CN101460612A - Phytase variants - Google Patents

Phytase variants Download PDF

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CN101460612A
CN101460612A CNA2007800207715A CN200780020771A CN101460612A CN 101460612 A CN101460612 A CN 101460612A CN A2007800207715 A CNA2007800207715 A CN A2007800207715A CN 200780020771 A CN200780020771 A CN 200780020771A CN 101460612 A CN101460612 A CN 101460612A
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phytase
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animal
variant
feed
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CN101460612B (en
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伦纳多·德玛利亚
卡斯滕·安德森
拉斯·K·斯科夫
迈克尔·B·索伦森
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Novo Nordisk AS
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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
The sequence table reference
The application comprises the sequence table that exists with computer-reader form.Computer-reader form is incorporated herein by reference here.
Invention field
The present invention relates to have at least 74% identity and compare the phytase (that is, being its variant) that comprises at least one variation with this phytase with the phytase that is derived from Bu Shi citric acid bacillus (Citrobacter braakii) ATCC 51113.The invention still further relates to the DNA of these phytases of coding, their preparation method, and their purposes, for example purposes in animal-feed and animal feedstuff additive.The maturing part of Bu Shi citric acid bacillus ATCC 51113 phytases is included in the sequence table as SEQ ID NO:2.
Background of invention
Background technology
Submitted to the EMBL/GenBank/DDBJ database by Zinin etc. from the sequence of the phyA gene of Bu Shi citric acid bacillus, accession number is AY390262.Corresponding phytinic acid enzyme amino acid sequence is present in the UniProt/TrEMBL database with accession number Q676V7.The maturing part of Q676V7 expection is included in the sequence table of the present invention as SEQ ID NO:4.
It is the aminoacid sequence of phytase of the Bu Shi citric acid bacillus YH-15 of KCCM 10427 that WO-2004/085638 discloses from preservation with SEQ ID NO:7.The maturing part of this aminoacid sequence is included in this as SEQ ID NO:3.This sequence also is present among the 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, promptly as SEQ ID NO:6.
WO 2006/038062 and WO 2006/038128 disclose the aminoacid sequence of citrobacter freundii (Citrobacter freundii) the P3-42 phytase gene with accession number NCIMB 41247 preservations.This aminoacid sequence is included in this as SEQ ID NO:9.
An object of the present invention is to provide and have modification, 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, proteolytic enzyme susceptibility 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 compares with SEQ ID NO:2 and comprises at least one variation at least one position in being selected from group down: 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 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 variation: 1H, K, R, 60P, 105E, 106A, G, 155F, 157F, 173P, 175L, 188P, 205P, 215M, 231P, 254Y, 280P, 330D and/or 371P; Condition is that this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, is not SEQ ID NO:6, and is not SEQ ID NO:9 and its variant listed in Fig. 1.
The invention still further relates to the DNA of these phytases of coding, their preparation method, and their purposes, for example purposes in animal-feed and animal feedstuff additive.
The accompanying drawing summary
Fig. 1 is corresponding to the table 2 of WO 2006/038062 and disclose the multiple variant of citrobacter freundii NCIMB 41247 phytases with SEQ ID NO:9 aminoacid sequence;
Fig. 2 is the comparison of the phytase of SEQ ID NO:2 and 9.
Location number among Fig. 1 is with reference to the numbering of SEQ ID NO:9.Corresponding SEQ ID NO:2 position can obtain (for example, the meaning of the variant P229S of numbering Fig. 1 of application the application is variant P207S) by deducting 22.
Detailed Description Of The Invention
First aspect the present invention relates to phytase, and it has at least 74% homogeneity with SEQ ID NO:2 and compare at least one position that is selected from lower group with SEQ ID NO:2 and comprises at least one variation: 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 phytase is not SEQ ID NO:3, is not SEQ ID NO:4, and is not SEQ ID NO:6.
As described in " phytase polypeptides, homogeneity percentage " joint, determining homogeneity percentage.
Location number is with reference to the Position Number mode of SEQID NO:2, described in saving in " Position Number mode ". The position corresponding with these SEQID NO:2 location numbers determined described in " determine relevant position number " joint in other phytase.
Phytase of the present invention is the variant of the phytase of SEQID NO:2, and promptly they are different with SEQ ID NO:2, comprises at least one variation because it is compared with SEQ ID NO:2.
In an embodiment, phytase of the present invention is compared with SEQ ID NO:2 and comprise at least one variation at least one position of organizing under being selected from: 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 embodiment further, phytase of the present invention is not the variant of the SEQ ID NO:9 that lists of Fig. 1.
In a preferred embodiment, phytase of the present invention comprises at least one following variation: 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.
The nomenclature that this paper uses for variation " is changing, as replacing disappearance, insertion." describe in detail in the joint.
Phytase of the present invention preferably comprises at least one following variation: 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 position 179,180,181,182,183,184,185 and 186 amino acid by KEKHQ, KEKQQ, KEKKV or KTDKL replace.
In another preferred embodiment, position 179,180,181,182,183,184,185 and 186 amino acid is by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace.
The invention still further relates to the phytase that has at least 74% identity with SEQ ID NO:2 and comprise at least one following variation: 1H, K, R, 60P, 105E, 106A, G, 155F, 157F, 173P, 175L, 188P, 205P, 215M, 231P, 254Y, 280P, 330D and/or 371P; Condition is that this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, is not SEQ ID NO:6, and is not SEQ ID NO:9 and its variant listed in Fig. 1.One preferred embodiment the mysoinositol six-phosphatase comprise and change 1K.In other preferred implementation, phytase comprises following variation combination: 280P/282P/283P, 155F/254Y and/or 155F/157F/254Y.
