrary combination of these changes of arbitrary number.Term " insertion " is intended to the extension also comprising N-end and/or C-end in this article.

As follows to the general nomenclature that change is separately used herein: XDcY, wherein " X " and " Y " represents the amino acid code of single-letter independently of one another, or " * " (amino acid whose disappearance), " D " represents numeral, and " c " represents lexicographic order counting (a, b, c etc.), it is present in insertion.With table 1 below as a reference, which depict the example supposed completely that this nomenclature is applied to various types of change.

chart 1

Type	Describe	Example
			Replace	Amino acid in position c vacancy Y=variant in amino acid D=template in X=template	G80A 80AALNNSIG VLGVAPSAELYAVKVLGASGSG|||||||：||||||||||||||||||||||AALNNSIA VLGVAPSAELYAVKVLGASGSG
Insert	In X=＂ * ＂ D=template, position c=＂ a ＂ before insertion represents first insertion in this position, and ＂ b ＂ represents next, etc.	＊80aT ＊80bY ＊85aS 80 85AALNNSIG..VLGVA.PSAELYAVKVLGASG|||||||| ||||| |||||||||||||||AALNNSIGTYVLGVASPSAELYAVKVLGASG
			Delete	Position c vacancy Y=＂ * ＂ in amino acid D=template in X=template	V81 ＊ 80AALNNSIGVLGVAPSAELYAVKVLGASGSG|||||||| |||||||||||||||||||||AALNNSIG.LGVAPSAELYAVKVLGASGSG
N-end extends	In the insertion of position ＂ 0 ＂	＊0aA ＊0bT ＊0cG 1...AQSVPWGISRVQ ||||||||||||ATGAQSVPWGISRVQ
			C-end extends	Insertion after-terminal amino acid	＊275aS ＊275bT 270 275ATSLGSTNLYGSGLVNAEAATR..||||||||||||||||||||||ATSLGSTNLYGSGLVNAEAATRST

As explained above, positional number (" D ") counts from first amino-acid residue of SEQ ID NO:2.

Several change "/" (oblique line) in same sequence are separated, such as, identify " 1 ^*/ 2 ^*/ 3 ^*" represent at location number 1,2 withthe amino acid of 3 all lacks, and mark " 104A/105F " represents that the amino acid of location number 104 is replaced by A, and the amino acid of location number 105 is replaced by F.

Selectable change ", " (comma) is separated, and such as, mark " 119R, K " represents that the amino acid on position 119 is replaced by R or K.

Herein in other possibilities too numerous to enumerate various, comma () used represents their effects grammatically usually, namely normally and/or.Such as, in list " 53V, Q, 121D and/or 167Q ", first comma represents in two and selects (a V orq), two commas and are below interpreted as and/orselection: 53V or Q, and/or 121D, and/or 167Q.

In this article, " at least one " (such as changing) represents one or more, such as 1,2,3,4,5,6,7,8,9 or 10 change; Or 12,14,15,16,18,20,22,24,25,28 or 30 changes; Etc., until maximum variable number 125,130,140,150,160,170,180,190 or 200.But, inositol six-phosphatase variants of the present invention also must with SEQID NO:2 have at least 74% identity, this per-cent is determined as mentioned above.

Do not carry out any replacement of specifying or extension and refer to replacing with what or extending and insert any natural, or non-natural amino acid, except occupying the amino acid of this position in template.

Embodiment 13 provides further explain explanation for how applying this nomenclature.

Identify corresponding location number

As above explain, using the ripe phytase of Bu Shi citric acid bacillus ATCC 51113 (SEQ ID NO:2) as the standard of Position Number, is therefore also the standard of nomenclature.

For other phytases, especially inositol six-phosphatase variants of the present invention, method comparison two sequences passing through to describe in detail in " phytase polypeptides, identity per-cent " by name one saves corresponding to the position of position D in SEQ ID NO:2 find.From comparison, position corresponding with the position D of SEQ ID NO:2 in sequence of the present invention can clearly and expressly identify out (those two positions mutually at the other side top in comparison).

Embodiment 13 is comparison examples of the phytase of SEQ ID NO:2 and the phytase of SEQ ID NO:9, and how the corresponding position in these two main chains of this examples show identifies.

In some other example supposed completely is also included within, these example source are from table 1 above, and table 1 comprises the comparison of a collection of two sequences at the 3rd row:

The 3rd lattice of the first row with reference to table 1: sequence is above template, below be variant.Location number 80 refers to the amino-acid residue G in template.Amino acid A occupies the corresponding position in variant.Therefore, this replacement is expressed as G80A.

The 3rd lattice referring now to the second row of table 1: sequence above or template and be variant below.Location number 80 is still the amino-acid residue G in finger print plate.Variant has two insertions, that is, TY, after the G80 of template before V81.And T and Y certainly have they oneself in Variant amino acid sequences the location number of " reality ", with regard to present case, we are always with reference to the location number in template, so be called by T and Y respectively at location number 80a and 80b.

Finally, with reference to last column the 3rd lattice of table 1: location number 275 refers to last amino acid of template.The extension ST of C-end is called at location number 275a and 275b, although they also have the location number in Variant amino acid sequences of their oneself " reality " certainly equally.

The characteristic of amendment, with reference to phytase

In an embodiment, phytase of the present invention has amendment, the characteristic preferably improved.Term " amendment " means compared with another kind of phytase with " improvement ".These other for reference to or the phytase example that compares be: SEQ ID NO:3 and/or SEQ IDNO:4.Further example with reference to phytase can be SEQ ID NO:2 and/or SEQID NO:6.Can be SEQ ID NO:9 with reference to the further example of phytase, and its variant disclosed in FIG.

Amendment, the non-limiting example being preferably the characteristic improved is as follows: thermostability, pH curve, specific activity, the performance in animal-feed, protease sensitive and/or glycosylation pattern.Phytase of the present invention can also have amendment, advantageous embodiment, temperature curve, and/or it can mix the change of potential proteolytic cleavage site.

thermostability

Thermostability, or temperature stability can be measure as described in " mensuration of temperature stability " part as embodiment 1 exercise question.Therefore, in a preferred embodiment, phytase of the present invention has the remaining activity higher than reference phytase, wherein remaining activity measures as follows: fermentation supernatant is divided into two portions, incubation 30 minutes at the temperature that a part improves in expection, another part 5 DEG C of incubations 30 minutes, for the activity of p-nitrophenyl phosphoric acid both then measuring in 37 DEG C and pH 5.5, by the activity of the sample of the incubation at elevated temperatures activity divided by the same sample at 5 DEG C of incubations.The temperature of preferred raising is 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C or 85 DEG C.If necessary, the sample containing enzyme can dilute in 0.1M NaAc pH 5.5.The remaining activity of phytase of the present invention is preferably with reference to phytase remaining activity at least 105%, or at least 110%, 120%, 130%, 140%, 150%.In further embodiment, the remaining activity of phytase of the present invention is with reference to phytase remaining activity at least 200%, or at least 250%, 300%, 400% or at least 500%.In further embodiment, the remaining activity of phytase of the present invention is at least 2 times with reference to phytase remaining activity, 3 times, 4 times, 5 times, 6 times, 7 times, 10 times, 15 times, 20 times or at least 25 times.

Thermostability can also measure by described in embodiment 5.Therefore, in a preferred embodiment, phytase of the present invention has the remaining activity higher than reference phytase, wherein remaining activity measures as follows: fermentation supernatant is divided into two portions, a part was 50 DEG C of incubations 30 minutes, another part 5 DEG C of incubations 30 minutes, for the activity of p-nitrophenyl phosphoric acid both then measuring at 37 DEG C of pH 5.5, by the activity of the sample of the incubation at elevated temperatures activity divided by the same sample at 5 DEG C of incubations.If needed, the sample containing enzyme can dilute in 0.1M NaAc pH 5.5.The remaining activity of phytase of the present invention is preferably at least 2 times of the reference phytase remaining activity of SEQ ID NO:3,3 times, 4 times, 5 times, 6 times, 7 times, 10 times, 15 times, 20 times or at least 25 times.The remaining activity of phytase of the present invention is preferably at least 105% of the reference phytase remaining activity of SEQ ID NO:2, or at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or at least 200%.Following replacement is especially preferred, because they make thermostability improve to some extent (see table 3) compared with the phytase of SEQ ID NO:3 and the phytase of SEQ ID NO:2: 4P, 5P, 111P, 1 ^*, 1 ^*/ 2 ^*, 1 ^*/ 2 ^*/ 3 ^*, 273L and/or 286Q.

Thermostability can also measure as described in example 8 above.Therefore, one preferred embodiment in, phytase of the present invention has the remaining activity higher than reference phytase, wherein remaining activity measures as follows: fermentation supernatant is divided into two portions, a part was 60 DEG C of incubations 30 minutes, another part 5 DEG C of incubations 30 minutes, for the activity of p-nitrophenyl phosphoric acid both then measuring at 37 DEG C of pH 5.5, by the activity of the sample of the incubation at elevated temperatures activity divided by the sample at 5 DEG C of incubations.If needed, the sample containing enzyme can dilute in the 0.1M NaAcpH 5.5 optionally comprising 0.005%Tween-20.Phytase of the present invention and reference phytase can be expressed in subtilis (Bacillus subtilis) host strain.Host strain can 100ml PS1 substratum (100g/L sucrose, 40g/L soybean flakes (Soy flakes), 10g/LNa in 500ml shaking flask ₂hPO ₄.12H ₂o, 0.1ml/L Dowfax 63N10 (Dow)) in 300rpm 30 DEG C cultivate four days.The remaining activity of phytase of the present invention is preferably at least 32% of the remaining activity of the reference phytase of SEQ ID NO:2, or at least 34%, 36%, 38% or at least 40%.More preferably, the remaining activity of phytase of the present invention is at least 50% of the remaining activity of the reference phytase of SEQ ID NO:2, or at least 60%, 70%, 80%, 90% or at least 100%.Also more preferably the remaining activity of phytase of the present invention is at least 120%, 140%, 160% of the remaining activity of the reference phytase of SEQ ID NO:2,180% or at least 200%.Most preferably, the remaining activity of phytase of the present invention is at least 2 times of the remaining activity of the reference phytase of SEQ ID NO:2, or at least 3 times, 4 times or at least 5 times.Following replacement especially preferred (see table 5):

(i)409E，136P；

(ii)411K，331K/55D，167Q，179K/180T/181D/182K/183L/184 ^*/185 ^*/186 ^*，107E；

(iii)196Q，276R，285G，299L，200K；

(iv)119R，121D，107D，179K/180E/181K/182H/183Q/184 ^*/185 ^*/186 ^*；

(v)314N，161P，410D，141C，179K/180E/181K/182Q/183Q/184 ^*/185 ^*/186 ^*，285N；

(vi)164E，411R，52C，137P，314G；

(vii)1K，1 ^*/2 ^*/3 ^*，121T，406A，82E，109A；

(iix)5P，57Y，379R，1 ^*/2 ^*；

(ix)410E，1 ^*，119K，52E；

(x)4P，362K，202N，276K，385D；

(xi)111P/241Q，162C，179K/180E/181K/182K/183V/184 ^*/185 ^*/186 ^*，241Q；

(xii)223E，286Q，107G，114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L，379K，273L；

(xiii)31C，53V，59C/100C；

(xiv)46E，111P，114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L，

76G，362R；

(xv)141C/199C，52C/99C。

Thermostability can also measure as described in example 9 above, namely applies the denaturation temperature Td that dsc measurement method measures the phytinic acid zymoprotein of purifying.Td has indicative for the thermostability of albumen: Td is higher, and then thermostability is higher.Therefore, in a preferred embodiment, phytase of the present invention has the Td higher than reference phytase, wherein for purifying phytase sample use dsc from 20-90 DEG C with the scan rate of 90 DEG C/h at 20mM sodium acetate buffer, measuring Td in pH 4.0 (preferably uses purity to be at least 95%, measured by SDS-PAGE), in 20mM sodium acetate pH4.0, (step of preferably then spending the night after the step of 2-3 hour) was dialysed before this measures, carry out 0.45um filtration again and with dialysis buffer liquid protein concentration be diluted to being equivalent to about 2 absorbance unit (A ₂₈₀).In a preferred embodiment, the Td of phytase of the present invention is higher than the Td of the phytase of SEQID NO:4, is more preferably its at least 101%, or at least 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109% or it at least 110%.Also more preferably, the Td of phytase of the present invention is at least 120%, 130%, 140% of the phytase Td of SEQ ID NO:4,150%, 160%, 170%, 180% or at least 190%.Following replacement especially preferred (see table 6): 362K, 362R, 111P and/or 273L.In further specific embodiment, as used the dsc (DSC) described in embodiment 2 (namely at 20mM sodium acetate, in pH 4.0) measure, heat-staple phytase of the present invention has the melting temperature(Tm) Tm (or denaturation temperature, Td) of at least 50 DEG C.In further embodiment, Tm is at least 51,52,53,54,55,56,57,58,59,60,61,62,62.5,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or at least 100 DEG C.Dsc measurement method can also be carried out as described in embodiment 1 (" dsc measurement method "), or carries out as described in embodiment 2 (" measuring thermostability by DSC ").

Thermostability can also measure as described in example 12 above.Therefore, one preferred embodiment in, under 70 DEG C and pH 4.0 condition, incubation is after 60 minutes, phytase of the present invention has the remaining activity of improvement compared with the reference phytase according to the same manner process, and for often kind of phytase, described remaining activity is that the activity existed relative to (at the 0th minute) before incubation calculates.Remaining activity preferred pin is measured when pH 5.5 and 37 DEG C sodium phytate.Incubation preferably at 0.1M sodium acetate, in pH 4.0.Phytase is preferably purifying, is more preferably purified to the purity being determined as at least 95% by SDS-PAGE.Preferred phytase activity assay buffer is 0.25M sodium acetate pH 5.5.Application present method, the remaining activity of phytase of the present invention preferably with reference to phytase remaining activity at least 105%, more preferably at least 110%, 115%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or at least 200%.Or the remaining activity of the activity relative to the 0th minute is preferably at least 31% or at least 32%.The following replacement providing the thermostability of improvement is preferred (see table 9): 273L, 46E, 362R and/or 53V.

In an embodiment, it is more stable that inositol six-phosphatase variants of the present invention and reference phytase compare heat, and wherein thermostability measures (Case-based Reasoning 1,5 by any test of applying in above-mentioned four kinds of tests, 8,9 or 12).

In a specific embodiment, the following variant of the phytase of expection SEQ ID NO:2 has the thermostability (in each grouping, with preferred order) of improvement:

(i)K141C/V199C，Q91C/W46C，G52C/A99C，N31C/E176C，N31C/T177C，G59C/F100C，S162C/S247C；

(ii)D41P，Q91P，N136P，T137P，L154P，S161P，T355P，Q111P，K240P，G282P，T283P，T284P，G289P，N4P，G5P；

(iii)G52E，V55I，E57Y，L104A/A105F，K107D，G，Q109A，G，T76G，A84Y，N121T，I362K，M273L，Q，E285G，R，N286Q，V294T，I299L，E331K/V55D，F351Y；

(iv)E1 ^*，E1 ^*/E2 ^*，E1 ^*/E2 ^*/Q3 ^*；

V () replaces the ring comprised between C178 and C187 with shorter ring, described shorter ring is selected from such as, QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV, KTDKL;

(vi)E119R，K，E411R，K；

(vii)K107E，R164E，D；

(iix)I362R，K，T276R，K，I379R，K，V409D，E，Q223E，N385D，W46D，E，T410D，E，Q82E。

(ix) with being selected from such as, HQEKMGTMDPT, HQQDIKQVDSL, HQPEIGKMDPV, TQADTSSPDPL, HQQDIKQADPL, the ring of TQTDTSSPDPL, NQADLKKTDPL replaces the ring (YQKDEEKNDPL) in the face of avtive spot between residue 114 and 124;

(x)R339D。

temperature curve

Phytase of the present invention can be determined as described in example 10 above with the temperature whether compared with phytase with amendment.Therefore, phytase of the present invention and with reference to the temperature curve compared with phytase with amendment in an embodiment, when wherein temperature curve being determined as (with 10 DEG C for one-level) pH 5.5 within the scope of 20-90 DEG C phytase for the activity of sodium phytate relative to the function of temperature.Preferred damping fluid is at 0.25M sodium acetate buffer, in pH 5.5.The activity of each temperature is preferably expressed as relative to the relative reactivity (representing with %) after the value stdn of optimum temps.Optimum temps is temperature when activity is the highest in the temperature (namely be those temperature of once crossing over 10 DEG C) of test.

In a preferred embodiment, phytase of the present invention has at least 18% 70 DEG C time, or at least 19%, 20%, 21%, 22%, 23%, 24%, or the relative reactivity of at least 25%.As above explain, this is relative to the activity when optimum temps.More preferably, phytase of the present invention has at least 26%, 27%, 28%, 29%, 30%, 31% 70 DEG C time, or the relative reactivity of at least 32%.The preferred replacement of the temperature curve of amendment (to have the form of higher relative reactivity at 70 DEG C) is provided to be (see table 7): 57Y, 76G, 107G, 273L, 362K, 46E, 362R, 53V and/or 241Q.Their relative reactivities 70 DEG C time and SEQ ID NO:3 with 4 reference phytase compare higher, and (57Y, 76G in some cases, 107G, 273L, 362K, 362R and/or 53V) be also like this compared with the reference phytase of SEQ ID NO:2.

pH curve

Phytase of the present invention can as measured with the pH curve whether compared with phytase with amendment described in embodiment 11.Therefore, phytase of the present invention and with reference to the pH curve compared with phytase with amendment in an embodiment, to be wherein defined as 37 DEG C time in pH 2.0 to 7.5 scope (using 0.5 pH unit for one-level) phytase activity for sodium phytate as the function of pH by pH curve.Preferred damping fluid is 50mM glycine, the mixed solution (cocktail) of 50mM acetic acid and 50mM Bis-Tris.Another preferred damping fluid is 0.25M sodium acetate.The relative reactivity (representing with %) after relative to the value stdn at Optimal pH is preferably expressed as in the activity of each pH.

The example of the pH curve of amendment is that pH curve (relative reactivity is as the function of pH) offsets to higher or lower pH.With SEQ ID NO:2, the reference phytase of 3 or 4 is compared, and provides the preferred replacement to higher pH skew 0.5pH unit to be (see table 8): 46E and/or 218Q.

Other examples of the pH curve of amendment are that wherein Optimal pH changes upward or downwards.With SEQID NO:2,3 or 4 compare, and provide the preferred replacement of lower Optimal pH to be (see table 8): 46E, 121D and/or 200K.With SEQ ID NO:2,3 or 4 compare, and provide the preferred replacement of higher Optimal pH to be (see table 8): 218Q and/or 241Q.

The pH curve of amendment can also as described in embodiment 1 (" the pH curve of amendment: the mensuration of pH 3.5/5.5 specific activity ") mensuration, that is, by comparing the phosphatase activity pH 3.5 and 5.5 time.Alternatively, activity during pH 3.5 can with pH 4.0,4.5 or 5.0 time expression activitiy.In further alternative embodiment, that compares is phytase activity but not phosphatase activity.

In an embodiment, phytase of the present invention and the pH curve compared with phytase with amendment.More specifically, pH curve is revised within the scope of pH3.5-5.5.Further more specifically, the activity pH 4.0,4.5,5.0 and/or 5.5 time is at least 50%, 55%, 60%, 65%, 70%, 75%, 80% of the activity when Optimal pH (pH 3.5), the level of 85%, 90% or at least 95%.

The pH curve of polypeptide, and Optimal pH can be measured by this polypeptide of incubation under various pH value, described incubation application determines the substrate of concentration in advance and carries out under fixing heated culture temperature.PH curve is the diagram of phytase activity relative to pH, and Optimal pH is determined from this pH curve.In an embodiment, apply Phosphoric acid esterase or the phytinic acid enzyme assay of embodiment 1, such as substrate is 5mM sodium phytate, temperature of reaction 37 DEG C, and measure in each pH value active, such as pH 2-12, replace pH 5.5 acetate buffer with suitable damping fluid.The example of suitable damping fluid is: 0.1M glycine/HCl (pH 2.0-3.5), 0.1M NaAc/Ac (pH 4.0-5.0), 0.1MBis-Tris/HCl (pH 5.5-6.5), 0.1M Tris/HCl (pH 7.0).Other examples of damping fluid are: the 100mM succsinic acid being adjusted to pH value 2.0,2.5,3.0,3.5,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0 and 12.0 with HCl or NaOH, 100mM HEPES, 100mM CHES, 100mMCABS.

In a particular embodiment, the following variant of the phytase of expection SEQ ID NO:2 has the pH curve (in each grouping, with preferred order) of amendment:

(i)E218Q，D324N，T200R，K，N121D，E196Q，D202N，E406A，E167Q，E53V，Q，E241Q，D314N，G，E239Q，E285N；

(ii) with being selected from such as HQEKMGTMDPT, HQQDIKQVDSL, HQPEIGKMDPV, TQADTSSPDPL, the ring of HQQDIKQADPL, TQTDTSSPDPL, NQADLKKTDPL replaces the ring (YQKDEEKNDPL) in the face of avtive spot between residue 114 and 124.

specific activity

In one embodiment, phytase of the present invention is relative to the specific activity with reference to phytase with improvement.More specifically, the specific activity of phytase of the present invention be with respect to same steps measure reference phytase specific activity at least 105%.In further embodiment, relative specific activity is at least 110,115,120,125,130,140,145,150,160,170,180,190,200,220,240,260,280,300,350 or even 400%, still with respect to the specific activity of the reference phytase of same steps mensuration.

In alternative situation, the specific activity that term is high refers to the specific activity of at least 200FYT/mg zymoprotein (EP).In a specific embodiment, specific activity is at least 300,400,500,600,700,800,900,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,2000,2100,2200,2300,2400,2500,2600,2700,2800,2900 or 3000FYT/mgEP.

Specific activity is measured for highly purified sample (sds page should show only have a kind of composition to exist).The concentration of zymoprotein can be measured by amino acid analysis, with the phytase activity of FYT unit representation by measuring as described in example 1 above.Specific activity is a kind of feature of discussed concrete inositol six-phosphatase variants, and it can be used as the phytase activity measured with following unit to calculate, i.e. the FYT units of every milligram of inositol six-phosphatase variants zymoprotein.Further details see Example 7.

In a specific embodiment, the following variant of the phytase of expection SEQ ID NO:2 has the specific activity of amendment, wherein with preferred order, by between residue 114 and 124 in the face of the ring (YQKDEEKNDPL) of avtive spot replaces with being selected from following ring, such as, HQEKMGTMDPT, HQQDIKQVDSL, HQPEIGKMDPV, TQADTSSPDPL, HQQDIKQADPL, TQTDTSSPDPL, NQADLKKTDPL.

performance in animal-feed

In a particular implementation, phytase of the present invention has the performance of improvement in animal-feed compared with reference phytase.Performance in animal-feed can be measured by the external model in embodiment 6.Therefore, phytase of the present invention has the performance of improvement in animal-feed in a preferred embodiment, wherein said performance measures in model in vitro, by preparing the Feed Sample be made up of 30% soyflour (soybean meal) and 70% Semen Maydis powder (maize meal), add CaCl to this Feed Sample ₂to the concentration of every kilogram of feed 5 grams of calcium; Under 40 DEG C and pH 3.0 condition preincubation they 30 minutes, then add stomach en-(3000U/g feed) and phytase; Preincubation sample 60 minutes then under pH 4.0 condition 30 minutes under 40 DEG C and pH 3.0 condition; Termination reaction; 40 DEG C of incubations 2 hours by adding HCl to final concentration 0.5M, then carry out a freeze-thaw cycle and within 1 hour, extract phytinic acid and phosphoinositide at 40 DEG C of incubations; Phytinic acid and phosphoinositide is separated by high-effect ionic chromatography (high performance ion chromatography); Measure the amount of remaining phytinic acid phosphorus (IP6-P); Calculate through phytase treatment and the difference (this difference be degraded IP6-P) of remaining IP6-P between the blank sample without phytase treatment; Relative to the IP6-P of degraded with reference to phytase (such as there is the phytase of SEQ ID NO:3 and 4) to represent the IP6-P of the degraded of phytase of the present invention.

Certain phytase of the present invention is with identical amount with reference phytase, is preferably based on phytase activity unit (FYT) and carrys out administration.Preferred dosage is 125FYT/ kilogram of feed.Another preferred dosage is 250FYT/ kilogram of feed.Phytase can with the phytase form of purifying, or with the form administration of fermentation supernatant.The phytase of purifying preferably has the purity of at least 95%, as measured by SDS-PAGE.

In a preferred embodiment, the IP6-P value of the degraded of purifying phytase of the present invention, relative to the IP6-P value of the degraded with reference to phytase, is at least 101%, or at least 102%, 103%, 104%, 105%, 110%, 115%, or at least 120%.In further preferred implementation, the IP6-P value of the degraded of purifying phytase of the present invention, relative to the IP6-P value of the degraded with reference to phytase, is at least 125%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or at least 200%.Preferably, the IP6-P value of the degraded of phytase of the present invention, relative to the IP6-P value of the degraded of SEQ ID NO:2 phytase, is at least 105%, 110%, 113%, 115%, 120%, 125%, or at least 130%.

Compared with the phytase of SEQ ID NO:3, the performance (see table 4A) in that following replacement provides improvement or outstanding at least equally animal-feed in vitro: 4P, 5P, 111P, 1 ^*, 1 ^*/ 2 ^*, 1 ^*/ 2 ^*/ 3 ^*, 273L, 286Q.

Compared with the phytase of SEQ ID NO:3, performance (see table 4A) in that following replacement also provides improvement or outstanding at least equally animal-feed in vitro: 57Y, 76G, 107G, 362K, 362R, 121D, 196Q, 200K, 202N, 314N, 406A and 114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L.

The replacement be more preferably about animal-feed performance is: 57Y, 76G, 362K, 362R, 121D, 196Q, 200K, 202N and 406A.

The relative performance of phytase of the present invention can also calculate as the per-cent by the phosphorus with reference to phytinic acid enzyme r e lease.

In further embodiment, the relative performance of phytase of the present invention can calculate relative to the per-cent by the phosphorus amount with reference to phytinic acid enzyme r e lease as the phosphorus by phytinic acid enzyme r e lease of the present invention.

In further embodiment, the relative performance of phytase of the present invention is at least 105%, preferably at least 110,120,130,140,150,160,170,180,190, or at least 200%.

the protease sensitive reduced

In an embodiment, phytase of the present invention has the protease sensitive of reduction.More specifically, it has the susceptibility of reduction to Kex2 proteolytic enzyme, means and occurs to be reduced by the trend of the cutting of this proteolytic enzyme.

Variant 339D, preferred R339D are examples of the phytase of the present invention of the protease sensitive with reduction.

glycosylation pattern

Glycosylation is the phenomenon only observed during expressing protein in eukaryotic cell is as fungi and transgenic plant, but does not observe in prokaryotic cell prokaryocyte is as bacterium.Have various types of glycosylation, but maximally related be in this article N-glycosylation, namely l-asparagine connect glycosylation, wherein initial in l-asparagine from N-acetyl-glucosamine attaching molecules, make sugar attach to albumen.Have been found that N-glycosylation only betides the l-asparagine of the part being following tripeptides in the sequence: N-X-T or N-X-S, wherein X represents any amino acid.

Surprisingly, when the phytase of SEQ ID NO:2 is expressed in fungi (yeast) pichia pastoris phaff (Pichia pastoris), with the ratio of Expression temporal in subtilis, find lower thermostability, seen embodiment 2.

This discovery causes proposal of the present invention, namely by changing potential glycosylation site, can improve the thermostability of the phytase of expressing in fungi.

Therefore the invention still further relates to the glycosylation pattern with amendment, the inositol six-phosphatase variants of the N-glycosylation site of preferred modification.Expect when expressing in fungi, the glycosylation of amendment can give the thermostability of inositol six-phosphatase variants improvement.

The example of phytase is bacterial phytases, such as Gram-negative (Gram-negative) phytase, as intestinal bacteria (E.coli) and Citrobacter (Citrobacter) phytase and variant thereof, comprise phytase of the present invention and this paper SEQ ID NO:2, SEQ ID NO:3, the phytase of SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:9.The example of the expressive host of fungi is Pichia (Pichia), yeast belong (Saccharomyces) and Aspergillus (Aspergillus) bacterial classification.

In a particular embodiment, following phytase of the present invention (such as the variant of SEQ ID NO:2) expects the glycosylation pattern with amendment, with preferred order: N31T, N74A, N171T, N203T, N281H, N316D, N308A.Following is replace N-X-T pattern formula: N31T, N74A, N281H.Following is replace N-X-S pattern formula: N171T, N203T, N308A, N316D.

hypoallergenic variant

In an embodiment, phytase of the present invention (also) is hypoallergenic variant, plan when and animal, comprise people when contacting, the immunne response of reduction can be caused.Term immunne response should be understood to any reaction produced by the immunity system of the animal of contact inositol six-phosphatase variants.The immunne response of a type is allergic response, and it causes increasing of IgE level in the animal body that is touched.Hypoallergenic variant can use technology known in the art to prepare.The epi-position of the inositol six-phosphatase variants that such as inositol six-phosphatase variants can relate to polymeric groups masked segment (polymer moieties shielding portions) or immunne response is combined.The iii vitro chemical coupling of polymkeric substance to inositol six-phosphatase variants can be related to, such as, as described in WO 96/17929, WO 98/30682, WO98/35026 and/or WO 99/00489 with the combination of polymkeric substance.In conjunction with additionally or selectively relating to polymkeric substance and inositol six-phosphatase variants coupling in vivo.This combination can be realized by following method: the nucleotide sequence of genetically engineered process encoding inositol six-phosphatase variants, the consensus sequence of other glycosylation sites of coding is inserted in inositol six-phosphatase variants, with can make to express inositol six-phosphatase variants in the glycosylated host of inositol six-phosphatase variants, see such as WO 00/26354.There is provided the another kind of approach of hypoallergenic variant to be with the nucleotide sequence of genetically engineered process encoding inositol six-phosphatase variants thus cause inositol six-phosphatase variants from oligomerization, cause the epi-position of described other inositol six-phosphatase variants monomers of inositol six-phosphatase variants monomer maskable and reduce the antigenicity of oligomer thus.These products and being prepared in such as WO 96/16177 of they describe.Can by the phage display of various method as described in WO 00/26230 and WO 01/83559, or the random device described in EP 561907 identifies the epi-position participating in immunne response.Once identify epi-position, by known gene manipulation techniques if directed mutagenesis is (see such as WO 00/26230, WO 00/26354 and/or WO 00/22103) change it aminoacid sequence with the immune property of mutagenic inositol six-phosphatase variants, and/or can in the combination enough carrying out polymkeric substance close to epi-position place, to make epi-position described in polymer shield.

Nucleotide sequence and construct

The invention still further relates to the nucleotide sequence of the nucleotide sequence comprising code book invention inositol six-phosphatase variants.

Term " nucleotide sequence of separation " refers to substantially (essentially) not containing the nucleotide sequence of other nucleotide sequences, such as measured by agarose gel electrophoresis, pure at least about 20%, preferably pure at least about 40%, more preferably pure at least about 60%, even more preferably pure at least about 80%, most preferably pure at least about 90%.Such as, the nucleotide sequence of separation can be obtained by Standard cloning methods used in genetically engineered, and being repositioned onto it to make nucleotide sequence from its natural place will carry out the different positions copied.Cloning process can relate to excision and the expectation nucleic acid fragment being separated the nucleotide sequence comprising coded polypeptide, by fragment insertion vector molecule, and recombinant vectors is incorporated to host cell, will copies multiple copy or the clone of this nucleotide sequence in this host cell.Nucleotide sequence can be genomic, and cDNA, RNA are semisynthetic, the source of synthesis, or their arbitrary combination.

Nucleotide sequence of the present invention can be prepared in template phytase encoding sequence or its subsequence by introducing at least one sudden change, the nucleic acid sequence encoding variant phytase wherein suddenlyd change.Sudden change is introduced in nucleotide sequence, to replace another nucleic acid with a nucleic acid, this can be completed by any known method in this area, such as pass through directed mutagenesis, pass through random mutagenesis, or (doped) by mixing, (spiked) of wedging, or the random mutagenesis (localized random mutagenesis) of local.

Random mutagenesis be suitable for as local or the random mutagenesis of regiospecificity carry out at least three parts of gene translating into described aminoacid sequence, or to carry out in complete genome.When by using oligonucleotide to carry out mutagenesis, in the building-up process of oligonucleotide, in the position that will change, described three non-parent (non-parent) Nucleotide can be mixed (doped) or wedging (spiked) this oligonucleotide.Carry out mixing or wedging thus avoid undesired amino acid whose codon.Oligonucleotide that is that mix or wedging can be incorporated in the DNA of encoding inositol six-phosphatase by any technology, such as, and PCR, LCR or any archaeal dna polymerase considered appropriate and ligase enzyme.

Preferably, mixing use " constant mix at random (constant random doping) " and carry out, is wherein predefined in the wild-type of each position and the per-cent of sudden change.In addition, can make to mix and be devoted to preferably to introduce specific nucleotide, and therefore preferably introduce one or more specific amino-acid residues.Mix and can make such as, make it allow to introduce 90% wild-type and 10% sudden change in each position.Another in the selection of mixing design considers it is the restriction based on heredity and protein structure.

Random mutagenesis advantageously can be confined to the part of discussed parent phytase six-phosphatase.Such as, when some region identifying enzyme is even more important for the particular characteristics of this enzyme, this mode may be favourable.

Alternative method of variant of the present invention is provided to comprise gene shuffling (shuffling), such as described in WO95/22625 or WO 96/00343, and the consensus sequence deriving method (consensus derivation process) as described in EP 897985.

Nucleic acid construct

Nucleic acid construct comprises nucleotide sequence of the present invention, and this sequence is operably connected with one or more regulating and controlling sequence, and described regulating and controlling sequence instructs the expression of encoding sequence in suitable host cell under the condition compatible with this regulating and controlling sequence.By expressing any step being interpreted as during comprising polypeptide generates and relating to, should include but not limited to, transcribing, post transcriptional modificaiton, translation, posttranslational modification and secretion.

Terminology used here " nucleic acid construct " refers to the nucleic acid molecule of strand or double-strand, described nucleic acid molecule is separated from naturally occurring gene, or modifies described nucleic acid molecule and contain the fragment of nucleic acid to make it in the mode that originally can not be present in occurring in nature (would not otherwise exist in nature).When nucleic acid construct comprises the regulating and controlling sequence of expressing needed for encoding sequence of the present invention, term nucleic acid construct and term " expression cassette " (expression cassette) synonym.

Term " regulating and controlling sequence " is defined as the polynucleotide expression comprised for coding polypeptide of the present invention be herein required or favourable all the components.For the nucleotide sequence of coded polypeptide, each regulating and controlling sequence can be natural or external source.Such regulating and controlling sequence includes, but not limited to leader sequence, Polyadenylation sequences, propeptide sequence, promotor, signal peptide sequence and transcription terminator.Minimum situation, regulating and controlling sequence comprises promotor, and transcribe with translation termination signal.Regulating and controlling sequence can provide together with the joint for introducing specific restriction sites, and described specific restriction sites promotes the connection in the nucleotide sequence coded district of regulating and controlling sequence and coded polypeptide.

Term " is operably connected " and represents such configuration herein, wherein regulating and controlling sequence is placed in the appropriate position of the encoding sequence relative to polynucleotide sequence, thus makes regulating and controlling sequence instruct the expression of polypeptid coding sequence.

Term " encoding sequence " (CDS) represents the nucleotide sequence of the aminoacid sequence of directly specifying its protein product when used herein.The border of encoding sequence generally determines by opening frame, usually starts with ATG initiator codon or other initiator codon such as GTG and TTG.Encoding sequence can be DNA, cDNA or recombinant nucleotide sequence.

Expression vector

Term " expression " comprises during polypeptide generates any step related to, but is not limited to, and transcribes, post transcriptional modificaiton, translation, posttranslational modification and secretion.

Term " expression vector " is defined as DNA molecular that is linear or ring-type herein, it comprises the polynucleotide of polypeptide of the present invention of encoding, and described polynucleotide be provided for its additional nucleotides of expressing and be operably connected.

The nucleotide sequence of inositol six-phosphatase variants of the present invention of encoding can use expression vector to express, and described expression vector generally includes regulating and controlling sequence, its encoded protomers, operon, ribosome bind site, translation initiation signal, and optionally suppressor gene or various activating gene.

The recombinant expression vector carrying the DNA sequence dna of inositol six-phosphatase variants of the present invention of encoding can be any carrier, and it can carry out recombinant DNA method easily, and its host cell that will import often is depended in the selection of carrier.Carrier can be so a kind of carrier, and when being imported host cell, this vector integration enters host cell gene group and copies together with incorporating its karyomit(e).

Inositol six-phosphatase variants can also together with other enzyme of at least one interested in animal-feed coexpression, other enzyme such as phytase described, Phosphoric acid esterase, zytase, Galactanase, alpha-galactosidase, proteolytic enzyme, Phospholipid hydrolase, amylase, and/or beta-glucanase.Enzyme from different carriers coexpression, from a carrier coexpression, or can apply the combination of two kinds of technology.When using different carriers, carrier can have different selective markers, and different replication orgin.When only using a carrier, each gene can from one or more promoter expression.If clone (two or polycistronic) under the regulation and control of a promotor, the order of gene clone may affect the expression level of albumen.Inositol six-phosphatase variants can also be expressed as fusion rotein, namely by the gene of encoding inositol six-phosphatase variants with meeting frame (in frame) be fused to coding another albumen gene in.This albumen can be other enzymes or the functional domain from other enzymes.

Host cell

Term " host cell ", as used herein, comprises for using the conversion comprising the nucleic acid construct of polynucleotide of the present invention, transfection, any cell type of the susceptibles such as transduction.

The invention still further relates to the recombinant host cell of the recombinant production being suitable for polypeptide, it comprises polynucleotide of the present invention.To the vector introduction of polynucleotide of the present invention be comprised to host cell, thus this carrier is kept as chromosomal integrant or as the outer carrier of self replication karyomit(e) as described earlier.Term " host cell " comprises any filial generation of parental cell, and it is incomplete same with parental cell due to the sudden change occurred between replicative phase.The selection of host cell will depend on gene and the source thereof of coded polypeptide to a great extent.

Host cell can be unicellular microorganism, such as prokaryotic cell prokaryocyte, or non-unicellular micro-organism, such as eukaryotic cell.

Useful unicellular microorganism is bacterial cell, such as gram positive bacterium, comprise, but be not limited to, bacillus (Bacillus) cell, such as, Alkaliphilic bacillus (Bacillus alkalophilus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus brevis (Bacillus brevis), Bacillus circulans (Bacillus circulans), Bacillus clausii (Bacillus clausii), Bacillus coagulans (Bacillus soagulans), bacillus lautus (Bacillus lautus), bacillus lentus (Bacillus lentus), Bacillus licheniformis (Bacillus licheniformis), bacillus megaterium (Bacillusmegaterium), bacstearothermophilus (Bacillus stearothermophilus), subtilis and bacillus thuringiensis (Bacillus thuringiensis), or streptomyces (Streptomyces) cell, such as, shallow Streptomyces glaucoviolaceus (Streptomyces lividans) and mouse ash streptomycete (Streptomycesmurinus), or gram negative bacterium, such as intestinal bacteria (E.coli) and Rhodopseudomonas bacterial classification (Pseudomonas sp).In preferred, bacterial host cell is bacillus lentus, Bacillus licheniformis, bacstearothermophilus or B. subtilis cell.In another is preferred, bacillus cell is Alkaliphilic bacillus (alkalophilic Bacillus).

To bacterial host cell import carrier can come by the following method: such as protoplast transformation (see, such as, Chang and Cohen, 1979, Molecular General Genetics 168:111-115), utilization experience polypeptide cell (see, such as, Young and Spizizin, 1961, Journal of Bacteriology81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56:209-221), electroporation (see, such as, Shigekawa and Dower, 1988, Biotechniques 6:742-751), or joint (conjugation) (see, Koehler and Thorne, 1987, Journal of Bacteriology 169:5771-5278).

Host cell can also be eukaryote, such as Mammals, insect, plant or fungal cell.

In preferred, host cell is fungal cell." fungi " of using here comprises with Xiamen: Ascomycota (Ascomycota), Basidiomycota (Basidiomycota), chytrid door (Chytridiomycota) and Zygomycota (Zygomycota) are (as by Hawksworth etc., at Ainsworth and Bisby ' sDictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, defined in UK) and oomycetes door (Oomycota) (as at Hawksworth etc., 1995, the same, quoted in 171 pages), with all mitosporic fungi (mitosporic fungi) (Hawksworth etc., 1995, the same).

In preferred, the host cell of fungi is yeast cell." yeast " is as used herein comprises ascosporogenous yeast (ascosporogenous yeast) (Endomycetale (Endomycetales)), produce load yeast (basidiosporogenous yeast), and belong to the yeast of imperfect fungi (Fungi Imperfecti) (gemma guiding principle (Blastomyetes)).Because being sorted in of yeast may change in the future, for the present invention, yeast should be defined as at biology of yeast and active (Biology and Activities of Yeast) (Skinner, F.A., Passmore, S.M. and Davenport, R.R., eds, Soc.App.Bacteriol.Symposium Series No.9,1980) described in.

In in preferred, yeast host cell is Candida (Candida), Hansenula (Hansenula), genus kluyveromyces (Kluyveromyces), Pichia (Pichia), yeast belong (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces) or the mould genus of Western alpine yarrow (Yarrowia) cell.

In in most preferred, yeast host cell is pichia pastoris phaff (Pichia pastoris), pichia methanolica (Pichia methanolica), saccharomyces carlsbergensis (Saccharomyces carlsbergensis), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), saccharomyces diastaticus (Saccharomyces diastaticus), Doug Laplace yeast (Saccharomyces douglasii), kluyveromyces (Saccharomyceskluyveri), promise ground yeast (Saccharomyces norbensis) or ellipsoideus yeast (Saccharomycesoviformis) cell.In in another is most preferred, yeast host cell is Kluyveromyces lactis (Kluyveromyces lactis) cell.Another most preferably in, yeast host cell be separate fat the West alpine yarrow mould (Yarrowia lipolytica) cell.

In in another is preferred, fungal host cells is filamentous fungal cells." filamentous fungus " comprises whole filamentous form in fungi (Eumycota) and oomycetes (Oomycota) subphylum (as Hawksworth etc., 1995, the samely to define).The feature of filamentous fungus is usually by chitin, Mierocrystalline cellulose, dextran, chitosan, mannosans, and the mycelia body wall that other complicated polysaccharide form.Nourish and grow is to be realized by hyphal elongation and carbon katabolism is obligate aerobic.On the contrary, yeast such as yeast saccharomyces cerevisiae nourish and grow be by unicellular thallus sprout realize and carbon katabolism can be fermentation.

In in preferred, filamentous fungal host cell is the mould genus of branch top spore (Acremonium), Aspergillus, aureobasidium genus (Aureobasidium), smoke pipe Pseudomonas (Bjerkandera), intend wax Pseudomonas (Ceriporiopsis), Coprinus (Coprinus), Coriolus Qu61 (Coriolus), genera cryptococcus (Cryptococcus), net spore Pseudomonas (Filobasidium), fusarium (Fusarium), Humicola (Humicola), seasonal febrile diseases Pseudomonas (Magnaporthe), Mucor (Mucor), myceliophthora (Myceliophthora), the mould genus of Xin Kaoma fat (Neocallimastix), Neurospora (Neurospora), paecilomyces (Paecilomyces), Penicillium (Penicillium), flat lead fungi belongs to (Phanerochaete), penetrate arteries and veins bacterium (Phlebia), cud Chytridium (Piromyces), pleurotus (Pleurotus), Schizophyllum (Schizophyllum), Talaromyces (Talaromyces), thermophilic ascomycete belongs to (Thermoascus), Thielavia (Thielavia), Tolypocladium (Tolypocladium), trametes (Trametes) or Trichoderma (Trichoderma) cell.

In in most preferred, filamentous fungal host cell is Aspergillus awamori, Aspergillus fumigatus (Aspergillusfumigatus), smelly aspergillus (Aspergillus foetidus), aspergillus japonicus (Aspergillus japonicus), Aspergillus nidulans (Aspergillus nidulans), aspergillus niger (Aspergillus niger) or aspergillus oryzae (Aspergillus oryzae) cell.In in another is most preferred, filamentous fungal host cell is bar spore shape sickle spore (Fusarium bactridioides), F.graminearum schw (Fusarium cerealis), storehouse prestige sickle spore (Fusarium crookwellense), machete sickle spore (Fusarium culmorum), fusarium graminaria (Fusarium graminearum), the red sickle spore of standing grain (Fusarium graminum), different spore sickle spore (Fusariumheterosporum), albizzia sickle spore (Fusarium negundi), point sickle spore (Fusarium oxysporum), racemosus sickle spore (Fusarium reticulatum), pink sickle spore (Fusarium roseum), Williams Elder Twig sickle spore (Fusarium sambucinum), colour of skin sickle spore (Fusarium sarcochroum), intend branch spore sickle spore (Fusarium sporotrichioides), sulphur look sickle spore (Fusarium sulphureum), circle sickle spore (Fusariumtorulosum), intend silk spore sickle spore (Fusarium trichothecioides) or empiecement sickle spore (Fusariumvenenatum) cell.In another is most preferred, filamentous fungal host cell is black thorn smoke pipe bacterium (Bjerkandera adusta), dry plan wax bacterium (Ceriporiopsis aneirina), dry plan wax bacterium, Ceriporiopsiscaregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa or worm intend wax bacterium (Ceriporiopsis subvermispora), Coprinus cinereus (Coprinus cinereus), hairy fungus (Coriolus hirsutus), Humicola insolens (Humicolainsolens), dredge cotton like humicola lanuginosa (Humicola lanuginose), rice black wool mould (Mucor miehei), thermophilic fungus destroyed wire (Myceliophthora thermophila), Neuraspora crassa (Neurospora crassa), penicillium purpurogenum (Penicillium purpurogenum), Phanerochaete chrysosporium (Phanerochaetechrysosporium), arteries and veins bacterium (Phlebia radiata) is penetrated in radiation, pleurotus eryngii (Pleurotus eryngii), autochthonal shuttle spore mould (Thielavia terrestris), long wool Trametes trogii (Trametes villosa), variegated bolt bacterium (Trametes versicolor), trichoderma harziarum (Trichoderma harzianum), healthy and free from worry wood mould (Trichoderma koningii), long shoot wood mould (Trichoderma longibrachiatum), the cell of Trichodermareesei (Trichoderma reesei) or viride (Trichoderma viride) bacterial strain.

Fungal cell can be formed by relating to protoplastis, and the method for protoplast transformation and cell wall-deficient mutant transforms in a way known.The appropriate method transforming Aspergillus and Trichoderma host cell, at EP 238 023 and Yelton etc., has description in 1984, Proceedings of the National Academy of SciencesUSA 81:1470-1474.The appropriate method of transforming Fusarium bacterial classification is by Malardier etc., and 1989, Gene 78:147-156 and WO96/00787 describes.Can Becker and Guarente be utilized, at Abelson, J.N. and Simon, the Guide to Yeast Genetics and MolecularBiology that M.I. edits, Methods in Enzymology, volume 194,182-187 page, Academic Press, Inc., New York; Ito etc., 1983, Journal of Bacteriology 153:163; With Hinnen etc., the method described in 1978, Proceedings of the National Academy of Sciences USA 75:1920 is by yeast conversion.

Preparation method

The invention still further relates to the method for the preparation of phytase of the present invention, it comprises (a) and cultivate host cell under the condition being of value to phytase generation; And (b) reclaims phytase.

In the preparation process in accordance with the present invention, culturing cell in the nutritional medium that method known in the art produces at applicable polypeptide is applied.Such as; shake-flask culture can be passed through; and small-scale in laboratory or industrial fermentation tank or large scale fermentation (comprise continuous print; in batches; fed-batch (fed-batch); or solid state fermentation), to carry out under the condition allowing described expression of polypeptides and/or separation in suitable substratum.Utilize methods known in the art, cultivate in suitable nutritional medium, described substratum comprises Carbon and nitrogen sources and inorganic salt.Suitable substratum can obtain from suppliers, or can according to disclosed composition preparation (such as, in the catalogue of American type culture collection (American Type CultureCollection)).If polypeptide is secreted in nutritional medium, then directly can reclaim polypeptide from substratum.If polypeptide is not secreted, then can reclaim from cell lysate.

The polypeptide obtained can reclaim by means known in the art.Such as, can include, but not limited to centrifugal by conventional method, filter, extract, spraying dry, evaporation or precipitation, reclaim polypeptide from nutritional medium.

Polypeptide of the present invention can carry out purifying by multiple methods known in the art, include but not limited to, chromatography (such as, ion-exchange, affine, hydrophobic, chromatofocusing, and size exclusion), electrophoresis method (such as, preparative isoelectrofocusing), differential solubility (such as, ammonium sulfate precipitation), SDS-PAGE or extraction (see, such as, Protein Purification, J.-C.Janson and Lars Ryden, editor, VCHPublishers, New York, 1989).

Transgenic plant

The invention still further relates to transgenic plant, plant part or vegetable cell, its nucleotide sequence having used coding to have the polypeptide of phytase activity of the present invention transforms, thus reaches with callable scale and produce described polypeptide.Polypeptide can reclaim from plant or plant part.Alternatively, the plant or the plant part that comprise recombinant polypeptide can be applied as such, in order to improve food or quality of the fodder, such as, improve nutritive value, palatability and rheological property, or destroy anti-nutrition (antinutritive) factor.

In a specific embodiment, polypeptide target is to endosperm storage bubble (endosperm storagevacuole) in seed.This can have appropriate signals propeptide to realize by being synthesized, see Horvath etc., at PNAS, and on February 15th, 2000,97 volumes, No. 4, in 1914-1919 page.

Transgenic plant can be dicots (dicotyledons) or monocotyledonous (monocotyledons) or their genetically engineered variants.Monocotyledonous example is grass, such as English grass (meadowgrass) (bluegrass (blue grass), Gramineae (Poa)), herbage (forage grass) such as festuca (Festuca), lolium (Lolium), temperate zone grass is Agrostis (Agrostis) such as, and cereal, such as, wheat, oat, rye, barley, rice, Chinese sorghum, triticale (triticale) (the stable heterozygote of wheat (Triticum (Triticum)) and rye (Secale (Secale))), and corn (maize) (corn (corn)).The example of dicotyledons is tobacco, beans, such as Sunflower Receptacle (Helianthus (Helianthus)), cotton (Gossypium (Gossypium)), lupine, potato, sugar beet (sugar beet), pea, Kidney bean (bean) and soybean, and cress (Cruciferae (family Brassicaceae)), such as Cauliflower, Semen Brassicae campestris (rape seed), and the model animals Arabidopis thaliana (Arabidopsisthaliana) of tight association.Low-phytate salt plant as such as United States Patent (USP) no.5,689,054 and United States Patent (USP) no.6,111, described in 168 is the example of genetically engineered plant.

The example of plant part is stem, callus, leaf, root, fruit, seed and stem tuber, and the independent body comprising these parts, such as epidermis, mesophyll, parenchyma, vascular tissue, meristematic tissue.Specific palnt cell compartments, such as chloroplast(id), apoplast (apoplast), plastosome, vacuole, peroxysome, and tenuigenin is also considered to plant part.In addition, any vegetable cell, no matter how tissue-derived, all think plant part.Equally, plant part, such as, such as, for promoting that the particular organization that application of the present invention is separated and cell are also considered to plant part, embryo, endosperm, aleuron and seed coat (seed coats).

Also comprise these plants within the scope of the present invention, the filial generation of plant part and vegetable cell.

Can according to the transgenic plant of methods known in the art construction expression polypeptide of the present invention or vegetable cell.Briefly, following build transgenic plant or vegetable cell, by the expression construct of one or more code book invention polypeptide is mixed plant host genome, and make the modified plant that obtains or vegetable cell propagation become transgenic plant or vegetable cell.

Easily, expression construct is nucleic acid construct, and it comprises the nucleotide sequence of polypeptide of the present invention of encoding, and described nucleotide sequence is operably connected with the suitable regulating and controlling sequence of expressing in selected plant or plant part needed for this nucleotide sequence.In addition, expression construct can comprise selective marker for the identification of incorporate expression construct host cell and for construct being imported the necessary DNA sequence dna of described plant (the latter depends on applied DNA introduction method).

Regulating and controlling sequence, the selection of such as promotor and terminator sequence and optionally signal or transit sequence, based on when such as expecting, where and how express polypeptide decides.Such as, the expression of the gene of polypeptide of the present invention of encoding can be composing type or induction type, can be maybe to grow, the stage or tissue-specific, and gene product can target specific cells compartment, and tissue or plant part are as seed or leaf.Regulating and controlling sequence is, such as, by Tague etc., described by 1988, Plant Physiology 86:506.

For constitutive expression, following promotor can be used: 35S-CaMV promotor (Franck etc., 1980, Cell21:285-294), maize ubiquitin 1 (Christensen AH, Sharrock RA and Quail1992.Maize polyubiquitin genes:structure, thermal perturbation of expressionand transcript splicing, and promoter activity following transfer to protoplasts byelectroporation), or rice Actin muscle 1 promotor (Plant Mo.Biol.18, 675-689., Zhang W, McElroy D. and Wu R1991, Analysis of rice Act1 5 ' region activity in transgenicrice plants.Plant Cell 3,1155-1165).Organ specific promotor can be, such as, depots tissue (storage sink tissue) such as seed, promotor (the Edwards & Coruzzi of potato tuber and fruit, 1990, Ann.Rev.Genet.24:275-303), or from metabolic pool tissue (metabolicsink tissue) such as merismatic promotor (Ito etc., 1994, Plant Mol.Biol.24:863-878), the promotor of seed-specific is such as from the gluten of rice, prolamine, sphaeroprotein or albuminous promotor (Wu etc., 1998, Plant and Cell Physiology 39:885-889), from the broad bean promotor (Conrad etc. of legumin B4 and the unknown seed protein gene from broad bean (Vicia faba), 1998, Journal of Plant Physiology 152:708-711), from the promotor (Chen etc. of seed oil bodies albumen (seed oil bodyprotein), 1998, Plant and Cell Physiology 39:935-941), from the storage protein napA promotor of colea (Brassica napus), or the promotor of other any seed-specifics known in the art, such as, as described in WO 91/14772.In addition, promotor can be that leaf specific promoter is such as from the rbcs promotor (Kyozuka etc. of rice or tomato, 1993, PlantPhysiology 102:991-1000), chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26:85-93), or from the aldP gene promoter (Kagaya etc. of rice, 1995, Molecular and General Genetics 248:668-674), or wound inducement type promotor such as potato pin2 promotor (Xu etc., 1993, Plant Molecular Biology22:573-588).Equally, promotor can by abiotic (abiotic) processing example as temperature, arid or salinity altercation are induced, the material of the activation promotor maybe can used by external source is induced, described material such as ethanol, oestrogenic hormon, plant hormone is as ethene, dormin, gibberic acid and/or heavy metal.

Promotor strengthens element and can also be used in plant, obtain the higher expression of polypeptide.Such as, it can be intron that promotor strengthens element, and it is placed in promotor and between the nucleotide sequence of polypeptide of the present invention of encoding.Such as, above-mentioned Xu etc., 1993, disclose and utilize the First Intron of rice Actin muscle 1 gene to carry out Enhanced expressing.

Further, can select relative to the floristics optimizing codon of research to improve expression (see Horvath above-mentioned etc.).

Any other part of selectable marker gene and expression construct those obtainablely can be chosen from this area.

According to routine techniques known in the art, nucleic acid construct is incorporated to into Plant Genome, these technology comprise the conversion that Agrobacterium (Agrobacterium) mediates, virus-mediated conversion, microinjection, particle bombardment, biolistics transforms and electroporation (Gasser etc., 1990, Science 244:1293; Potrykus, 1990, Bio/Technology8: 535; Shimamoto etc., 1989, Nature 338:274).

At present, the transgenosis that agrobacterium tumefaciens (Agrobacterium tumefaciens) mediates be for the preparation of transgenic dicots preferred method (see summary Hooykas and Schilperoort, 1992, Plant Molecular Biology 19:15-38), and it can also be used for transforming monocots, although more generally use other method for transformation for these plants.At present, prepare the preferred method of transgenic monocot plant, except Agrobacterium approach, particle bombardment (meticulous gold (microscopic gold) or tungsten particle with transfering DNA the coating) (Christou of embryo callus or developmental embryo, 1992, PlantJournal 2:275-281; Shimamoto, 1994, Current Opinion Biotechnology 5:158-162; Vasil etc., 1992, Bio/Technology 10:667-674).Alternative monocot transformation methods based on protoplast transformation, as Omirulleh etc., described in 1993, Plant Molecular Biology21:415-428.

After conversion, according to method choice known in the art be incorporated with expression construct transformant and regeneration become whole plant.Usually be designed to by method for transformation optionally to remove Select gene in regenerative process or in ensuing offspring, it such as uses the cotransformation of two T-DNA constructs be separated by utilizing or carries out site specific excision by specific recombinase to Select gene.

The invention still further relates to the method preparing polypeptide of the present invention, it cultivates transgenic plant or vegetable cell under being included in the condition being of value to the generation of described polypeptide, and described transgenic plant or vegetable cell comprise the nucleotide sequence that coding has the polypeptide of phytase activity of the present invention.

Transgenic animal

The invention still further relates to genetically modified non-human animal and their product or composition, the example is body fluid such as breast and blood, organ, meat and zooblast.For the technology of expressing protein, the such as technology of expressing protein in mammalian cell, known in the art, see such as handbook Protein Expression:A Practical Approach (protein expression: practical approach), Higgins and Hames (editor), OxfordUniversity Press (Oxford University Press) (1999), and this series relates to genetic transcription, three handbooks of RNA processing and post translational processing.As a rule, in order to prepare transgenic animal, the nucleotide sequence with coding with the polypeptide of phytase activity of the present invention transforms cell selected by selected animal, thus expresses and prepare polypeptide.Polypeptide can be reclaimed from animal body, such as, from the Ruzhong of jenny, maybe can by expression of polypeptides for being of value to animal itself, such as auxiliary animal digestion.The example of animal below title is mention in the part of animal-feed.

In order to prepare by the transgenic animal of reclaiming from animal Ruzhong for the purpose of polypeptide, can the gene of coded polypeptide be inserted in the zygote of discussed animal, such as, by using the transgene expression vector comprising the gene of suitable milk protein promoter and coding said polypeptide.Zygote is entered in transgene expression vector microinjection, and preferred permanent integration enters karyomit(e).Once ovum starts growth and division, just by the parent (surrogate mother) of potential Embryonic limb bud cell substitute, and identify and carry this genetically modified animal.The animal obtained can be raised by routine immediately and breed.Polypeptide can from animal Ruzhong purifying, see such as Meade, H.M. (1999) are waited: Expression of recombinant proteins in the milk oftransgenic animals, Gene expression systems:Using nature for the art ofexpression. (expresses recombinant protein in transgenic animal Ruzhong, gene expression system: natural property is used for expression technology) .J.M.Fernandez and J.P.Hoeffler (editor), Academic Press.

On the other hand, in order to be prepared in its somatocyte and/or stem cell genome in carry the non-human transgenic animal of the nucleotide sequence comprising heterologous transgene construct, wherein said heterologous transgene construct comprises the transgenosis of coded polypeptide, the first specific expressed to this transgenosis and the sialisterium being used for polypeptide regulating and controlling sequence can be operably connected, disclosed in WO 00/064247.

Composition and purposes

In a still further aspect, the present invention relates to the composition comprising polypeptide of the present invention, and apply the method for these compositions.

Peptide composition can be prepared according to methods known in the art, and can with the form of liquid or dry composition.Such as, peptide composition can exist with particle or microparticulate form.The polypeptide that will comprise in the composition can carry out stabilization according to methods known in the art.

Phytase of the present invention can in any industrial environment for degraded, such as, phytate, phytinic acid and/or list, two, three, four and/or five phosphoinositides.The cat ions of the phosphate portion chelating divalent and trivalent of knowing these compounds, as metal ion, includes but not limited to (i.a.) nutritional necessary ionized calcium, iron, zinc and magnesium, and trace-metal manganese, copper and molybdenum.In addition, phytinic acid is also to a certain extent by electrostatic interaction associated proteins.

Therefore, the preferable use of polypeptide of the present invention in animal-feed prepared product, (comprises human foods) or for the additive of these prepared products.

In an embodiment, polypeptide of the present invention may be used for the nutritive value improving animal-feed.The non-limiting example improving the nutritive value of animal-feed (comprising human foods) is: improve feed digestion; Promote growth of animal; Improve feed applications; Improve the bioavailability of albumen; Raising can digest phosphatic level; Improve release and/or the degraded of phytate; Improve the bioavailability of trace minerals; Improve the bioavailability of large mineral substance; Eliminate the phosphoric acid salt adding complementarity, the needs of trace minerals and/or large mineral substance; And/or improve chorion quality.Therefore the nutritive value of feed increases, and can improve growth velocity and/or weight increase (weight gain) and/or the feed conversion rate (feed relative namely taken in increases relative to weight) of animal.

And then polypeptide of the present invention may be used for reducing the phytinic acid salt level in ight soil.

Animal, animal-feed and animal feedstuff additive

Term animals comprises all animals, comprises the mankind.The example of animal is non-ruminant animal, and ruminating animal.Ruminating animal comprises, such as sheep, goat and ox (cattle), and such as milk cow (cow) is as beef cattle (beefcattle) and milk cow (dairy cow).In an embodiment, animal is the non-animal ruminated.Non-ruminant animal comprises the animal of simple stomach, such as pig (pig) or pig (swine) (include but not limited to, piggy (piglet), the pig (growing pig) in growth and sow (sow)); Poultry is turkey such as, duck and chicken (including but not limited to chick (broiler chick), laying hen (layers)); Fish (including but not limited to salmon (salmon), trout (trout), tilapia (tilapia), catfish (catfish) and carp (carp)); With crustaceans (including but not limited to river prawn (shrimp) and prawn (prawn)).

Term feed or feed composition refer to any compound, prepared product, mixture or the composition that are suitable for animal and take in or be intended to be taken in by animal.

According in purposes of the present invention, can before the meal, after the meal or with dining simultaneously to feeding animal polypeptide.The latter is preferred.

In an embodiment, polypeptide, adds the form in feed to it, or when it is included in fodder additives, is that (substantially) is pure substantially.In an embodiment, it is (well-defined) that determine.Term " determines " to mean that phytase prepared product is at least 50% pure, as by size exclusion chromatography measure (embodiment 12 see WO01/58275).In the embodiment that other are concrete, as measured by this method, phytase prepared product is at least 60,70,80,85,88,90,92,94 or at least 95% pure.

Substantially pure, and/or the polypeptide preparation thing determined is favourable.Such as, there is no that the polypeptide of the interference of other polypeptide or pollution is much easier with correct dose administration.Term specifically refers to the object obtaining constant (consistent) and constant (constant) result with correct dose administration, and the ability of effect optimization dosage based on expectation.

But for the application in animal-feed, phytase polypeptides of the present invention does not need so pure; Such as it can comprise other polypeptide, can be called phytase prepared product in the case.

Phytase prepared product (a) can directly add feed (or being directly used in the treatment process of albumen) to, or (b) may be used for the production of one or more intermediate component, described intermediate component is as added fodder additives or the premix (premix) of (or applying in treatment process) in feed subsequently to.The degree of above-mentioned purity refers to the purity of original polypeptide prepared product, no matter according to (a) above or (b) application.

The polypeptide preparation thing with the purity of this order of magnitude especially can be applied recombinant method for production to obtain, otherwise they are not be easy to and have much higher difference between batch (batch-to-batch variation) by traditional fermentation methods acquisition.

Such polypeptide preparation thing can mix with other polypeptide certainly.

Polypeptide can add in feed in any form, as relatively pure polypeptide (be it as arelatively pure polypeptide), or be intended to other components of adding in animal-feed and mix, namely with the form of animal feedstuff additive, the premix (pre-mixes) of such as so-called animal-feed.

The composition used in animal-feed is the present invention relates to, such as animal-feed, and animal feedstuff additive, such as premix further.

Except polypeptide of the present invention, animal additive of the present invention contains at least one liposoluble vitamin, and/or at least one water-soluble vitamins, and/or at least one trace minerals.Animal additive can also comprise the large mineral substance of at least one (macro mineral).

In addition, optionally, fodder additives composition is tinting material, and such as carotenoid is as β-carotene, astaxanthin and xenthophylls; Aromatic compound; Stablizer; Antimicrobial peptide; Polyunsaturated fatty acid; Active oxygen resultant (reactive oxygen generating species); And/or be selected from following among other polypeptide of at least one: phytase (EC 3.1.3.8 or 3.1.3.26); Phosphoric acid esterase (EC 3.1.3.1; EC 3.1.3.2; EC 3.1.3.39); Zytase (EC 3.2.1.8); Galactanase (EC 3.2.1.89); Alpha-galactosidase (EC 3.2.1.22); Proteolytic enzyme (EC 3.4.-.-); Phospholipase A1 (EC 3.1.1.32); Phospholipase A2 (EC 3.1.1.4); Lysophospholipase (EC 3.1.1.5); Phospholipase C (3.1.4.3); Phospholipase D (EC3.1.4.4); Amylase is α-amylase (EC 3.2.1.1) such as; And/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

In an embodiment, these other polypeptide is (as above in the face of phytase prepared product defines) determined.

Phytase of the present invention can also combine with other phytase, such as sac fungi (ascomycete) phytase is as Aspergillus phytase, such as be derived from Aspergillus ficuum (Aspergillus ficuum), aspergillus niger or Aspergillus awamori; Or club fungi (basidiomycete) phytase, such as be derived from Peniophora lycii, flat ricetulus rodents (Agrocybe pediades), suede Trametes trogii (Trametes pubescens) or involute paxillus (Paxillus involutus); Or there is their derivative, fragment or the variant of phytase activity.

Thus, in the preferred implementation of the purposes in animal-feed of the present invention, and in the preferred implementation of animal feedstuff additive of the present invention and animal-feed, phytase of the present invention and these phytases are combined.

Antimicrobial peptide (AMP ' example s) is CAP18, LeucocinA, Tritrpticin, Protegrin-1, Thanatin, defensin (Defensin), lactoferrin (Lactoferrin), Lactoferricin and Ovispirin is as Novispirin (Robert Lehrer, 2000), Plectasins and statins (Statins), be included in compound disclosed in WO 03/044049 and WO 03/048148 and polypeptide, and keep above-mentioned every variant or the fragment of antimicrobial acivity.

Anti-fungus polypeptide (AFP ' example s) is huge aspergillus (Aspergillus giganteus) and aspergillus niger peptide, and keep their variant and the fragment of anti-mycotic activity, as disclosed in WO 94/01459 and WO02/090384.

The example of polyunsaturated fatty acid is C18, C20 and C22 polyunsaturated fatty acid, as arachidonic acid, and docosahexenoic acid (docosohexaenoic acid), eicosapentaenoic acid and gamma-linoleic acid.

The example of active oxygen resultant is chemical reagent such as perborate, persulphate or antihypo; And polypeptide such as oxydase, oxygenase or synthetic enzyme.

Usually fat-soluble and water-soluble vitamins, and trace minerals forms the part being intended to the so-called premix added to feed, and large mineral substance then adds to separately in feed usually.Any one in these types of compositions just becomes animal feedstuff additive of the present invention when adding and having polypeptide of the present invention.

In an embodiment, be intended to by animal feedstuff additive of the present invention with 0.01 to 10.0% level be contained in (or regulation must be contained in) animal diet followed or feed; More specifically with 0.05 to 5.0% level; Or the level of 0.2 to 1.0% (% represents the grams of additive in every 100 grams of feeds).In premix especially like this.

Following is the nonexcludability example list of these components:

The example of liposoluble vitamin is vitamin A, Vitamin D3 500,000 I.U/GM, vitamin-E and vitamin K, such as vitamin K3.

The example of water-soluble vitamins is vitamin B12, vitamin H and choline, VITMAIN B1, Lin Suanna Vitamin B2 Sodium Phosphate, vitamin B6, nicotinic acid, folic acid and pantothenate (panthothenate), such as D-VB5 calcium (Ca-D-panthothenate).

The example of trace minerals is manganese, zinc, iron, copper, iodine, selenium and cobalt.

The example of large mineral substance is calcium, phosphorus and sodium.

The nutritional requirement of these components (be example with poultry and piggy/pig) is listed in the Table A of WO 01/58275.Nutritional requirement means that these components should provide with the concentration of regulation in the diet.

On the other hand, animal feedstuff additive of the present invention comprises the individual components that at least one is specified in the Table A of WO 01/58275.At least one means, one or more, one, or two, or three, or four and whole 13 of as many as by that analogy, or any one of whole 15 individual components of as many as.More specifically, the individual components of this at least one is included in additive of the present invention with such amount, this amount is provided in the 4th row of Table A, or the 5th row, or the 6th row in illustrate scope in feed in concentration (in-feed-concentration).

The invention still further relates to the composition of animal-feed.Animal feedstuff compositions or diet have relatively high protein content.The feature of the diet of poultry and pig can be shown described in B 2-3 row as WO 01/58275.The feature of the diet of fish can as described in the 4th row of this table B.In addition such fish diets has the crude fat content of 200-310g/kg usually.

WO 01/58275 corresponds to US 09/779334, it is incorporated to by reference.

Animal feedstuff compositions of the present invention has the crude protein content of 50-800g/kg, and comprises the claimed polypeptide of at least one in addition herein.

, or in alternative situation (crude protein content for above-mentioned), animal feedstuff compositions of the present invention has the Metabolizable energy that content is 10-30MJ/kg in addition; And/or the calcium contents of 0.1-200g/kg; And/or the available phosphorus of 0.1-200g/kg (available phosphorus) content; And/or the methionine contents of 0.1-100g/kg; And/or the methionine plus cysteine content of 0.1-150g/kg; And/or the lysine content of 0.5-50g/kg.

In a specific embodiment, Metabolizable energy, crude protein, calcium, phosphorus, methionine(Met), methionine plus cysteine, and/or the content of Methionin is within arbitrary in scope 2,3,4 or 5 (R.2-5) in the table B of WO01/58275.

Crude protein is multiplied by the factor 6.25 with nitrogen (N) and calculates, i.e. crude protein (g/kg)=N (g/kg) x6.25.Nitrogen content is by Kjeldahl nitrogen determination (A.O.A.C., 1984, Official Methods of Analysis14th ed., Association of Official Analytical Chemists, Washington DC).

The calculating of Metabolizable energy can based on the nutritional needs of NRC publication pig, the 9th revised edition 1988, pig trophology sub-committee, the animal nutrition council, the Ministry of Agriculture, state science commission.National Academy Press, Washington D.C., 2-6 page (Nutrient requirements in swine, ninthrevised edition 1988, subcommittee on swine nutrition, committee on animalnutrition, board of agriculture, national research council.National Academy Press, Washington, D.C., pp.2-6), and European poultry feed Energy value table, the research of Spelderholt poultry and the center of expansion, 7361 DA Beekbergen, Holland.Grafisch bedrijf Ponsen & looijenbv，Wageningen.ISBN 90-71463-12-5(European Table of Energy Values forPoultry Feed-stuffs，Spelderholt centre for poultry research and extension，7361DA Beekbergen，The Netherlands.Grafisch bedrijf Ponsen & looijen bv，Wageningen.ISBN 90-71463-12-5)。

Calcium in intact animal diet, the calculating of available phosphorus and amino acid whose dietary content is based on feed table, such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6,8219pk Lelystad.ISBN 90-72839-13-7.

In an embodiment, animal feedstuff compositions of the present invention comprises at least one albumen.Described albumen can be animal proteinum, such as meat and bone meal, and/or fish meal; Or it can be vegetable-protein.Term vegetable-protein used herein refers to any compound, composition, prepared product or mixture, and it comprises the albumen of at least one from plant derivation or origin, comprises modified albumen and protein derivatives.In a specific embodiment, the protein content of vegetable-protein is at least 10,20,30,40,50 or 60% (w/w).

Vegetable-protein can be derived from plant protein source, such as beans and cereal, such as from pulse family (Fabaceae) (pulse family (Leguminosae)), Cruciferae (Cruciferaceae), the material of the plant of Chenopodiaceae (Chenopodiaceae) and Gramineae (Poaceae), such as soyflour, feather fan bean powder (lupin meal) and canola (rapeseed meal).

In an embodiment, plant protein source is the material of one or more plants from pulse family, such as soybean, lupine, pea or Kidney bean.

In another embodiment, plant protein source is the material of one or more plants from Chenopodiaceae, such as beet, sugar beet, spinach or goosefoot (quinoa).

Other examples of plant protein source are Semen Brassicae campestris, sunflower seed (sunflower seed), cottonseed (cottonseed) and wild cabbage (cabbage).

Soybean is preferred plant protein source.

Other examples of plant protein source are cereal such as barleys, wheat, rye, oat, corn (corn), rice, triticale and Chinese sorghum.

In further embodiment, animal feedstuff compositions of the present invention comprises 0-80% corn; And/or 0-80% Chinese sorghum; And/or 0-70% wheat; And/or 0-70% barley; And/or 0-30% oat; And/or 0-40% soyflour; And/or 0-25% fish meal; And/or 0-25% digested tankage or bone meal; And/or 0-20% whey.

Animal diet followed such as can be produced as mass (mash feed) (non-particulate (nonpelleted)) or granulated feed (pelleted feed).Normally, by the feed ground mixing and according to adding enough essential vitamins and mineral substance to the specification (specification) of discussed species.Polypeptide can add as the form of solid or liquid polypeptide formulations.Such as, solid polypeptide preparation usually before mixing step or period add; Liquid polypeptide formulations adds usually after granulation step.Polypeptide also can be blended in fodder additives or premix.

In diet, the final concentration of polypeptide is within the scope of every kilogram of diet 0.01-200mg polypeptide protein, such as the scope of every kilogram of animal diet 5-30mg polypeptide protein.

Phytase of the present invention should effectively to measure application, namely enough to improve the amount of dissolving and/or improving feed nutritive value.Current consideration polypeptide is used with one or more following amounts (dosage range): 0.01-200; 0.01-100; 0.5-100; 1-50; 5-100; 10-100; 0.05-50 or 0.10-10---these all scopes are the milligram number (ppm) of the phytase polypeptides albumen in every kilogram of feed.

For the milligram number of the phytase polypeptides albumen of the every kilogram of feed of mensuration, be purified from feed composition by phytase, the specific activity application relevant assay of the phytase of purifying measures.The phytase activity of feed composition like this is also applied identical assay method to determine, and based on these two kinds mensuration, calculates the milligram dosage of every kilogram of feed mysoinositol six-phosphatase albumen.

Identical mechanism is applicable to the milligram number of the phytase polypeptides albumen measured in fodder additives.Certainly, if the sample of the phytase for the preparation of fodder additives or feed can be obtained, then from this sample, specific activity (without the need to purifying phytase from feed composition or additive) is measured.

Embodiment

The invention still further relates to following detailed description:

I. phytase, itself and SEQ ID NO:2 have at least 74% identity, and it is included at least one being selected from least one following position and changes compared with SEQ ID NO:2: 1, 2, 3, 4, 5, 31, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410 and 411,

Preferably be selected from least one following position: 1,2,3,4,5,31,46,52,53,55,57,59,76,82,99,100,107,109,111,114,115,116,117,118,119,120,121,122,123,124,137,141,161,162,164,167,179,180,181,182,183,184,185,186,196,199,200,202,218,223,241,273,276,285,286,299,314,331,339,362,379,385,406,410 and 411;

Condition is this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, and is not SEQ ID NO:6.

II. phytase, itself and SEQ ID NO:2 have at least 74% identity, and it is included at least one being selected from least one following position and changes compared with SEQ ID NO:2: 1, 2, 3, 4, 5, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 324, 339, 351, 355, 362, 379, 385, 406, 409, 410 and 411,

Preferably be selected from least one following position: 1,2,3,4,5,46,52,53,55,57,59,76,82,99,100,107,109,111,114,115,116,117,118,119,120,122,123,124,137,141,161,162,164,167,179,180,181,182,183,184,185,186,196,199,200,202,218,223,241,273,276,285,286,299,314,339,362,379,385,406,410 and 411.

III. phytase, itself and SEQ ID NO:2 have at least 74% identity, and it is included at least one being selected from least one following position and changes compared with SEQ ID NO:2: 1, 2, 3, 4, 5, 31, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410 and 411,

Preferably preferably be selected from least one following position being selected from least one following position: 1, 2, 3, 4, 5, 31, 46, 52, 53, 55, 57, 59, 76, 82, 99, 100, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 137, 141, 161, 162, 164, 167, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 218, 223, 241, 273, 276, 285, 286, 299, 314, 331, 339, 362, 379, 385, 406, 410 and 411,

Condition is this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, is not SEQID NO:6, and is not its variant listed in SEQ ID NO:9 and Fig. 1.

IV. phytase, itself and SEQ ID NO:2 have at least 74% identity, and it is included at least one being selected from least one following position and changes compared with SEQ ID NO:2: 1, 2, 3, 4, 5, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 324, 339, 351, 355, 362, 379, 385, 406, 409, 410 and 411

Preferably be selected from least one following position: 1,2,3,4,5,46,52,53,55,57,59,76,82,99,100,107,109,111,114,115,116,117,118,119,120,122,123,124,137,141,161,162,164,167,179,180,181,182,183,184,185,186,196,199,200,202,218,223,241,273,276,285,286,299,314,339,362,379,385,406,410 and 411;

Condition is this phytase is not its variant listed in SEQ ID NO:9 and Fig. 1.

V. phytase, itself and SEQ ID NO:2 have at least 74% identity, and it is included at least one being selected from least one following position and changes compared with SEQ ID NO:2: 4, 5, 41, 46, 59, 82, 84, 91, 99, 105, 107, 109, 111, 115, 116, 117, 119, 122, 123, 124, 136, 137, 141, 161, 162, 164, 167, 171, 176, 179, 180, 186, 196, 199, 200, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 289, 294, 299, 308, 314, 324, 339, 351, 355, 379, 385, 406, 409, 410 and 411,

Preferably be selected from least one following position: 4,5,46,59,82,99,107,109,111,115,116,117,119,122,123,124,137,141,161,162,164,167,179,180,186,196,199,200,218,223,241,273,276,299,314,339,379,385,406,410 and 411.

VI. phytase, itself and SEQ ID NO:2 have at least 74% identity and it comprises at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, T, 41P, 46C, D, E, 52C, E, 53V, Q, 55D, I, 57Y, 59C, 74A, 76G, 82E, 84Y, 91C, P, 99C, 100C, 104A, 105F, 107D, E, G, 109A, G, 111P, 114H, N, T, 115Q, 116A, E, P, T, Q, 117D, E, K118I, L, M, T, 119G, K, R, S, 120K, S, T, Q, 121A, D, M, P, T, V, 122D, 123P, S, 124L, T, V, 136P, 137P, 141C, 154P, 161P, 162C, 164D, E, 167Q, 171T, 176C, 177C, 179G, I, K, N, Q, 180A, E, G, T, 181D, G, I, K, 182H, K, S, Q, 183A, L, P, S, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K, R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L, Q, 276K, R, 281H, 282P, 283P, 284P, 285G, N, R, 286K, Q, 289P, 294T, 299L, 308A, 314G, N, 316D, 324N, 331K, 339D, 351Y, 355P, 362K, R, 379K, R, 385D, 406A, 409D, E, 410D, E and/or 411R, K, and/or the amino acid of wherein position 179,180,181,182,183,184,185 and 186 replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL,

Preferably at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, 46E, 52C, E, 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D, E, G, 109A, 111P, 114T, 115Q, 116AT, 117D, 118T, 119K, R, S, 120S, 121D, P, T, 122D, 123P, 124L, 137P, 141C, 161P, 162C, 164E, 167Q, 179K, 180E, T, 181D, K, 182H, K, Q, 183L, V, Q, 184*, 185 ^*, 186 ^*, 196Q, 199C, 200K, 202N, 218Q, 223E, 241Q, 273L, 276K, R, 285G, R, 286Q, 299L, 314G, N, 331K, 339D, 362K, R, 379K, R, 385D, 406A, 410D, E and/or 411R, K; And/or wherein replaced by KEKHQ, KEKQQ, KEKKV or KTDKL at the amino acid of position 179,180,181,182,183,184,185 and 186;

Condition is this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, and is not SEQ ID NO:6.

VII. phytase, itself and SEQ ID NO:2 have at least 74% identity and it comprises at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, T, 41P, 46C, D, E, 52C, E, 53V, Q, 55I, D, 57Y, 59C, 74A, 76G, 82E, 84Y, 91C, P, 99C, 100C, 104A, 105F, 107D, E, G, 109A, G, 111P, 114H, N, T, 115Q, 116A, E, P, T, Q117D, E, K, 118I, M, L, T, 119G, K, R, S, 120K, S, T, Q, 121A, D, M, P, V, 122D, 123P, S, 124L, T, V, 136P, 137P, 141C, 154P, 161P, 162C, 164D, E, 167Q, 171T, 176C, 177C, 179G, I, K, N, Q, 180A, E, G, T, 181D, G, I, K, 182H, K, S, Q, 183A, L, P, S, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K, R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L, Q, 276K, R, 281H, 282P, 283P, 284P, 285G, N, R, 286K, Q, 289P, 294T, 299L, 308A, 314G, N, 316D, 324N, 339D, 351Y, 355P, 362K, R, 379K, R, 385D, 406A, 409D, E, 410D, E and/or 411K, R, and/or wherein replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL at the amino acid of position 179,180,181,182,183,184,185 and 186,

Preferably at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, 46E, 52C, E, 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D, E, G, 109A, 111P, 114T, 115Q, 116AT, 117D, 118T, 119K, R, S, 120S, 121D, P, 122D, 123P, 124L, 137P, 141C, 161P, 162C, 164E, 167Q, 179K, 180E, T, 181D, K, 182H, K, Q, 183L, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K202N, 218Q, 223E, 241Q, 273L, 276K, R, 285G, R, 286Q, 299L, 314G, N, 339D, 362K, R, 379K, R, 385D, 406A, 410D, E and/or 411R, K; And/or wherein replaced by KEKHQ, KEKQQ, KEKKV or KTDKL at the amino acid of position 179,180,181,182,183,184,185 and 186.

IIX. phytase, itself and SEQ ID NO:2 have at least 74% identity and it comprises at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, T, 41P, 46C, D, E, 52C, E, 53V, Q, 55D, I, 57Y, 59C, 74A, 76G, 82E, 84Y, 91C, P, 99C, 100C, 104A, 105F, 107D, E, G, 109A, G, 111P, 114H, N, T, 115Q, 116A, E, P, T, Q, 117D, E, K, 118I, L, M, T, 119G, K, R, S, 120K, S, T, Q, 121A, D, M, P, T, V, 122D, 123P, S, 124L, T, V, 136P, 137P, 141C, 154P, 161P, 162C, 164D, E, 167Q, 171T, 176C, 177C, 179G, I, K, N, Q, 180A, E, G, T, 181D, G, I, K, 182H, K, S, Q, 183A, L, P, S, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K, R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L, Q, 276K, R, 281H, 282P, 283P, 284P, 285G, N, R, 286K, Q, 289P, 294T, 299L, 308A, 314G, N, 316D, 324N, 331K, 339D, 351Y, 355P, 362K, R, 379K, R, 385D, 406A, 409D, E, 410D, E and/or 411R, K, and/or wherein replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL at the amino acid of position 179,180,181,182,183,184,185 and 186,

Preferably at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, 46E, 52C, E, 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D, E, G, 109A, 111P, 114T, 115Q, 116AT, 117D, 118T, 119K, R, S, 120S, 121D, P, 122D, 123P, 124L, 137P, 141C, 161P, 162C, 164E, 167Q, 179K, 180E, T, 181D, K, 182H, K, Q, 183L, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K202N, 218Q, 223E, 241Q, 273L, 276K, R, 285G, R, 286Q, 299L, 314G, N, 339D, 362K, R, 379K, R, 385D, 406A, 410D, E and/or 411R, K; And/or wherein replaced by KEKHQ, KEKQQ, KEKKV or KTDKL at the amino acid of position 179,180,181,182,183,184,185 and 186.

Condition is this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, is not SEQID NO:6, and is not its variant listed by SEQ ID NO:9 and Fig. 1.

IX. phytase, itself and SEQ ID NO:2 have at least 74% identity and it comprises at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, T, 41P, 46C, D, E, 52C, E, 53V, Q, 55I, D, 57Y, 59C, 74A, 76G, 82E, 84Y, 91C, P, 99C, 100C, 104A, 105F, 107D, E, G, 109A, G, 111P, 114H, N, T, 115Q, 116A, E, P, T, Q117D, E, K, 118I, M, L, T, 119G, K, R, S, 120K, S, T, Q, 121A, D, M, P, V, 122D, 123P, S, 124L, T, V, 136P, 137P, 141C, 154P, 161P, 162C, 164D, E, 167Q, 171T, 176C, 177C, 179G, I, K, N, Q, 180A, E, G, T, 181D, G, I, K, 182H, K, S, Q, 183A, L, P, S, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K, R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L, Q, 276K, R, 281H, 282P, 283P, 284P, 285G, N, R, 286K, Q, 289P, 294T, 299L, 308A, 314G, N, 316D, 324N, 339D, 351Y, 355P, 362K, R, 379K, R, 385D, 406A, 409D, E, 410D, E and/or 411K, R, and/or wherein replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL at the amino acid of position 179,180,181,182,183,184,185 and 186,

Preferably at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, 46E, 52C, E, 53V, 55D, 57Y, 59C, 76G, 82E, 99C, 100C, 107D, E, G, 109A, 111P, 114T, 115Q, 116AT, 117D, 118T, 119K, R, S, 120S, 121D, P, 122D, 123P, 124L, 137P, 141C, 161P, 162C, 164E, 167Q, 179K, 180E, T, 181D, K, 182H, K, Q, 183L, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K202N, 218Q, 223E, 241Q, 273L, 276K, R, 285G, R, 286Q, 299L, 314G, N, 339D, 362K, R, 379K, R, 385D, 406A, 410D, E and/or 411R, K; And/or wherein replaced by KEKHQ, KEKQQ, KEKKV or KTDKL at the amino acid of position 179,180,181,182,183,184,185 and 186.

Condition its variant that to be this phytase be not listed by SEQ ID NO:9 and Fig. 1.

X. phytase, itself and SEQ ID NO:2 have at least 74% identity and it comprises at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, T, 41P, 46C, D, E, 52C, E, 53Q, 55D, 57Y, 59C, 74A, 82E, 84Y, 91C, P, 99C, 100C, 104A, 105F, 107D, E, G, 109A, G, 111P, 114H, T, 115Q, 116A, E, P, T, Q, 117D, E, K, 118I, M, L, T, 119G, K, R, S, 120K, S, T, Q, 121A, D, M, V, 122D, 123P, S, 124L, T, V, 136P, 137P, 141C, 154P, 161P, 162C, 164D, E, 167Q, 171T, 176C, 177C, 179G, I, K, N, Q, 180A, E, G, T, 181D, G, K, 182K, S, Q, 183A, L, S, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K, R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L, Q, 276K, R, 281H, 282P, 283P, 284P, 285G, N, R, 286K, Q, 289P, 294T, 299L, 308A, 314G, N, 316D, 324N, 339D, 351Y, 355P, 362K, R, 379K, R, 385D, 406A, 409D, E, 410D, E and/or 411K, R, and/or wherein replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL at the amino acid of position 179,180,181,182,183,184,185 and 186,

Preferably at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, 46E, 52C, E, 55D, 57Y, 59C, 82E, 99C, 100C, 107D, E, G, 109A, 111P, 114T, 115Q, 116AT, 117D, 118T, 119K, R, S, 120S, 121D, 122D, 123P, 124L, 137P, 141C, 161P, 162C, 164E, 167Q, 179K, 180E, T, 181D, K, 182K, Q, 183L, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K202N, 218Q, 223E, 241Q, 273L, 276K, R, 285G, R, 286Q, 299L, 314G, N, 339D, 362K, R, 379K, R, 385D, 406A, 410D, E and/or 411R, K; And/or wherein replaced by KEKHQ, KEKQQ, KEKKV or KTDKL at the amino acid of position 179,180,181,182,183,184,185 and 186.

XI. phytase, itself and SEQ ID NO:2 have at least 74% identity and it comprises at least one following change: 1H, K, R, 60P, 105E, 106A, G, 155F, 157F, 173P, 175L, 188P, 205P, 215M, 231P, 254Y, 280P, 330D and/or 371P;

Preferred 1K;

Condition is this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, is not SEQID NO:6, and is not its variant listed in SEQ ID NO:9 and Fig. 1.

XII. phytase, itself and SEQ ID NO:2 have at least 74% identity and it comprises at least one following change: 1H, R, 60P, 105E, 106A, G, 157F, 173P, 175L, 188P, 205P, 215M, 231P, 254Y, 280P.

XIII. phytase, itself and SEQ ID NO:2 have at least 74% identity and it comprises at least one following change: 52C, 141C, 162C, 31C, 52C, 99C, 59C, 100C, 141C/199C, 4P, 5P, 111P, 137P, 161P, 52E, 57Y, 76G, 107D, 107G, 109A, 1 ^*, 1 ^*/ 2 ^*, 1 ^*/ 2 ^*/ 3 ^*, 121T, 273L, 285G, 286Q, 299L, 362K, 331K/55D, 107E, 46E, 82E, 119R, 119K, 164E, 223E, 276R, 276K, 362R, 379R, 379K, 385D, 410D, 410E, 411R, 411K, 53V, 121D, 167Q, 196Q, 200K, 202N, 218Q, 241Q, 285N, 314N, 314G, 406A

179K/180E/181K/182H/183Q/184 ^*/185 ^*/186 ^*，

179K/180E/181K/182Q/183Q/184 ^*/185 ^*/186 ^*，

179K/180E/181K/182K/183V/184 ^*/185 ^*/186 ^*，

179K/180T/181D/182K/183L/184 ^*/185 ^*/186 ^*，

111P/241Q，1K，

114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L，

114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L。

XIV. the phytase any one of embodiment 1-13, it is the variant of the phytase of SEQ ID NO:2.

XV. the phytase any one of embodiment 1-13, it is the variant of the phytase of SEQ ID NO:3.

XVI. the phytase any one of embodiment 1-13, it is the variant of the phytase of SEQ ID NO:4.

XVII. the phytase any one of embodiment 1-13, it is the variant of the phytase of SEQ ID NO:6.

IIXX. the phytase any one of embodiment 1-13, it is the variant of the phytase of SEQ ID NO:9.

IXX. the phytase any one of embodiment 1-13, it is relevant to SEQ ID NO:9 and in the inositol six-phosphatase variants listed in FIG the variant of any one.

XX. the phytase any one of embodiment 1-19, itself so that comprise and be selected from replacement that in Fig. 1, every ranks go out and the replacement replaced in combination or replace combination.

XXI. the phytase any one of embodiment 1-20, it has the thermostability of improvement, the pH curve improved, the specific activity improved, the glycosylation pattern of amendment, the temperature curve improved, the performance in animal-feed of improvement, and/or it contains the change of (incorporate) potential proteolytic cleavage site.

XXII. the nucleotide sequence be separated, it comprises the nucleotide sequence of the phytase any one of code embodiment I-XXI.

XXIII. nucleic acid construct, it comprises the nucleotide sequence of embodiment XXII, and this nucleotide sequence is operably connected to the one or more regulating and controlling sequences instructing and produce phytase in appropriate expressive host.

XXIV. recombinant expression vector, it comprises the nucleic acid construct of embodiment XXIII.

XXV. recombinant host cell, it comprises the nucleic acid construct of embodiment XXIII and/or the expression vector of embodiment XXIV.

XXVI. prepare the method for the phytase any one of embodiment I-XXI, it comprises

A () cultivates the host cell of embodiment XXV to produce the supernatant liquor comprising phytase; (b) phytase is reclaimed.

XXVII. transgenic plant, or plant part, it can express the phytase any one of embodiment I-XXI.

IIXXX. genetically modified, non-human animal, or its product, or component (element), it can express the phytase any one of embodiment I-XXI.

IXXX. composition, it comprises at least one phytase any one of embodiment I-XXI, and

(a) at least one liposoluble vitamin;

(b) at least one water-soluble vitamins; And/or

(c) at least one trace minerals.

XXX. the composition of embodiment IXX, it comprises at least one enzyme in the enzyme being selected from lower group further: amylase, phytase, Phosphoric acid esterase, zytase, Galactanase, alpha-galactosidase, proteolytic enzyme, Phospholipid hydrolase and/or beta-glucanase.

XXXI. the composition any one of embodiment IXX-XXX, it is animal feedstuff additive.

XXXII. animal feedstuff compositions, it has the crude protein content of 50 to 800g/kg and the phytase comprised any one of embodiment I-XXI or the composition any one of embodiment IXXX-XXXI.

XXXIII. for improvement of the method for animal feed nutritive value, wherein the phytase any one of embodiment I-XXI or the composition any one of embodiment IXXX-XXXI are added in feed.

XXXIV. for reducing the method for animal excrement mysoinositol six phosphate level, it comprises with the forage feed animal of the embodiment XXXII of significant quantity.

XXXV. for the treatment of the method for vegetable-protein, it comprises the step of adding the phytase any one of embodiment I-XXI or the composition any one of embodiment IXXX-XXXI at least one vegetable-protein or protein source.

XXXVI. the phytase any one of embodiment I-XXI or the purposes of the composition any one of embodiment IXXX-XXXI in animal-feed; Purposes in the preparation of animal-feed; For improvement of the purposes of the nutritive value of animal-feed; For reducing the purposes of the phytinic acid salt level in animal excrement; For the purposes of the process of vegetable-protein; Or for discharging the purposes of phosphorus from phytinic acid enzyme substrates.

A). phytase, it has at least 70% identity with SEQ ID NO:2 and it comprises and is selected from following at least one and changes compared with SEQ ID NO:2: 1, 2, 3, 4, 5, 31, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410 and 411, condition is this phytase is not SEQ ID NO:3, is not SEQ IDNO:4, and is not SEQ ID NO:6.

A1). phytase, it has at least 70% identity with SEQ ID NO:2 and it comprises and is selected from following at least one and changes compared with SEQ ID NO:2: 1, 2, 3, 4, 5, 31, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 316, 324, 331, 339, 351, 355, 362, 379, 385, 406, 409, 410 and 411, condition is that this variant does not comprise (i) 31D/121T/316N/331E, and does not comprise (ii) 31D/121N/316K/331E, and does not comprise (iii) 31N/121N/316N/331K.

A2). phytase, it has at least 70% identity with SEQ ID NO:2 and it comprises and is selected from following at least one and changes compared with SEQ ID NO:2: 1, 2, 3, 4, 5, 41, 46, 52, 53, 55, 57, 59, 74, 76, 82, 84, 91, 99, 100, 104, 105, 107, 109, 111, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 136, 137, 141, 154, 161, 162, 164, 167, 171, 176, 177, 179, 180, 181, 182, 183, 184, 185, 186, 196, 199, 200, 202, 203, 218, 223, 239, 240, 241, 247, 273, 276, 281, 282, 283, 284, 285, 286, 289, 294, 299, 308, 314, 324, 339, 351, 355, 362, 379, 385, 406, 409, 410 and 411.

A3). embodiment a2) phytase, it comprises at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, T, 41P, 46C, D, E, 52C, E, 53V, Q, 55I, D, 57Y, 59C, 74A, 76G, 82E, 84Y, 91C, P, 99C, 100C, 104A, 105F, 107D, E, G, 109A, G, 111P, 114H, N, T, 115Q, 116A, E, P, T, Q117D, E, K, 118I, M, L, T, 119G, K, R, S, 120K, S, T, Q, 121A, D, M, P, V, 122D, 123P, S, 124L, T, V, 136P, 137P, 141C, 154P, 161P, 162C, 164D, E, 167Q, 171T, 176C, 177C, 179G, I, K, N, Q, 180A, E, G, T, 181D, G, I, K, 182H, K, S, Q, 183A, L, P, S, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K, R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L, Q, 276K, R, 281H, 282P, 283P, 284P, 285G, N, R, 286K, Q, 289P, 294T, 299L, 308A, 314G, N, 316D, 324N, 339D, 351Y, 355P, 362K, R, 379K, R, 385D, 406A, 409D, E, 410D, E and/or 411K, R, and/or wherein replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL at the amino acid of position 179,180,181,182,183,184,185 and 186.

A4). embodiment a2)-a3) and any one of phytase, it comprises at least one following change:

(, i) 31C, 46C, 52C, 59C, 91C, 99C, 100C, 141C, 162C, 176C, 177C, 199C and/or 247C;

(ii) 4P, 5P, 41P, 91P, 111P, 136P, 137P, 154P, 161P, 240P, 282P, 283P, 284P, 289P and/or 355P;

(iii) 52E, 55D, I, 57Y, 76G, 84Y, 104A, 105F, 107D, G, 109A, G, 273L, Q, 285G, R, 286Q, 294T, 299L, 351Y and/or 362K;

(iv) 1 ^*, 1 ^*/ 2 ^*or 1 ^*/ 2 ^*/ 3 ^*;

V () be K179 wherein, T180, T181, E182, K183, S184, T185 and K186 replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL;

(vi) 119K, R and/or 411K, R;

(vii) 107E and/or 164D, E;

(viii) 46D, E, 82E, 223E, 276K, R, 362K, R, 379K, R, 385D, 409D, E and/or 410D, E;

(ix) 53V, Q, 121D, 167Q, 196Q, 200K, R, 202N, 218Q, 239Q, 241Q, 285N, 314G, N, 324N and/or 406A;

(x) 114H, N, T115Q, 116A, E, P, T, Q, 117D, E, K, 118I, L, M, T119G, K, S, 120K, S, T, Q, 121A, M, P, V, 122D, 123P, S and/or 124L, T, V

(xi) 31T, 74A, 171T, 203T, 281H, 308A and/or 316D; And/or

(xii)339D。

A5). embodiment a2)-a4) and any one of phytase, it comprises at least one following change:

(i) 141C/199C, 91C/46C, 52C/99C, 31C/176C, 31C/177C, 59C/100C and/or 162C/247C;

(ii) 41P, 91P, 136P, 137P, 154P, 161P, 355P, 111P, 240P, 282P, 283P, 284P, 289P, 4P and/or 5P;

(iii) 52E, 55I, 57Y, 104A/105F, 107D, G, 109A, G, 76G, 84Y, 362K, 273L, Q, 285G, R, 286Q, 294T, 299L, 331K/55D and/or 351Y;

(iv) 1 ^*, 1 ^*/ 2 ^*or 1 ^*/ 2 ^*/ 3 ^*;

V () be K179 wherein, T180, T181, E182, K183, S184, T185 and K186 are replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL;

(vi) 119R, K and/or 411R, K;

(vii) 107E and/or 164E, D;

(viii) 362R, K, 276R, K, 379R, K, 409D, E, 223E, 385D, 46D, E, 410D, E and/or 82E;

(ix) 218Q, 324N, 200R, K, 121D, 196Q, 202N, 406A, 167Q, 53V, Q, 241Q, 314N, G, 239Q and/or 285N;

(x)114H/115Q/116E/117K/118M/119G/120T/121M/122D/123P/124T，

114H/115Q/116Q/117D/118I/119K/120Q/121V/122D/123S/124L，

114H/115Q/116P/117E/118I/119G/120K/121M/122D/123P/124V，

114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L，

114H/115Q/116Q/117D/118I/119K/120Q/121A/122D/123P/124L，

114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L or

114N/115Q/116A/117D/118L/119K/120K/121T/122D/123P/124L；

(xi) 31T, 74A, 171T, 203T, 281H, 308A, and/or 316D; And/or

(xii)339D。

B). embodiment a) or a1) phytase, it comprises at least one following change: 1 ^*, 2 ^*, 3 ^*, 4P, 5P, 31C, T, 41P, 46C, D, E, 52C, E, 53V, Q, 55D, I, 57Y, 59C, 74A, 76G, 82E, 84Y, 91C, P, 99C, 100C, 104A, 105F, 107D, E, G, 109A, G, 111P, 114H, N, T, 115Q, 116A, E, P, T, Q, 117D, E, K118I, L, M, T119G, K, R, S, 120K, S, T, Q, 121A, D, M, P, T, V, 122D, 123P, S, 124L, T, V, 136P, 137P, 141C, 154P, 161P, 162C, 164D, E, 167Q, 171T, 176C, 177C, 179G, I, K, N, Q, 180A, E, G, T, 181D, G, I, K, 182H, K, S, Q, 183A, L, P, S, V, Q, 184 ^*, 185 ^*, 186 ^*, 196Q, 199C, 200K, R, 202N, 203T, 218Q, 223E, 239Q, 240P, 241Q, 247C, 273L, Q, 276K, R, 281H, 282P, 283P, 284P, 285G, N, R, 286K, Q, 289P, 294T, 299L, 308A, 314G, N, 316D, 324N, 331K, 339D, 351Y, 355P, 362K, R, 379K, R, 385D, 406A, 409D, E, 410D, E and/or 411R, K, and/or wherein replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL at the amino acid of position 179,180,181,182,183,184,185 and 186.

C). the phytase above any one of embodiment, it comprises at least one following change:

(i) 31C, 46C, 52C, 59C, 91C, 99C, 100C, 141C, 162C, 176C, 177C, 199C and/or 247C;

(ii) 4P, 5P, 41P, 91P, 111P, 136P, 137P, 154P, 161P, 240P, 282P, 283P, 284P, 289P and/or 355P;

(iii) 52E, 55D, I, 57Y, 76G, 84Y, 104A, 105F, 107D, G, 109A, G, 121T, 273L, Q, 285G, R, 286Q, 294T, 299L, 331K, 351Y and/or 362K;

(iv) 1 ^*, 1 ^*/ 2 ^*or 1 ^*/ 2 ^*/ 3 ^*;

V () be K179 wherein, T180, T181, E182, K183, S184, T185 and K186 are replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL;

(vi) 119K, R and/or 411K, R;

(vii) 107E and/or 164D, E;

(viii) 46D, E, 82E, 223E, 276K, R, 362K, R, 379K, R, 385D, 409D, E and/or 410D, E;

(ix) 53V, Q, 121D, 167Q, 196Q, 200K, R, 202N, 218Q, 239Q, 241Q, 285N, 314G, N, 324N and/or 406A;

(x) 114H, N, T115Q, 116A, E, P, T, Q, 117D, E, K, 118I, L, M, T119G, K, S, 120K, S, T, Q, 121A, M, P, T, V, 122D, 123P, S and/or 124L, T, V;

(xi) 31T, 74A, 171T, 203T, 281H, 308A and/or 316D; And/or

(xii)339D。

C1). the phytase any one of above-mentioned embodiment, it comprises at least one following change:

(i) 31C, 46C, 52C, 59C, 91C, 99C, 100C, 141C, 162C, 176C, 177C, 199C and/or 247C, preferred 52C, 99C, 141C and/or 199C;

(ii) 4P, 5P, 41P, 91P, 111P, 136P, 137P, 154P, 161P, 240P, 282P, 283P, 284P, 289P and/or 355P, preferred 4P, 5P, 111P;

(iii) 52E, 55D, I, 57Y, 76G, 84Y, 104A, 105F, 107D, G, 109A, G, 121T, 273L, Q, 285G, R, 286Q, 294T, 299L, 331K, 351Y and/or 362K, preferred 57Y, 76G, 107G, 273L, 286Q and/or 362K;

(iv) 1 ^*, 1 ^*/ 2 ^*or 1 ^*/ 2 ^*/ 3 ^*;

V () be K179 wherein, T180, T181, E182, K183, S184, T185 and K186 are by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL, and preferred KEKKV replaces;

(vi) 119K, R and/or 411K, R, preferred 119K;

(vii) 107E and/or 164D, E;

(viii) 46D, E, 82E, 223E, 276K, R, 362K, R, 379K, R, 385D, 409D, E and/or 410D, E preferred 46E, 223E362K, R and/or 379K, R;

(ix) 53V, Q, 121D, 167Q, 196Q, 200K, R, 202N, 218Q, 239Q, 241Q, 285N, 314G, N, 324N and/or 406A, preferred 53V, 121D, 196Q, 200K, 202N, 218Q, 241Q, 314N and/or 406A;

(x) 114H, N, T115Q, 116A, E, P, T, Q, 117D, E, K, 118I, L, M, T, 119G, K, S, 120K, S, T, Q, 121A, M, P, T, V, 122D, 123P, S and/or 124L, T, V preferred 114T115Q, 116A, T, 117D, 118T119K, S, 120S, 121P, 122D, 123P and/or 124L;

(xi) 31T, 74A, 171T, 203T, 281H, 308A and/or 316D; And/or

(xii)339D。

D). the phytase above any one of embodiment, it has the characteristic of improvement.

E). embodiment c) or phytase c1), it comprises the characteristic (i) of embodiment 3, (ii), (iii), (iv), (v), (vi), (vii), (viii), (x), at least one in one or more changes of (xi) and/or (xii), and there is the thermostability of improvement.

F). embodiment c) or phytase c1), it comprises embodiment c) characteristic (ix) and/or (x) one or more changes at least one, and there is the pH curve of improvement.

G). embodiment c) or phytase c1), it comprises embodiment c) characteristic (x) one or more changes at least one, and there is the specific activity of improvement.

H). embodiment c) or phytase c1), it comprises embodiment c) characteristic (xi) one or more changes at least one, and there is the glycosylation pattern of amendment.

I). embodiment c) or phytase c1), it comprises embodiment c) the change of characteristic (xii), that it changes potential proteolytic cleavage site.

J). comprise a1)-a5) and embodiment a)-d c1)) any one of phytase, it comprises at least one following change:

(i) 141C/199C, 91C/46C, 52C/99C, 31C/176C, 31C/177C, 59C/100C and/or 162C/247C;

(ii) 41P, 91P, 136P, 137P, 154P, 161P, 355P, 111P, 240P, 282P, 283P, 284P, 289P, 4P and/or 5P;

(iii) 52E, 55I, 57Y, 104A/105F, 107D, G, 109A, G, 76G, 84Y, 121T, 362K, 273L, Q, 285G, R, 286Q, 294T, 299L, 331K/55D and/or 351Y;

(iv) 1 ^*, 1 ^*/ 2 ^*or 1 ^*/ 2 ^*/ 3 ^*;

V () be K179 wherein, T180, T181, E182, K183, S184, T185 and K186 are replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL;

(vi) 119R, K and/or 411R, K;

(vii) 107E and/or 164E, D;

(viii) 362R, K, 276R, K, 379R, K, 409D, E, 223E, 385D, 46D, E, 410D, E and/or 82E;

(ix) 218Q, 324N, 200R, K, 121D, 196Q, 202N, 406A, 167Q, 53V, Q, 241Q, 314N, G, 239Q and/or 285N;

(x)114H/115Q/116E/117K/118M/119G/120T/121M/122D/123P/124T，

114H/115Q/116Q/117D/118I/119K/120Q/121V/122D/123S/124L，

114H/115Q/116P/117E/118I/119G/120K/121M/122D/123P/124V，

114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L，

114H/115Q/116Q/117D/118I/119K/120Q/121A/122D/123P/124L，

114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L or

114N/115Q/116A/117D/118L/119K/120K/121T/122D/123P/124L；

(xi) 31T, 74A, 171T, 203T, 281H, 316D and/or 308A; And/or

(xii)339D。

K). comprise a1)-a5) and embodiment a)-d c1)) any one of phytase, comprise at least one following change:

(i) K141C/V199C, Q91C/W46C, G52C/A99C, N31C/E176C, N31C/T177C, G59C/F100C and/or S162C/S247C;

(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P, T283P, T284P, G289P, N4P and/or G5P;

(iii) G52E, V55I, E57Y, L104A/A105F, K107D, G, Q109A, G, T76G, A84Y, N121T, I362K, M273L, Q, E285G, R, N286Q, V294T, I299L, E331K/V55D and/or F351Y;

(iv) E1 ^*, E1 ^*/ E2 ^*or E1 ^*/ E2 ^*/ Q3 ^*;

V () be K179 wherein, T180, T181, E182, K183, S184, T185 and K186 replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL;

(vi) E119R, K and/or E411R, K;

(vii) K107E and/or R164E, D;

(viii) I362R, K, T276R, K, I379R, K, V409D, E, Q223E, N385D, W46D, E, T410D, E and/or Q82E;

(ix) E218Q, D324N, T200R, K, N121D, E196Q, D202N, E406A, E167Q, E53V, Q, E241Q, D314N, G, E239Q and/or E285N;

(x) Y114H/K116E/D117K/E118M/E119G/K120T/N121M/L124T, Y114H/K116Q/E118I/E119K/K120Q/N121V/P123S, Y114H/K116P/D117E/E118I/E119G/N121M/L124V, Y114T/K116A/E118T/E119S/K120S/N121P, Y114H/K116Q/E118I/E119K/K120Q/N121A, Y114T/K116T/E118T/E119S/K120S/N121P or Y114N/K116A/E118L/E119K/N121T;

(xi) N31T, N74A, N171T, N203T, N281H, N316D and/or N308A; And/or

(xii)R339D。

1). embodiment k) phytase, it is the variant of SEQ ID NO:2.

M). comprise a1)-a5) and embodiment a)-d c)) any one of phytase, it comprises at least one following change:

(i) T141C/V199C, Q91C/W46C, G52C/A99C, D31C/E176C, D31C/T177C, G59C/F100C and/or S162C/S247C;

(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P, T283P, T284P, G289P, N4P and/or G5P;

(iii) G52E, V55I, E57Y, L104A/A105F, K107D, G, Q109A, G, T76G, A84Y, I362K, M273L, Q, E285G, R, N286Q, V294T, I299L, E331K/V55D and/or F351Y;

(iv) E1 ^*, E1 ^*/ E2 ^*or E1 ^*/ E2 ^*/ Q3 ^*;

V () be K179 wherein, T180, T181, E182, K183, S184, T185 and K186 are by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL;

(vi) E119R, K and/or E411R, K;

(vii)K107E，R164E，D；

(viii)I362R，K，T276R，K，I379R，K，V409D，E，Q223E，N385D，W46D，E，T410D，E，Q82E；

(ix) E218Q, D324N, T200R, K, T121D, E196Q, D202N, E406A, E167Q, E53V, Q, E241Q, D314N, G, E239Q and/or E285N;

(x) Y114H/K116E/D117K/E118M/E119G/K120T/T121M/L124T, Y114H/K116Q/E118I/E119K/K120Q/T121V/P123S, Y114H/K116P/D117E/E118I/E119G/T121M/L124V, Y114T/K116A/E118T/E119S/K120S/T121P/, Y114H/K116Q/E118I/E119K/K120Q/T121A/, Y114T/K116T/E118T/E119S/K120S/T121P or Y114N/K116A/E118L/E119K;

(xi) N74A, N171T, N203T, N281H, N316D and/or N308A; And/or

(xii)R339D。

N). embodiment m) phytase, it is the variant of SEQ ID NO:4.

O). comprise a1)-a5) and embodiment a)-d c1)) any one of phytase, it comprises at least one following change:

(i) K141C/V199C, Q91C/W46C, G52C/A99C, D31C/E176C, D31C/T177C, G59C/F100C and/or S162C/S247C;

(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P, T283P, T284P, G289P, N4P and/or G5P;

(iii) G52E, V55I, E57Y, L104A/A105F, K107D, G, Q109A, G, T76G, A84Y, N121T, I362K, M273L, Q, E285G, R, N286Q, V294T, I299L, E331K/V55D and/or F351Y;

(iv) E1 ^*, E1 ^*/ E2 ^*or E1 ^*/ E2 ^*/ Q3 ^*;

V () be K179 wherein, T180, T181, E182, K183, S184, T185 and K186 replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL;

(vi) E119R, K and/or E411R, K;

(vii) K107E and/or R164E, D;

(viii) I362R, K, T276R, K, I379R, K, V409D, E, Q223E, N385D, W46D, E, T410D, E and/or Q82E;

(ix) E218Q, D324N, T200R, K, N121D, E196Q, D202N, E406A, E167Q, E53V, Q, E241Q, D314N, G, E239Q and/or E285N;

(x) Y114H/K116E/D117K/E118M/E119G/K120T/N121M/L124T, Y114H/K116Q/E118I/E119K/K120Q/N121V/P123S, Y114H/K116P/D117E/E118I/E119G/N121M/L124V, Y114T/K116A/E118T/E119S/K120S/N121P, Y114H/K116Q/E118I/E119K/K120Q/N121A, Y114T/K116T/E118T/E119S/K120S/N121P or Y114N/K116A/E118L/E119K/N121T;

(xi) N74A, N171T, N203T, N281H and/or N308A; And/or

(xii)R339D。

P). embodiment o) phytase, it is the variant of SEQ ID NO:3.

Q). comprise a1)-a5) and embodiment a)-d c1)) any one of phytase, it comprises at least one following change:

(i) K141C/V199C, Q91C/W46C, G52C/A99C, N31C/E176C, N31C/T177C, G59C/F100C and/or S162C/S247C;

(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P, T283P, T284P, G289P, N4P and/or G5P;

(iii) G52E, V55I, E57Y, L104A/A105F, K107D, G, Q109A, G, T76G, A84Y, N121T, I362K, M273L, Q, E285G, R, N286Q, V294T, I299L, V55D and/or F351Y;

(iv) E1 ^*, E1 ^*/ E2 ^*or E1 ^*/ E2 ^*/ Q3 ^*;

V () be K179 wherein, T180, T181, E182, K183, S184, T185 and K186 replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL;

(vi) E119R, K and/or E411R, K;

(vii) K107E and/or R164E, D;

(viii) I362R, K, T276R, K, I379R, K, V409D, E, Q223E, N385D, W46D, E, T410D, E and/or Q82E;

(ix) E218Q, D324N, T200R, K, N121D, E196Q, D202N, E406A, E167Q, E53V, Q, E241Q, D314N, G, E239Q and/or E285N;

(x) Y114H/K116E/D117K/E118M/E119G/K120T/N121M/L124T, Y114H/K116Q/E118I/E119K/K120Q/N121V/P123S, Y114H/K116P/D117E/E118I/E119G/N121M/L124V, Y114T/K116A/E118T/E119S/K120S/N121P, Y114H/K116Q/E118I/E119K/K120Q/N121A, Y114T/K116T/E118T/E119S/K120S/N121P or Y114N/K116A/E118L/E119K/N121T;

(xi) N31T, N74A, N171T, N203T, N281H, N316D and/or N308A; And/or

(xii)R339D。

R). embodiment q) phytase, it is the variant of SEQ ID NO:6.

S). comprise a1)-a5) and embodiment a)-d c1)) any one of phytase, it comprises at least one following change:

(i) K141C/V199C, Q91C/W46C, G52C/A99C, D31C/E176C, D31C/T177C, G59C/F100C and/or S162C/S247C;

(ii) D41P, Q91P, N136P, T137P, L154P, S161P, T355P, Q111P, K240P, G282P, T283P, T284P, G289P, N4P and/or G5P;

(iii) G52E, V55I, E57Y, L104A/A105F, K107D, G, Q109A, G, T76G, A84Y, I362K, M273L, Q, E285G, R, N286Q, V294T, I299L, E331K/V55D and/or F351Y;

(iv) E1 ^*, E1 ^*/ E2 ^*or E1 ^*/ E2 ^*/ P3 ^*;

V () be K179 wherein, T180, T181, E182, K183, S184, T185 and K186 replaced by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL;

(vi) E119R, K and/or E411R, K;

(vii) K107E and/or R164E, D;

(viii) I362R, K, T276R, K, I379R, K, V409D, E, Q223E, N385D, W46D, E, T410D, E and/or Q82E;

(ix) E218Q, D324N, T200R, K, T121D, E196Q, D202N, E406A, E167Q, E53V, Q, E241Q, D314N, G, E239Q and/or E285N;

(x)Y114H/K116E/D117K/E118M/E119G/K120T/T121M/L124T，

Y114H/K116Q/E118I/E119K/K120Q/T121V/P123S，

Y114H/K116P/D117E/E118I/E119G/T121M/L124V，

Y114T/K116A/E118T/E119S/K120S/T121P，

Y114H/K116Q/E118I/E119K/K120Q/T121A，

Y114T/K116T/E118T/E119S/K120S/T121P or

Y114N/K116A/E118L/E119K；

(xi) D31T, N74A, N171T, N203T, N281H, N316D and/or N308A; And/or

(xii)R339D。

T). embodiment s) phytase, it is the variant of SEQ ID NO:9.

U). the nucleotide sequence of separation, it comprises code embodiment a)-t) comprise a1)-a5) and c1) any one of the nucleotide sequence of phytase.

V). nucleic acid construct, it comprises embodiment u) nucleotide sequence, this nucleotide sequence is operably connected to the one or more regulating and controlling sequences instructing and produce phytase in suitable expressive host.

W). recombinant expression vector, it comprises embodiment v) nucleic acid construct.

v).

X). recombinant host cell, it comprises embodiment v) nucleic acid construct and/or embodiment w) expression vector.

Y). for generation of embodiment a)-t) comprise a1)-a5) and c1) any one of the method for phytase, it comprises

A () cultivates embodiment x) host cell to produce the supernatant liquor comprising phytase; (b) phytase is reclaimed.

Z). transgenic plant, or plant part, it can express embodiment a)-t) comprise a1)-a5) and c1) any one of phytase.

Ae). genetically modified, non-human animal, or its product, or component, it can express embodiment a)-t) comprise a1)-a5) and c1) any one of phytase.

Oe). composition, it comprises embodiment a)-t) comprise a1)-a5) and c1) any one of at least one phytase, and

(a) at least one liposoluble vitamin;

(b) at least one water-soluble vitamins; And/or

(c) at least one trace minerals.

Aa). embodiment oe) composition, it comprises at least one enzyme in the enzyme being selected from lower group further: amylase, phytase, Phosphoric acid esterase, zytase, Galactanase, alpha-galactosidase, proteolytic enzyme, Phospholipid hydrolase, and/or beta-glucanase.

Bb). embodiment oe)-aa) and any one of composition, it is animal feedstuff additive.

Cc). animal feedstuff compositions, it has the crude protein content of 50 to 800g/kg and comprises embodiment a)-t) comprise a1)-a5) and c1) any one of phytase or embodiment oe)-aa) and any one of composition.

Dd). for improvement of the method for animal feed nutritive value, wherein by embodiment a)-t) comprise a1)-a5) and c1) any one of phytase or embodiment oe)-aa) and any one of composition be added in feed.

Ee). for reducing the method for animal excrement mysoinositol six phosphate level, it comprises the embodiment cc by significant quantity) forage feed animal.

Ff). for the treatment of the method for vegetable-protein, it comprises embodiment a)-t) comprise a1)-a5) and c1) any one of phytase or embodiment oe)-aa) and any one of composition add step at least one vegetable-protein or protein source to.

Gg). embodiment a)-t) comprise a1)-a5) and c1) any one of phytase or embodiment oe)-aa) and any one of the purposes of composition in animal-feed; Preparing the purposes in animal-feed; For improvement of the purposes of the nutritive value of animal-feed; For reducing the purposes of the phytinic acid salt level in animal excrement; For the treatment of the purposes of vegetable-protein; Or for discharging the purposes of phosphorus from phytinic acid enzyme substrates.

The scope of description and claimed invention is not restricted to embodiment disclosed herein, because these embodiments are intended to the explanation as several aspect of the present invention herein.Any equivalent embodiment should be within the scope of the invention.Really, except herein show and except describing will be apparent to those skilled in the art to various modification of the present invention.These modifications also should be within the scope of the appended claims.In case of conflict, be as the criterion to comprise the disclosure defined herein.

The various reference papers quoted herein, are incorporated to by reference by its disclosure herein.

Embodiment

Chemical agent used is at least SILVER REAGENT commodity.

Embodiment 1: the preparation of variant, and the detection of temperature stability and pH curve

the preparation of inositol six-phosphatase variants

The DNA that coding has the inositol six-phosphatase variants of the aminoacid sequence of SEQ ID NO:2 is produced by methods known in the art, and by standard technique construct be blended in by the DNA of the coded signal peptide described in Biosci.Biotechnol.Biochem.56:1455 (1992) such as Takami by PCR and be integrated into by homologous recombination in the genome of Bacillus subtillis host cell (see (1990) such as Diderichsen, J.Bacteriol., 172,4315-4321).Gene is expressed and is purified by ordinary method the phytinic acid zymoprotein obtained under the control of three promoter systems (as described in WO 99/43835).

the mensuration of temperature stability

The temperature stability of inositol six-phosphatase variants can be measured by following manner: 500 microlitre protein solutions (the SEQ ID NO:2 of variant and reference protein, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:6) there are about 10 micrograms of protein every milliliter and be dissolved in 0.1M sodium acetate buffer, in pH5.5, this protein solution is divided into two portions, a part is in the temperature (such as 50 DEG C of the rising expected, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C or 85 DEG C) incubation in plastic containers, another part is stored in 5 DEG C.Protein solution is transferred to ice bath after 30 minutes by incubation at elevated temperatures, and is measured the activity of the sample of sample that (deduction buffer blank (buffer blind subtracted)) cool and heating by following phosphatase assay.Residual activity is defined as the activity (with % form) of the activity after thermal treatment divided by the sample of cooling.If the residual activity in Phosphoric acid esterase or phytinic acid enzyme assay is higher compared with reference, then think that variant is to temperature more stable (thermally-stabilised).

the mensuration of phosphatase activity

The enzyme solution that 75 microlitres contain phytase is assigned in micro titer plate well (microtiter platewell) such as NUNC 269620, and add 75 il of substrate (for preparing substrate, by two 5mg p-nitrophenyl phosphoric acid tablet (Sigma, catalog number (Cat.No.) N-9389) be dissolved in 10ml 0.1M sodium acetate buffer, pH 5.5).By plate seal then incubation 15 minutes, at 37 DEG C with 750rpm wave and culture.After incubation period, add 75 microliter termination reagent (stopping reagent is 0.1M disodium tetraborate in water) and on microtiter plate spectrophotometer, be determined at the absorbancy of 405nm.A phosphatase unit is defined as the enzymic activity (deduction buffer blank) that per minute under the given reaction conditions discharges 1 micromole's phosphate radical.The absorbancy measuring 1 micromole's p-nitrophenol is under test conditions 56AU (AU=absorbance units).

dsc measurement

Differential scanning calorimetry (DSC) can carry out with the VP-DSC of Micro Cal under various pH-value.Scanning is carried out under constant scan rate 1.5 DEG C/min, scope 20-90 DEG C.Before beginning DSC, phytase is used in appropriate buffer (such as 0.1M glycine-HCl, pH 2.5 or 3.0; 20mM sodium acetate pH 4.0; 0.1M sodium acetate, pH 5.5; 0.1M Tris-HCl, pH 7.0) middle NAP-5 post (Pharmacia) desalination balanced.Data processing uses MicroCal Origin software (MicroCal Originsoftware) (edition 4 .10) to carry out, and the tip temperature at peak in denaturation temperature Td (also referred to as melting temperature(Tm), Tm) being defined as thermolysis curve (thermogram).

the pH curve revised: the mensuration of pH 3.5/5.5 activity ratio

The correction of the pH curve of inositol six-phosphatase variants can be determined as follows: (the 0.1M acetate buffer when pH 3.5, pH 3.5) and pH5.5 time (0.1M acetate buffer, pH 5.5) measure activity, all deduct buffer blank in two kinds of situations.The activity measured when being used in pH 3.5, divided by the activity measured during pH 5.5, is divided by (see below) by two absorption values.For measuring activity, the supernatant liquor of variant and reference is suitably diluted (such as 1:5000) in respective damping fluid.75 microlitres enzyme solution is separately assigned in micro titer plate well such as NUNC 269620, and add 75 microlitres and there is the substrate of corresponding pH (by respectively at 10ml 0.1M sodium acetate buffer, pH 5.5 and 10ml 0.1M acetate buffer, dissolve two 5mg p-nitrophenyl phosphoric acid tablets (Sigma, catalog number (Cat.No.) N-9389) in pH 3.5 and prepare substrate).By plate seal and incubation 15 minutes, at 37 DEG C with 750rpm wave and culture.After incubation period, add 75 microliter termination reagent (the 0.1M disodium tetraborate aqueous solution in water), and measure absorbancy at 405nm in microtiter plate spectrophotometer.

the mensuration of phytase activity

To suitably dilute (such as at 0.25M sodium acetate, 0.005% (w/v) Tween-20.pH5.5 first) 75 microlitres containing the enzyme solution of phytase be assigned to micro-come in that titer plate well such as NUNC 269620, and add 75 il of substrate (by 10ml 0.25M sodium acetate buffer, dissolve 100mg in pH 5.5 and prepare from the sodium phytate (Aldrich catalog number (Cat.No.) 274321) of rice).By plate seal and incubation 15 minutes, at 37 DEG C with 750rpm wave and culture.Add 75 microliter termination reagent after incubation period and (stop reagent by mixing 10ml molybdate solution (10% (w/v) seven-ammonium molybdate is in 0.25% (w/v) ammonia solution); Prepared by 10ml ammonium vanadate (commerical prod of 0.24%Bie & Berntsen, catalog number (Cat.No.) LAB17650) and 21.7% (w/v) nitric acid), microtiter plate spectrophotometer is measured the absorbancy of 405nm.Phytase activity represents with FYT unit, and a FYT is that per minute discharges the enzyme amount of 1 micromole inorganic ortho-phosphoric acid root under these conditions.The typical curve prepared by reference to the suitable diluent from inorganic phosphate or with reference to (such standard enzyme prepared product with known activity can from Novozymes A/S from the phytase prepared product of known activity, Krogshoejvej 36, DK-2880 Bagsvaerd asks for acquisition) diluent make typical curve to obtain the absolute value of the phytase activity of measurement.

Embodiment 2: expressive host/glycosylation is on the impact of thermostability

expression in bacillus

The phytase of SEQ ID NO:2 is expressed as described in Example 1 in subtilis, and purify by ordinary method: centrifugal, microbe filter (germ filtration), ammonium sulfate precipitation (80% ammonium sulfate saturated solution), centrifugal, at buffer A (50mM sodium acetate, 1.5M ammonium sulfate pH 4.5) in resuspended precipitation, filter, hydrophobic interaction chromatography (hydrophobic interaction chromatography) (Phenyl Toyopearl, use buffer A loading, with buffer B (50mM sodium acetate pH 4.5) wash-out) and cation-exchange chromatography (SP-agarose, by 10mM Trisodium Citrate pH 4.0 loading, with linear salt gradient (10mM Trisodium Citrate pH4.0+1M NaCl wash-out)).

expression in Pichia

Further, the phytase of SEQ ID NO:2 is expressed in pichia pastoris phaff, as by Rodriguez etc. at Archives of Biochemistry and Biophysics, vol.382, no.1, to summarize in 2000, pp.105-112 and describe.As following purifying phytase from the supernatant of fermentation culture: by ammonium sulfate (80% saturated solution) precipitation, dissolve again in 10ml 25mM sodium acetate buffer pH4.5, relative to same buffer dialysis, and by 0.45mm frit.This solution of 150ml is added to 40ml SP Sepharose FF post (Pharmacia) balanced with same buffer pH 4.5, by albumen linear NaCI gradient (0-0.5M) wash-out.Analyze the phytase activity from the fraction of post.Detected the fraction with phytase activity by SDS-PAGE, and collect (pool) pure fraction.Protein concentration is measured by using BCA test kit (Pierce).

by the thermostability that DSC measures

The phytase of the SEQ ID NO:2 that Pichia and bacillus are expressed is carried out to the measurement of the thermostability by differential scanning calorimetry (DSC).

Sample preparation:

By sample (volume is less than 3ml), in cold house, (about 5 degrees Celsius) dialyse minimum 1 hour relative to the 20mM sodium acetate buffer pH 4.0 of 500ml.Sample is transferred to 500ml fresh, cold buffer formulation dialysed overnight.By sample 0.45 micrometer syringe frit, with dialysis buffer liquid by volume-adjustment extremely about 1.5ml, and record A ₂₈₀(absorbancy at 280nm place).Dialysis buffer liquid is used as a reference in DSC scanning.With vacuum take-off and stir about 10 minutes is sample vent gas.

During the sample preparation of the phytase of Pichia expression, (dialysing relative to 20mM sodium acetate (NaAc) pH 4.0) defines precipitation.Supernatant liquor is used for first experiment.Then the remainder of the liquid storage of purifying is dialysed relative to 20mM NaAc pH 4.0, under making some the low Mw contamination precipitations existed in this batch like this.This batch is used for second experiment, this experiment discloses the Tm (54 to 55 DEG C) closely similar with first experiment.

DSC tests:

Application MicroCal ^tMthe Setup Experiments of VP-DSC equipment: scanning speed: 90K/h.Scanning interval: 20-90 degree Celsius.Feedback model: nothing.The filtration phase (filtering period): 16 seconds.

The enzyme concn of sample is about 1-1.5mg/ml, as passed through A ₂₈₀estimate with Theoretical Calculation optical extinction coefficient (Vector NTI version 9.0.0) at 280nm.Temperature (Td is folded in pyrolysis; Thermal unfoldingtemperature) assess with MicroCal Origin software (edition 4 .10), and denaturation temperature is defined as the summit temperature of thermolysis curve.

Sum up in result table 2 below.

table 2

Table 2 clearly illustrates that the thermostability of the phytase that the phytase that Pichia is expressed is expressed than bacillus is very different.

The phytase that Pichia is expressed has severe glycosylation, and as used mass spectroscopy (Maldi-TOF) by seen by wide in range molecular weight ranges, the phytase that bacillus is expressed then does not have glycosylation.

Embodiment 3: inositol six-phosphatase variants R339D

Preparation has the phytase protein engineered variants of the SEQ ID NO:2 replacing R339D, and expresses by methods known in the art in aspergillus oryzae.The denaturation temperature Td using DSC as described in Example 2 to measure it is 62.5 DEG C (20mM acid sodium, pH 4.0).

In addition, R339D replaces for removing and express potential relevant Kex2 proteolytic cleavage site in Aspergillus.

Embodiment 4: the animal-feed and the animal feedstuff additive that comprise inositol six-phosphatase variants

animal feedstuff additive

The formulation of the inositol six-phosphatase variants R339D comprising the SEQ ID NO:2 of 0.15g phytase zymoprotein is added into following pre-composition (every kilogram of pre-composition):

5000000 IE vitamin A

1000000 IE Vitamin D3 500,000 I.U/GMs

13333 mg vitamin-Es

1000 mg vitamin K3s

750 mg VITMAIN B1

2500 mg Lin Suanna Vitamin B2 Sodium Phosphates

1500 mg vitamin B6s

7666 mcg vitamin B12

12333 mg nicotinic acid

33333 mcg vitamin Hs

300 mg folic acid

3000 mg D-VB5 calcium

1666 mg Cu

16666 mg Fe

16666 mg Zn

23333 mg Mn

133 mg Co

66 mg I

66 mg Se

5.8 % calcium

25 % sodium

animal-feed

This is the example of animal-feed (chicken feed (broilerfeed)), and this feed comprises the inositol six-phosphatase variants R339D (calculating with phytase zymoprotein) of 1.5mg/kg (1.5ppm) SEQ ID NO:2:

62.55% corn

33.8% soyflour (50% crude protein, CP)

1.0% soybean oil

0.2% DL-methionine(Met)

0.22% DCP (Si Liaodengji dicalcium phosphate feed grade)

0.76% CaCO ₃(calcium carbonate)

0.32% is husky

0.15% NaC1 (sodium-chlor)

1% above-mentioned pre-composition

Each composition is mixed, and the temperature (such as 60,65,75,80,85,90 or even 95 DEG C) that feed is being expected is made particle.

Embodiment 5: the mensuration of temperature stability

Prepare eight variants (change compared with SEQ ID NO:2 shows in the following Table 3) of SEQ ID NO:2 as described in example 1 above.Prepare in the same manner with reference to phytase for two with SEQ ID NO:2 and SEQ ID NO:3.

Temperature stability measures as follows:

Variant and reference protein 200 RI of supernatant are separately divided into two portions, and a part is at 50 DEG C of incubations in plastic containers, and another part is kept at 5 DEG C.After 30 minutes, protein solution is transferred to ice bath at 50 DEG C of incubations.With 1:100 at 0.1M sodium acetate buffer, in pH 5.5 after dilution, that measure cooling by the phosphatase assay in embodiment 1 (" mensuration of phosphatase activity ") with the activity of sample that is heating, deduction buffer blank.Result is shown as respectively 5 DEG C and the enzymic activity of 50 DEG C of incubations after 30 minutes (representing with absorbance units (AU)) in the following Table 3, and residual activity (RA) is calculated as with the activity of the activity of heat treated sample (50 DEG C of incubations) divided by cooling sample (5 DEG C of incubations), represent with %.

table 3: the inositol six-phosphatase variants with the thermostability of improvement

Embodiment 6: the performance in animal-feed in vitro in model

Compare in the performance of inositol six-phosphatase variants in animal-feed in vitro model with the performance of reference protein, described reference protein is as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:6.External model is simulated the gastrointestinal conditions of monogastric animal and is fully associated with the result that animal experiment in body obtains.Relatively be performed as follows:

Phytase activity in variant sample measures as described in embodiment 1 " mensuration of phytase activity ".

Then prepare the Feed Sample be made up of 30% soyflour and 70% Semen Maydis powder, add CaCl ₂be every kilogram of feed 5g calcium to concentration, and 40 DEG C and pH 3.0 preincubation 30 minutes, the phytase (all phytases that will detect are applied with dose to compare) of following interpolation stomach en-(3000U/g feed) and suitable dosage, such as 0.25 to 0.75 phytase unit FYT/g feed.Also comprise without phytase activity blank as a reference.Thereafter by sample at 40 DEG C and pH 3.0 incubation 60 minutes, then pH 4.0 incubation 30 minutes.

By adding HCl to final concentration 0.5M and 40 DEG C of incubations 2 hours, then carrying out a freeze-thaw cycle and 40 DEG C of incubations 1 hour, thus reaction terminating being extracted phytinic acid and phosphoinositide.

By high-effect ionic chromatography if Chen etc. is at Journal of Chromatography A (2003) vol.1018, described in pp.41-52, phytinic acid is separated with phosphoinositide, and if Skoglund etc. is in J.Agric.Food Chem. (1997), carry out described in vol.45, pp.431-436 quantitatively.

Then the phosphorus of release is calculated as the difference of the phosphorus (IP-P) that phosphoinositide combines between phytase treatment and untreated sample.The relative performance of variant is calculated as the per-cent of the phosphorus relative to contrast phytinic acid enzyme r e lease.

As mentioned above with the external performance of multiple inositol six-phosphatase variants of the dosimetry SEQ ID NO:2 of 125FYT/kg feed.

The result of the phytase of supernatant liquor and purifying is shown to show in 4A and 4B respectively below.Remaining IP6-P refer to incubation in vitro after the amount of remaining IP6-P (phytinic acid phosphorus), represent with mg/gDM (dry-matter).The IP6-P of degraded is determined as the difference between blank remaining IP6-P and the remaining IP6-P of each sample.Finally, in the end row, represent the IP6-P of degraded relative to the phytase with SEQ ID NO:2.In the averages that table 4A empty and reference (SEQ ID NO:2) value are multiple independent mensuration, and other values are based on unitary determination.In the averages that table 4B hollow white value is multiple independent mensuration, and other values are based on unitary determination.

the external performance of table 4A. inositol six-phosphatase variants supernatant liquor

Variant 015,016,018,040,041,042,044 and 050 look have with SEQ IDNO:2 with 3 phytase to compare be at least equally good or better external performance.

table 4B: the external performance of the inositol six-phosphatase variants of purifying

Variant 030,031,037,056,072,085,087,089,090,095,098 seems at least equally good with the phytase of SEQ ID NO:3 with 125 performances in vitro.

Embodiment 7: specific activity

Based on relative to 250mM sodium acetate, the highly purified sample that pH 5.5 dialyses measures the specific activity of inositol six-phosphatase variants.On sds page, detecting purity in advance, only there is a component in display.

Protein concentration is measured by following amino acid analysis: by the aliquots containig of sample in the Glass tubing of finding time at 6N HCl, in 0.1% phenol 110 DEG C hydrolysis 16 hours.Carry out the amino acid of quantitative gained according to the specification sheets operation of manufacturers with Applied Biosystems420A amino acid analysis system.From amino acid whose amount, total mass and the concentration of the albumen in the aliquots containig of hydrolysis can be calculated.

Phytase activity is measured with FYT unit as described in embodiment 1 (" mensuration of phytase activity "), and specific activity being calculated as phytase activity, described phytase activity is measured with FYT unit every mg inositol six-phosphatase variants zymoprotein.

The specific activity of the phytase of SEQ ID NO:2 and variant 072 (I362R of SEQ ID NO:2) is as said determination.The specific activity of variant 072 is 86% of the phytinic acid specific enzyme activity of SEQ ID NO:2.Uncertain (standard deviation) is estimated to be about 10%, mainly because phytase activity assay method is based on the substrate of complexity.

Embodiment 8: temperature stability

Prepare multiple variants of SEQ ID NO:2 as described in example 1 above, and by 100ml PS1 substratum (100g/L sucrose, 40g/L soybean flakes, the 10g/L Na of subtilis host strain in 500ml shaking flask ₂hPO ₄.12H ₂o, 0.1ml/L Dowfax63N10 (Dow)) in cultivate four days at 30 DEG C with 300rpm.

Prepare in the same manner with reference to phytase for two, namely there is the phytase (the variant N31D/Q139K/L197F/N316K corresponding to SEQ ID NO:2) of SEQ ID NO:3, and there is the phytase (the variant N31D/N121T/K132T/Q139K corresponding to SEQ ID NO:2) of SEQ ID NO:4.

Also comprise the phytase with SEQ ID NO:9 to carry out contrasting (the variant Q3P/N31D/N121T/K132T/Q139K corresponding to SEQ IDNO:2).

Variant and determine as follows with reference to the temperature stability of phytase:

By adding 20ul (microlitre) supernatant liquor to 180ul 0.1M sodium acetate buffer, supernatant liquor is diluted ten times by pH5.5+0.005%Tween-20.The enzyme of dilution is divided into two portions, and a part is at 60 DEG C of incubations in plastic containers, and another part is kept at 5 DEG C.Protein solution is transferred to ice bath at 60 DEG C of incubations after 30 minutes.At 0.1M sodium acetate buffer, with after 1:10 dilution in pH5.5 and 0.005%Tween-20, that measure cooling by the phosphatase assay of (determination of phosphatase activity) in embodiment 1 with activity that the is sample of heating, deduction buffer blank.

Table 5 is and the list of variants compared with phytase with the temperature stability of improvement.For each variant, this table also describes the change compared with SEQ ID NO:2.Determine respectively 5 DEG C and the enzymic activity of 60 DEG C of incubations after 30 minutes (representing with absorbance units (AU)), and residual activity (RA) is calculated as come with the activity of the activity of heat treated sample (60 DEG C of incubations) divided by cooling sample (5 DEG C of incubations).Next, by the residual activity stdn of the result of residual activity relative to the phytase of the SEQ ID NO:2 expressed in the same manner and process.The improvement factor (IF) drawn is displayed in Table 5.For the phytase of SEQ ID NO:2, IF is 1.0, with reference to phytases, then thermostability is poor compared with the phytase of SEQ ID NO:2 with 4 two for SEQ ID NO:3, and it is apparent that this IF from these two phytases is only the fact of 0.1 and 0.3 respectively.

table 5: the inositol six-phosphatase variants with the thermostability of improvement

Embodiment 9: according to the thermostability of DSC

As multiple purified variants of preparation SEQ ID NO:2 as described in summary in embodiment 1.Prepare two in the same manner with reference to phytase, namely there is the phytase (the variant N31D/Q139K/L197F/N316K corresponding to SEQ ID NO:2) of SEQ ID NO:3, and there is the phytase (the variant N31D/N121T/K132T/Q139K corresponding to SEQ ID NO:2) of SEQ ID NO:4.Also comprise there is SEQ ID NO:9 phytase for comparing (the variant Q3P/N31D/N121T/K132T/Q139K corresponding to SEQ ID NO:2).

The aliquots containig of protein sample is dialysed at 4 DEG C relative to 2 x 500ml20mM sodium acetate pH4.0 in the step of 2-3 hour, then dialysed overnight.Each sample filters through 0.45um and is diluted to about 2 A with damping fluid ₂₈₀unit.The definite absorption value recorded provides in result table.MicroCalVP-DSC carries out DSC, at 20mM sodium acetate buffer, in pH 4.0, carries out with 90 DEG C/h scanning speed within the scope of 20-90 DEG C.

The denaturation temperature (Td) obtained shows in the following Table 6, summarizes the result of three different experiments.

table 6: measured by the Td of DSC

Embodiment 10: purifying and temperature curve

Inositol six-phosphatase variants used herein and reference and comparative phytase carry out purifying as follows: the fermented supernatant fluid containing phytase is first centrifugal one hour with 7200rpm and 5 DEG C, then through four layers of Whatman glass microfibre filter paper (2.7,1.6,1.2 and 0.7 microns) multi-layered devices (sandwich) filter.Then solution is carried out sterile filtration (or through with the quick PES bottle top filter (Fast PES Bottle topfilter) of 0.22 μm of cutoff value (cut-off), or utilize pressure through Seitz-EKS depth filter (Seitz-EKS depth filter)).In solution, add solid-state ammonium sulfate, obtain final concentration 1.5M, with 6M HCl by pH regulator to 6.0.

Solution containing phytase is added on butyl-agarose post, about 50ml in XK26 post, use 25mM bis-tris (two-(2-hydroxyethyl) imino--three (methylol) methane (Bis-(2-hydroxyethyl) imino-tris (hydroxymethyl) methan))+1.5M ammonium sulfate pH6.0 as buffer A, use 25mM bis-tris pH6.0 as buffer B.Use phosphatase assay (see embodiment 1, " mensuration of phosphatase activity ") analysis from the activity of the fraction of pillar, and collect the activated fraction of tool.The fraction collected is dialysed relative to 10mM sodium acetate pH4.5 abundant (extensively).Then, the solution containing phytase is passed through chromatography purification on S agarose, and about 75ml is in XK26 post, and with 50mM sodium acetate pH4.5 as buffer A, and 50mM sodium acetate+1MNaCl pH4.5 is as buffer B.Equally, analyze the activity from the fraction of pillar and collect activated fraction.Finally, with the Amicon ultra-15 filtration unit of the film with 10kDa cutoff value, the solution of the phytase containing purifying is concentrated.

In all cases, the molecular weight (as estimated by SDS-PAGE) of all phytases is about 40kDa, and purity is greater than 95%.

The temperature curve (phytase activity is as the function of temperature) of variant substantially as described in Example 1 (" mensuration of phytase activity ") is determined the temperature range of 20-90 DEG C, but enzyme reaction (100 microlitres are containing enzyme solution+100 il of substrate of phytase) is not carried out on microtiter plate in PCR pipe.After 15 minute reaction period of desired temperature terminates, pipe is cooled to 20 DEG C and continues 20 seconds, 150 microlitre reaction mixtures are transferred on microtiter plate.Add 75 microliter termination reagent and in microtiter plate spectrophotometer, measure the absorbancy at 405nm.Result is summed up in the following Table 7.The numeral provided for each temperature (20-90 DEG C, with 10 DEG C for one-level) is relative to the standardized relative reactivity of optimum value (representing with %).

table 7: temperature curve

Variant 030,031,037,044,056,062,072,083 has higher relative reactivity with comparing with 102 with reference to phytase 026 with 093 70 DEG C time.

Embodiment 11:pH curve

The pH curve (phytase activity is as the function of pH) of the multiple variant used in preceding embodiment and same reference and comparative phytase 37 DEG C 2.0 to 7.5 pH scope (with 0.5 pH unit for one-level) as described in Example 1 (" mensuration of phytase activity ") determine, damping fluid mixed solution (cocktail) (50mM glycine, 50mM acetic acid and 50mMBis-Tris) is just used to instead of 0.25M sodium acetate pH5.5 damping fluid.Result is summed up in the following Table 8.The numeral provided for each pH (2.0-7.5) is relative to the standardized relative reactivity of optimum value (representing with %).

table 8:pH curve

To YC062 and YC091, pH curve (relative reactivity is as the function of pH), seem to offset by 0.5pH unit to higher pH.

And then, when for the majority of the phytase (comprising with reference to phytase 026 and 102) for table 8, optimum value is at pH3.5-pH4.0, the optimal pH observed for numbering 062,085 and 089 is 3.5, and the optimal pH observed for numbering 091 and 093 is 4.0.

Embodiment 12: temperature stability

The temperature stability of the variant being measured multiple purifying as in the previous examples by the activity measuring remaining phytase after 70 DEG C with pH 4.0 (0.1M sodium acetate) incubation and identical reference and comparative phytase.By phytase incubation, sample was withdrawn from and in cooled on ice after 0,10,30 and 60 minute.The method of (" mensuration of phytase activity ") described in embodiment 1 is used to measure the residual activity of pH 5.5.By activity normalized relative to 0 minute time of result, show in the following Table 9.

table 9: temperature stability

Above result display numbering 044,062,072 and 083 under these conditions (70 DEG C and pH4) may than with reference to phytase more stable (although this experiment in large deviation is observed for numbering 000).

Embodiment 13: calculate identity per-cent and qualification corresponding position

By the Needle program of EMBOSS software package 2.8.0 version by SEQ ID NO:9 and SEQ IDNO:2 comparison.Substitution matrix used is BLOSUM62, and it is 10.0 that breach opens point penalty, and gap extension penalty is 0.5.

The comparison result obtained represents in fig. 2.

Identity degree between SEQ ID NO:9 and SEQ ID NO:2 calculates as follows: the number of exact matching was 406 (all these represent with vertical draw (vertical stroke)).The length of most short data records is 411 (SEQ ID NO:2).The per-cent of identity is 406/411 x 100%=98.8%.

Also the comparison result of Fig. 2 is used for corresponding position of deriving, as follows: in this comparison, to be in corresponding position at the amino acid at top mutually.Amino acid Q such as on SEQ ID NO:2 position 3 corresponds to the amino acid P of SEQ ID NO:9 location number 25.For the present invention, we adopt the location number of SEQ ID NO:2.Therefore, SEQ ID NO:9 can be thought of as the variant comprising the SEQ ID NO:2 replacing Q3P.

Other difference existed with replacement form in the lap of comparison result is in position 31,121,132 and 139, i.e. N31D, N121T, K132T and Q139K.

In addition difference is present in N-end, has 22 amino acid whose extensions at N-end SEQ ID NO:9 compared with SEQ ID NO:2.

Therefore generally, SEQ ID NO:9 can be thought of as the variant of following SEQ ID NO:2:

^*0aM/ ^*0bS/ ^*0cT/ ^*0dF/ ^*0eI/ ^*0fI/ ^*0gR/ ^*0hL/ ^*0iL/ ^*OjF/ ^*0kF/ ^*0mS/ ^*0nL/ ^*0oL/ ^*0pC/ ^*0qG/ ^*0rS/ ^*0sF/ ^*0tS/ ^*0uI/ ^*0vH/ ^*0wA/Q3P/N31D/N121T/K132T/Q139K。

Sequence table

<110> Novozymes Company (Novozymes A/S)

 

<120> inositol six-phosphatase variants

 

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<170>PatentIn version 3.4

 

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His Gly Val Arg Ala Pro Thr Lys Phe Thr Pro Ile Met Lys Asn Val

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Thr Pro Asp Gln Trp Pro Gln Trp Asp Val Pro Leu Gly Trp Leu Thr

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Pro Arg Gly Gly Glu Leu Val Ser Glu Leu Gly Gln Tyr Gln Arg Leu

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Trp Phe Thr Ser Lys Gly Leu Leu Asn Asn Gln Thr Cys Pro Ser Pro

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ggg cag gtt gct gtt att gca gac acg gat caa cgc acc cgt aaa acg 288

Gly Gln Val Ala Val Ile Ala Asp Thr Asp Gln Arg Thr Arg Lys Thr

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ggt gag gcg ttt ctg gct ggg tta gca cca aaa tgt caa att caa gtg 336

Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Lys Cys Gln Ile Gln Val

100 105 110

cat tat cag aag gat gaa gaa aaa aat gat cct ctt ttt aat ccg gta 384

His Tyr Gln Lys Asp Glu Glu Lys Asn Asp Pro Leu Phe Asn Pro Val

115 120 125

aaa atg ggg aaa tgt tcg ttt aac aca ttg cag gtt aaa aac gct att 432

Lys Met Gly Lys Cys Ser Phe Asn Thr Leu Gln Val Lys Asn Ala Ile

130 135 140

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Leu Glu Arg Ala Gly Gly Asn Ile Glu Leu Tyr Thr Gln Arg Tyr Gln

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tct tca ttt cgg acc ctg gaa aat gtt tta aat ttc tca caa tcg gag 528

Ser Ser Phe Arg Thr Leu Glu Asn Val Leu Asn Phe Ser Gln Ser Glu

165 170 175

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Thr Cys Lys Thr Thr Glu Lys Ser Thr Lys Cys Thr Leu Pro Glu Ala

180 185 190

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Leu Pro Ser Glu Leu Lys Val Thr Pro Asp Asn Val Ser Leu Pro Gly

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Ala Trp Ser Leu Ser Ser Thr Leu Thr Glu Ile Phe Leu Leu Gln Glu

210 215 220

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Ala Gln Gly Met Pro Gln Val Ala Trp Gly Arg Ile Thr Gly Glu Lys

225 230 235 240

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Glu Trp Arg Asp Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Leu

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caa aga act cca gaa gtt gcc cgt agt agg gcc aca cca tta ctc gat 816

Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp

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Met Ile Asp Thr Ala Leu Leu Thr Asn Gly Thr Thr Glu Asn Arg Tyr

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ggc ata aaa tta ccc gta tct ctg ttg ttt att gct ggt cat gat acc 912

Gly Ile Lys Leu Pro Val Set Leu Leu Phe Ile Ala Gly His Asp Thr

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aat ctt gca aat tta agc ggg gct tta gat ctt aac tgg tcg cta ccc 960

Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp Leu Asn Trp Ser Leu Pro

305 310 315 320

ggt caa ccc gat aat acc cct cct ggt ggg gag ctt gta ttc gaa aag 1008

Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Lys

325 330 335

tgg aaa aga acc agt gat aat acg gat tgg gtt cag gtt tca ttt gtt 1056

Trp Lys Arg Thr Ser Asp Asn Thr Asp Trp Val Gln Val Ser Phe Val

340 345 350

tat cag acg ctg aga gat atg agg gat ata caa ccg ttg tcg tta gaa 1104

Tyr Gln Thr Leu Arg Asp Met Arg Asp Ile Gln Pro Leu Ser Leu Glu

355 360 365

aaa cct gct ggc aaa gtt gat tta aaa tta att gca tgt gaa gag aaa 1152

Lys Pro Ala Gly Lys Val Asp Leu Lys Leu Ile Ala Cys Glu Glu Lys

370 375 380

aat agt cag gga atg tgt tcg tta aaa agt ttt tcc agg ctc att aag 1200

Asn Ser Gln Gly Met Cys Ser Leu Lys Ser Phe Ser Arg Leu Ile Lys

385 390 395 400

gaa att cgc gtg cca gag tgt gca gtt acg gaa 1233

Glu Ile Arg Val Pro Glu Cys Ala Val Thr Glu

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<213> Bu Shi citric acid bacillus ATCC 51113

 

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Glu Glu Gln Asn Gly Met Lys Leu Glu Arg Val Val Ile Val Ser Arg

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His Gly Val Arg Ala Pro Thr Lys Phe Thr Pro Ile Met Lys Asn Val

20 25 30

Thr Pro Asp Gln Trp Pro Gln Trp Asp Val Pro Leu Gly Trp Leu Thr

35 40 45

Pro Arg Gly Gly Glu Leu Val Ser Glu Leu Gly Gln Tyr Gln Arg Leu

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Trp Phe Thr Ser Lys Gly Leu Leu Asn Asn Gln Thr Cys Pro Ser Pro

65 70 75 80

Gly Gln Val Ala Val Ile Ala Asp Thr Asp Gln Arg Thr Arg Lys Thr

85 90 95

Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Lys Cys Gln Ile Gln Val

100 105 110

His Tyr Gln Lys Asp Glu Glu Lys Asn Asp Pro Leu Phe Asn Pro Val

115 120 125

Lys Met Gly Lys Cys Ser Phe Asn Thr Leu Gln Val Lys Asn Ala Ile

130 135 140

Leu Glu Arg Ala Gly Gly Asn Ile Glu Leu Tyr Thr Gln Arg Tyr Gln

145 150 155 160

Ser Ser Phe Arg Thr Leu Glu Asn Val Leu Asn Phe Ser Gln Ser Glu

165 170 175

Thr Cys Lys Thr Thr Glu Lys Ser Thr Lys Cys Thr Leu Pro Glu Ala

180 185 190

Leu Pro Ser Glu Leu Lys Val Thr Pro Asp Asn Val Ser Leu Pro Gly

195 200 205

Ala Trp Ser Leu Ser Ser Thr Leu Thr Glu Ile Phe Leu Leu Gln Glu

210 215 220

Ala Gln Gly Met Pro Gln Val Ala Trp Gly Arg Ile Thr Gly Glu Lys

225 230 235 240

Glu Trp Arg Asp Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Leu

245 250 255

Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp

260 265 270

Met Ile Asp Thr Ala Leu Leu Thr Asn Gly Thr Thr Glu Asn Arg Tyr

275 280 285

Gly Ile Lys Leu Pro Val Ser Leu Leu Phe Ile Ala Gly His Asp Thr

290 295 300

Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp Leu Asn Trp Ser Leu Pro

305 310 315 320

Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Lys

325 330 335

Trp Lys Arg Thr Ser Asp Asn Thr Asp Trp Val Gln Val Ser Phe Val

340 345 350

Tyr Gln Thr Leu Arg Asp Met Arg Asp Ile Gln Pro Leu Ser Leu Glu

355 360 365

Lys Pro Ala Gly Lys Val Asp Leu Lys Leu Ile Ala Cys Glu Glu Lys

370 375 380

Asn Ser Gln Gly Met Cys Ser Leu Lys Ser Phe Ser Arg Leu Ile Lys

385 390 395 400

Glu Ile Arg Val Pro Glu Cys Ala Val Thr Glu

405 410

 

<210>3

<211>411

<212>PRT

<213> Bu Shi citric acid bacillus YH-15

 

<220>

<221> mature peptide

<222>(1)..(411)

 

<400>3

 

Glu Glu Gln Asn Gly Met Lys Leu Glu Arg Val Val Ile Val Ser Arg

1 5 10 15

His Gly Val Arg Ala Pro Thr Lys Phe Thr Pro Ile Met Lys Asp Val

20 25 30

Thr Pro Asp Gln Trp Pro Gln Trp Asp Val Pro Leu Gly Trp Leu Thr

35 40 45

Pro Arg Gly Gly Glu Leu Val Ser Glu Leu Gly Gln Tyr Gln Arg Leu

50 55 60

Trp Phe Thr Ser Lys Gly Leu Leu Asn Asn Gln Thr Cys Pro Ser Pro

65 70 75 80

Gly Gln Val Ala Val Ile Ala Asp Thr Asp Gln Arg Thr Arg Lys Thr

85 90 95

Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Lys Cys Gln Ile Gln Val

100 105 110

His Tyr Gln Lys Asp Glu Glu Lys Asn Asp Pro Leu Phe Asn Pro Val

115 120 125

Lys Met Gly Lys Cys Ser Phe Asn Thr Leu Lys Val Lys Asn Ala Ile

130 135 140

Leu Glu Arg Ala Gly Gly Asn Ile Glu Leu Tyr Thr Gln Arg Tyr Gln

145 150 155 160

Ser Ser Phe Arg Thr Leu Glu Asn Val Leu Asn Phe Ser Gln Ser Glu

165 170 175

Thr Cys Lys Thr Thr Glu Lys Ser Thr Lys Cys Thr Leu Pro Glu Ala

180 185 190

Leu Pro Ser Glu Phe Lys Val Thr Pro Asp Asn Val Ser Leu Pro Gly

195 200 205

Ala Trp Ser Leu Ser Ser Thr Leu Thr Glu Ile Phe Leu Leu Gln Glu

210 215 220

Ala Gln Gly Met Pro Gln Val Ala Trp Gly Arg Ile Thr Gly Glu Lys

225 230 235 240

Glu Trp Arg Asp Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Leu

245 250 255

Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp

260 265 270

Met Ile Asp Thr Ala Leu Leu Thr Asn Gly Thr Thr Glu Asn Arg Tyr

275 280 285

Gly Ile Lys Leu Pro Val Ser Leu Leu Phe Ile Ala Gly His Asp Thr

290 295 300

Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp Leu Lys Trp Ser Leu Pro

305 310 315 320

Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Lys

325 330 335

Trp Lys Arg Thr Ser Asp Asn Thr Asp Trp Val Gln Val Ser Phe Val

340 345 350

Tyr Gln Thr Leu Arg Asp Met Arg Asp Ile Gln Pro Leu Ser Leu Glu

355 360 365

Lys Pro Ala Gly Lys Val Asp Leu Lys Leu Ile Ala Cys Glu Glu Lys

370 375 380

Asn Ser Gln Gly Met Cys Ser Leu Lys Ser Phe Ser Arg Leu Ile Lys

385 390 395 400

Glu Ile Arg Val Pro Glu Cys Ala Val Thr Glu

405 410

<210>4

<211>411

<212>PRT

<213> citrobacter freundii (Citrobacter freundii)

 

<220>

<221> mature peptide

<222>(1)..(411)

 

<400>4

 

Glu Glu Gln Asn Gly Met Lys Leu Glu Arg Val Val Ile Val Ser Arg

1 5 10 15

His Gly Val Arg Ala Pro Thr Lys Phe Thr Pro Ile Met Lys Asp Val

20 25 30

Thr Pro Asp Gln Trp Pro Gln Trp Asp Val Pro Leu Gly Trp Leu Thr

35 40 45

Pro Arg Gly Gly Glu Leu Val Ser Glu Leu Gly Gln Tyr Gln Arg Leu

50 55 60

Trp Phe Thr Ser Lys Gly Leu Leu Asn Asn Gln Thr Cys Pro Ser Pro

65 70 75 80

Gly Gln Val Ala Val Ile Ala Asp Thr Asp Gln Arg Thr Arg Lys Thr

85 90 95

Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Lys Cys Gln Ile Gln Val

100 105 110

His Tyr Gln Lys Asp Glu Glu Lys Thr Asp Pro Leu Phe Asn Pro Val

115 120 125

Lys Met Gly Thr Cys Ser Phe Asn Thr Leu Lys Val Lys Asn Ala Ile

130 135 140

Leu Glu Arg Ala Gly Gly Asn Ile Glu Leu Tyr Thr Gln Arg Tyr Gln

145 150 155 160

Ser Ser Phe Arg Thr Leu Glu Asn Val Leu Asn Phe Ser Gln Ser Glu

165 170 175

Thr Cys Lys Thr Thr Glu Lys Ser Thr Lys Cys Thr Leu Pro Glu Ala

180 185 190

Leu Pro Ser Glu Leu Lys Val Thr Pro Asp Asn Val Ser Leu Pro Gly

195 200 205

Ala Trp Ser Leu Ser Ser Thr Leu Thr Glu Ile Phe Leu Leu Gln Glu

210 215 220

Ala Gln Gly Met Pro Gln Val Ala Trp Gly Arg Ile Thr Gly Glu Lys

225 230 235 240

Glu Trp Arg Asp Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Leu

245 250 255

Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp

260 265 270

Met Ile Asp Thr Ala Leu Leu Thr Asn Gly Thr Thr Glu Asn Arg Tyr

275 280 285

Gly Ile Lys Leu Pro Val Ser Leu Leu Phe Ile Ala Gly His Asp Thr

290 295 300

Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp Leu Asn Trp Ser Leu Pro

305 310 315 320

Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Lys

325 330 335

Trp Lys Arg Thr Ser Asp Asn Thr Asp Trp Val Gln Val Ser Phe Val

340 345 350

Tyr Gln Thr Leu Arg Asp Met Arg Asp Ile Gln Pro Leu Ser Leu Glu

355 360 365

Lys Pro Ala Gly Lys Val Asp Leu Lys Leu Ile Ala Cys Glu Glu Lys

370 375 380

Asn Ser Gln Gly Met Cys Ser Leu Lys Ser Phe Ser Arg Leu Ile Lys

385 390 395 400

Glu Ile Arg Val Pro Glu Cys Ala Val Thr Glu

405 410

 

<210>5

<211>1236

<212>DNA

The variant of <213>SEQ ID NO:2

 

<220>

<221>CDS

<222>(1)..(1233)

 

<220>

<221> mature peptide

<222>(1)..(1233)

 

<400>5

gaa gag cag aat ggt atg aaa ctt gag cgg gtt gtg ata gtg agt cgt 48

Glu Glu Gln Asn Gly Met Lys Leu Glu Arg Val Val Ile Val Ser Arg

1 5 10 15

cat ggr gta aga gca cct acg aag ttc act cca ata atg aaa aat gtc 96

His Xaa Val Arg Ala Pro Thr Lys Phe Thr Pro Ile Met Lys Asn Val

20 25 30

aca ccc gat caa tgg cca caa tgg gat gtg ccg tta gga tgg cta acg 144

Thr Pro Asp Gln Trp Pro Gln Trp Asp Val Pro Leu Gly Trp Leu Thr

35 40 45

cct cgt ggg gga gaa ctt gtt tct gaa tta ggt cag tat caa cgt tta 192

Pro Arg Gly Gly Glu Leu Val Ser Glu Leu Gly Gln Tyr Gln Arg Leu

50 55 60

tgg ttc acg agc aaa ggt ctg ttg aat aat caa acg tgc cca tct cca 240

Trp Phe Thr Ser Lys Gly Leu Leu Asn Asn Gln Thr Cys Pro Ser Pro

65 70 75 80

ggg cag gtt gct gtt att gca gac acg gat caa cgc acc cgt aaa acg 288

Gly Gln Val Ala Val Ile Ala Asp Thr Asp Gln Arg Thr Arg Lys Thr

85 90 95

ggt gag gcg ttt ctg gct ggg tta gca cca aaa tgt caa att caa gtg 336

Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Lys Cys Gln Ile Gln Val

100 105 1l0

cat tat cag aag gat gaa gaa aaa aat gat cct ctt ttt aat ccg gta 384

His Tyr Gln Lys Asp Glu Glu Lys Asn Asp Pro Leu Phe Asn Pro Val

115 120 125

aaa atg ggg aaa tgt tcg ttt aac aca ttg cag gtt aaa aac gct att 432

Lys Met Gly Lys Cys Ser Phe Asn Thr Leu Gln Val Lys Asn Ala Ile

130 135 140

ctg gaa cgg gcc gga gga aat att gaa ctg tat acc caa cgc tat caa 480

Leu Glu Arg Ala Gly Gly Asn Ile Glu Leu Tyr Thr Gln Arg Tyr Gln

145 150 155 160

tct tca ttt cgg acc ctg gaa aat gtt tta aat ttc tca caa tcg gag 528

Ser Ser Phe Arg Thr Leu Glu Asn Val Leu Asn Phe Ser Gln Ser Glu

165 170 175

aca tgt aag act aca gaa aag tct acg aaa tgc aca tta cca gag gct 576

Thr Cys Lys Thr Thr Glu Lys Ser Thr Lys Cys Thr Leu Pro Glu Ala

180 185 190

tta ccg tct gaa ctt aag gta act cct gac aat gta tca tta cct ggt 624

Leu Pro Ser Glu Leu Lys Val Thr Pro Asp Asn Val Ser Leu Pro Gly

195 200 205

gcc tgg agt ctt tct tcc acg ctg act gag ata ttt ctg ttg caa gag 672

Ala Trp Ser Leu Ser Ser Thr Leu Thr Glu Ile Phe Leu Leu Gln Glu

210 215 220

gcc cag gga atg cca cag gta gcc tgg ggg cgt att acg gga gaa aaa 720

Ala Gln Gly Met Pro Gln Val Ala Trp Gly Arg Ile Thr Gly Glu Lys

225 230 235 240

gaa tgg aga gat ttg tta agt ctg cat aac gct cag ttt gat ctt ttg 768

Glu Trp Arg Asp Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Leu

245 250 255

caa aga act cca gaa gtt gcc cgt agt agg gcc aca cca tta ctc gat 816

Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp

260 265 270

atg ata gac act gca tta ttg aca aat ggt aca aca gaa aac agg tat 864

Met Ile Asp Thr Ala Leu Leu Thr Asn Gly Thr Thr Glu Asn Arg Tyr

275 280 285

ggc ata aaa tta ccc gta tct ctg ttg ttt att gct ggt cat gat acc 912

Gly Ile Lys Leu Pro Val Ser Leu Leu Phe Ile Ala Gly His Asp Thr

290 295 300

aat ctt gca aat tta agc ggg gct tta gat ctt aac tgg tcg cta ccc 960

Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp Leu Asn Trp Ser Leu Pro

305 310 315 320

ggt caa ccs gat aay acc ccg ccg ggc gac aag ctt gta ttc gaa aag 1008

Gly Gln Xaa Asp Asn Thr Pro Pro Gly Asp Lys Leu Val Phe Glu Lys

325 330 335

tgg aaa aga acc agt gat aat acg gat tgg gtt cag gtt tca ttt gtt 1056

Trp Lys Arg Thr Ser Asp Asn Thr Asp Trp Val Gln Val Ser Phe Val

340 345 350

tat cag acg ctg aga gat atg agg gat ata caa ccg ttg tcg tta gaa 1104

Tyr Gln Thr Leu Arg Asp Met Arg Asp Ile Gln Pro Leu Ser Leu Glu

355 360 365

aaa cct gct ggc aaa gtt gat tta aaa tta att gca tgt gaa gag aas 1152

Lys Pro Ala Gly Lys Val Asp Leu Lys Leu Ile Ala Cys Glu Glu Lys

370 375 380

aat agt cag gga atg tgt tcg tta aaa agt ttt tcc agg ctc att aag 1200

Asn Ser Gln Gly Met Cys Ser Leu Lys Ser Phe Ser Arg Leu Ile Lys

385 390 395 400

gaa att cgc gtg cca gag tgt gca gtt acg gaa taa 1236

Glu Ile Arg Val Pro Glu Cys Ala Val Thr Glu

405 410

 

<210>6

<211>411

<212>PRT

The variant of <213>SEQ ID NO:2

 

<220>

<221>misc_ feature

<222>(18)..(18)

<223> position 18 ' Xaa ' represents Gly.

 

<220>

<221>misc_ feature

<222>(323)..(323)

<223> position 323 ' Xaa ' represents Pro.

 

<400>6

 

Glu Glu Gln Asn Gly Met Lys Leu Glu Arg Val Val Ile Val Ser Arg

1 5 10 15

His Xaa Val Arg Ala Pro Thr Lys Phe Thr Pro Ile Met Lys Asn Val

20 25 30

Thr Pro Asp Gln Trp Pro Gln Trp Asp Val Pro Leu Gly Trp Leu Thr

35 40 45

Pro Arg Gly Gly Glu Leu Val Ser Glu Leu Gly Gln Tyr Gln Arg Leu

50 55 60

Trp Phe Thr Ser Lys Gly Leu Leu Asn Asn Gln Thr Cys Pro Ser Pro

65 70 75 80

Gly Gln Val Ala Val Ile Ala Asp Thr Asp Gln Arg Thr Arg Lys Thr

85 90 95

Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Lys Cys Gln Ile Gln Val

100 105 110

His Tyr Gln Lys Asp Glu Glu Lys Asn Asp Pro Leu Phe Asn Pro Val

115 120 125

Lys Met Gly Lys Cys Ser Phe Asn Thr Leu Gln Val Lys Asn Ala Ile

130 135 140

Leu Glu Arg Ala Gly Gly Asn Ile Glu Leu Tyr Thr Gln Arg Tyr Gln

145 150 155 160

Ser Ser Phe Arg Thr Leu Glu Asn Val Leu Asn Phe Ser Gln Ser Glu

165 170 175

Thr Cys Lys Thr Thr Glu Lys Ser Thr Lys Cys Thr Leu Pro Glu Ala

180 185 190

Leu Pro Ser Glu Leu Lys Val Thr Pro Asp Asn Val Ser Leu Pro Gly

195 200 205

Ala Trp Ser Leu Ser Ser Thr Leu Thr Glu Ile Phe Leu Leu Gln Glu

2l0 215 220

Ala Gln Gly Met Pro Gln Val Ala Trp Gly Arg Ile Thr Gly Glu Lys

225 230 235 240

Glu Trp Arg Asp Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Leu

245 250 255

Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp

260 265 270

Met Ile Asp Thr Ala Leu Leu Thr Asn Gly Thr Thr Glu Asn Arg Tyr

275 280 285

Gly Ile Lys Leu Pro Val Ser Leu Leu Phe Ile Ala Gly His Asp Thr

290 295 300

Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp Leu Asn Trp Ser Leu Pro

305 310 315 320

Gly Gln Xaa Asp Asn Thr Pro Pro Gly Asp Lys Leu Val Phe Glu Lys

325 330 335

Trp Lys Arg Thr Ser Asp Asn Thr Asp Trp Val Gln Val Ser Phe Val

340 345 350

Tyr Gln Thr Leu Arg Asp Met Arg Asp Ile Gln Pro Leu Ser Leu Glu

355 360 365

Lys Pro Ala Gly Lys Val Asp Leu Lys Leu Ile Ala Cys Glu Glu Lys

370 375 380

Asn Ser Gln Gly Met Cys Ser Leu Lys Ser Phe Ser Arg Leu Ile Lys

385 390 395 400

Glu Ile Arg Val Pro Glu Cys Ala Val Thr Glu

405 410

<210>7

<211>66

<212>DNA

<213> Bu Shi citric acid bacillus ATCC 51113

 

<220>

<221> signal peptide

<222>(1)..(66)

 

<220>

<221>CDS

<222>(1)..(66)

 

<400>7

atg agt aca ttc atc att cgt tta tta ttt ttt tct ctc tta tgc ggt 48

Met Ser Thr Phe Ile Ile Arg Leu Leu Phe Phe Ser Leu Leu Cys Gly

1 5 10 15

tct ttc tca ata cat gct 66

Ser Phe Ser Ile His Ala

20

<210>8

<211>22

<212>PRT

<213> Bu Shi citric acid bacillus ATCC 51113

 

<400>8

 

Met Ser Thr Phe Ile Ile Arg Leu Leu Phe Phe Ser Leu Leu Cys Gly

1 5 10 15

Ser Phe Ser Ile His Ala

20

 

<210>9

<211>433

<212>PRT

<213> citrobacter freundii NCIMB 41247

 

<220>

<221> mature peptide

<222>(1)..(433)

 

<400>9

 

Met Ser Thr Phe Ile Ile Arg Leu Leu Phe Phe Ser Leu Leu Cys Gly

1 5 10 15

Ser Phe Ser Ile His Ala Glu Glu Pro Asn Gly Met Lys Leu Glu Arg

20 25 30

Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Phe Thr

35 40 45

Pro Ile Met Lys Asp Val Thr Pro Asp Gln Trp Pro Gln Trp Asp Val

50 55 60

Pro Leu Gly Trp Leu Thr Pro Arg Gly Gly Glu Leu Val Ser Glu Leu

65 70 75 80

Gly Gln Tyr Gln Arg Leu Trp Phe Thr Ser Lys Gly Leu Leu Asn Asn

85 90 95

Gln Thr Cys Pro Ser Pro Gly Gln Val Ala Val Ile Ala Asp Thr Asp

100 105 110

Gln Arg Thr Arg Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro

115 120 125

Lys Cys Gln Ile Gln Val His Tyr Gln Lys Asp Glu Glu Lys Thr Asp

130 135 140

Pro Leu Phe Asn Pro Val Lys Met Gly Thr Cys Ser Phe Asn Thr Leu

145 150 155 160

Lys Val Lys Asn Ala Ile Leu Glu Arg Ala Gly Gly Asn Ile Glu Leu

165 170 175

Tyr Thr Gln Arg Tyr Gln Ser Ser Phe Arg Thr Leu Glu Asn Val Leu

180 185 190

Asn Phe Ser Gln Ser Glu Thr Cys Lys Thr Thr Glu Lys Ser Thr Lys

195 200 205

Cys Thr Leu Pro Glu Ala Leu Pro Ser Glu Leu Lys Val Thr Pro Asp

210 215 220

Asn Val Ser Leu Pro Gly Ala Trp Ser Leu Ser Ser Thr Leu Thr Glu

225 230 235 240

Ile Phe Leu Leu Gln Glu Ala Gln Gly Met Pro Gln Val Ala Trp Gly

245 250 255

Arg Ile Thr Gly Glu Lys Glu Trp Arg Asp Leu Leu Ser Leu His Asn

260 265 270

Ala Gln Phe Asp Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg

275 280 285

Ala Thr Pro Leu Leu Asp Met Ile Asp Thr Ala Leu Leu Thr Asn Gly

290 295 300

Thr Thr Glu Asn Arg Tyr Gly Ile Lys Leu Pro Val Ser Leu Leu Phe

305 310 315 320

Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp

325 330 335

Leu Asn Trp Ser Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly

340 345 350

Glu Leu Val Phe Glu Lys Trp Lys Arg Thr Ser Asp Asn Thr Asp Trp

355 360 365

Val Gln Val Ser Phe Val Tyr Gln Thr Leu Arg Asp Met Arg Asp Ile

370 375 380

Gln Pro Leu Ser Leu Glu Lys Pro Ala Gly Lys Val Asp Leu Lys Leu

385 390 395 400

Ile Ala Cys Glu Glu Lys Asn Ser Gln Gly Met Cys Ser Leu Lys Ser

405 410 415

Phe Ser Arg Leu Ile Lys Glu Ile Arg Val Pro Glu Cys Ala Val Thr

420 425 430

Glu

Claims (15)

1. phytase, it has the thermostability of improvement, thermostability is represented by remaining activity, at least 105% of the remaining activity that the remaining activity of described phytase is phytase shown in SEQ ID NO:2, wherein remaining activity measures as follows: fermentation supernatant is divided into two portions, a part was 60 DEG C of incubations 30 minutes, another part was 5 DEG C of incubations 30 minutes, then for the activity of p-nitrophenyl phosphoric acid both measuring at 37 DEG C of pH5.5, the activity of activity divided by the same sample at 5 DEG C of incubations of the sample of 60 DEG C of incubations is used in, compared with SEQ ID NO:2, the change of described phytase is selected from the one in lower group: 4P, 46E, 107G, 111P, 119K, 162C, 223E, 241Q, 273L, 276K, 379K, 385D, 52C/99C, 59C/100C, 141C/199C, 111P/241Q, 31C, 202N, 286Q, 362K, 362R, 1*, 52E, 53V, 76G, 410E, 179K/180E/181K/182K/183V/184*/185*/186*, 114T/115Q/116A/117D/118T/119S/120S/121P/122D/123P/124L, or 114T/115Q/116T/117D/118T/119S/120S/121P/122D/123P/124L,

Above-mentioned "/" represents connected amino acid whose common change; " * " represents amino acid whose disappearance.

2. the phytase of claim 1, wherein said change is selected from the one in lower group: 141C/199C, 52C/99C, 59C/100C or? 1P/241Q.

3. the nucleotide sequence be separated, it is made up of the nucleotide sequence of the phytase of encoding any one of claim 1-2.

4. nucleic acid construct, it comprises the nucleotide sequence of claim 3, and described nucleotide sequence is operably connected to one or more regulating and controlling sequence instructing phytase to produce in suitable expressive host.

5. comprise the recombinant expression vector of the nucleic acid construct of claim 4.

6. comprise the recombinant host cell of the nucleic acid construct of claim 4 and/or the expression vector of claim 5.

7. produce the method for the phytase any one of claim 1-2, it comprises

A () cultivates the host cell of claim 6 to produce the supernatant liquor comprising described phytase; With

B () reclaims described phytase.

8. composition, it comprises at least one phytase any one of claim 1-2, and

(a) at least one liposoluble vitamin;

(b) at least one water-soluble vitamins; And/or

C () at least one is selected from the trace minerals of manganese, zinc, iron, copper, iodine, selenium or cobalt.

9. the composition of claim 8, it comprises the enzyme that at least one is selected from lower group further: amylase, Phosphoric acid esterase, zytase, Galactanase, alpha-galactosidase, proteolytic enzyme, Phospholipid hydrolase and/or beta-glucanase.

10. the composition any one of claim 8-9, it is animal feedstuff additive.

11. animal feedstuff compositionss, it has the gross protein value of 50 to 800g/kg and comprises the phytase any one of claim 1-2 or the composition any one of claim 8-10.

Phytase any one of claim 1-2 or the composition any one of claim 8-10, for improvement of the method for the nutritive value of animal-feed, are wherein added into feed by 12..

The method of 13. reduction animal excrement mysoinositol six phosphate level, it comprises the animal feedstuff compositions nutrition purposes, especially for feeding animals of the claim 11 by significant quantity.

14. the method for the treatment of of vegetable protein, it comprises the step of the phytase any one of claim 1-2 or the composition any one of claim 8-10 being added at least one plant protein or protein source.

15. the phytase any one of claim 1-2 or the composition any one of claim 8-10 for following purposes: in animal-feed; In the preparation of animal-feed; For improvement of the nutritive value of animal-feed; For reducing the phytinic acid salt level in animal excrement; For the treatment of plant protein; Or for release from phytinic acid enzyme substrates containing the material of sub-phosphorus.