The preferred phytase of the present invention comprises and is selected from following variation a: 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 a variant any wild-type or the variant phytase.In concrete embodiment, it is SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, the variant of the phytase of SEQ ID NO:9, or with the variant of SEQ ID NO:9 any inositol six-phosphatase variants relevant and that in Fig. 1, list.
Phytase of the present invention can further comprise the combination of a variation (replacement) or a plurality of variation (replacement), and they are selected from variation (replacement) listed in each row of Fig. 1 or change the combination of (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:3,4 and 6 variation.
The especially preferred phytase of the present invention comprises at least one following variation: 339D, 4P, 5P, 111P, 1 *, 1 */ 2 *, 1 */ 2 */ 3 *, 273L and/or 286K.
The invention still further relates to the phytase that has at least 74% identity with SEQ ID NO:2 and comprise at least one following variation:
(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) K179 wherein, T180, T181, E182, K183, S184, T185 and K186 be by QADKP, GEDKP, and NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
(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。
The strategy of preparation variant
By homology modeling (modeling), the structure of utilizing intestinal bacteria (E.coli) AppA phytase is as template (Protein Data Bank id.:1DKO; Lim etc., Nat.Struct.Biol. (2000), vol.2, pp.108-113 (second volume, 108-113 page or leaf)) make up the structure of Bu Shi citric acid bacillus ATCC 51113 phytases.
This structure is carried out molecular dynamics (MD) simulation and electrostatic calculations.Also identified the disulfide linkage (disulfide bridges) of inferring and the position of proline(Pro), and other sites of the potential importance relevant with various desirable enzyme characteristics.At last, identified the glycosylation site (stretching, extension of NXT or NXS (stretches)) of inferring.
Within the framework of modeled structure and analog result, these all proposals are assessed, especially focused on temperature end assessment thermostability.
Corresponding inositol six-phosphatase variants is by method preparation known in the art and as testing described in the experimental section.
Phytase polypeptides, identity per-cent
Phytase is the polypeptide with phytase activity in this article, it is (phytase) its list of hydrolysis generation (1) inositol (myo-inositol) and/or (2) of (phytate (myo-inositol hexakisphosphate)) of catalysis phytinic acid (salt), 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 listed phosphoric acid salt in superincumbent (2).
ENZYME website on the network (http://www.expasy.ch/enzyme/) is the relevant information storage of nomenclature of enzyme.Its main suggestion according to international biological chemistry and NK of molecular biology association (IUB-MB) (Nomenclature Committee of the International Union of Biochemistryand Molecular Biology), it has described all kinds (the Bairoch A.The ENZYME database of the enzyme through characterizing that provides EC (the enzyme council) number, 2000, Nucleic Acids Res28:304-305).Also can be referring to the enzyme name handbook of NC-IUBMB, 1992.
According to the ENZYME website, known have three kinds of dissimilar phytases: so-called 3-Phytase (another name 1-phytase; Phytate 3-phosphohydrolase, EC3.1.3.8), (the another name 6-phytase is based on the title of 1L numbering system rather than 1D numbering for so-called 4-phytase, and so-called 5-phytase (EC 3.1.3.72) EC 3.1.3.26).For the present invention, with in the whole three kinds definition that are included in phytase.
In an embodiment, phytase of the present invention belongs to acid Histidine phosphatase family, comprises phytase such as Aspergillus awamori (Aspergillus awamorii) phytase A and the B (EC:3.1.3.8) (gene phyA and phyB) of intestinal bacteria pH2.5 acid phosphatase (gene appA) and fungi.Each Histidine acid phosphatase has the sequence similarity in two districts jointly, and each is distinguished round a conservative histidine residues center.These two Histidines look like the catalyst mechanism that has participated in enzyme.First Histidine is positioned at the N-terminal portions and forms phosphorus-Histidine intermediate, and second is positioned at the C-terminal portions and may plays a role as protophobe.
In embodiment more specifically, phytase of the present invention has conservative avtive spot motif, i.e. R-H-G-X-R-X-P, wherein X represents any amino acid (referring to SEQ ID NOs:2,3,4,6 amino acid/11 6 to 22 and the amino acid 38-44 of SEQ ID NO:9).In a preferred embodiment, conservative avtive spot motif is R-H-G-V-R-A-P, and promptly amino acid/11 6-22 (with reference to SEQ ID NO:2) is RHGVRAP.
For the present invention, the activity of phytase determines that with FYT unit a FYT is that per minute discharges the inorganic ortho-phosphoric enzyme amount of 1 micromole: pH5.5 under following condition; 37 ℃ of temperature; 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 in a kind of description of the embodiment of WO 00/20569.FTU is used for measuring the phytase activity of feed and premixture.Can also utilize assay method among the embodiment 1 (" mensuration of phosphatase activity " or " mensuration of phytase activity ") to determine the activity of phytase.
Phytase of the present invention is isolating in an embodiment.Term used herein " isolating " is meant that preferred at least 40% is pure as determining that by SDS-PAGE at least 20% is pure, more preferably at least 60% is pure, also more preferably at least 80% pure, most preferably at least 90% is pure, even at least 95% pure polypeptide most preferably.Concrete, preferred polypeptide is " (essentially) pure form basically ", that is, described polypeptide prepared product is substantially free of other polypeptide raw materials of (essentially free of) and its natural combination (nativelyassociated).For example, this can realize by preparing polypeptide with recombination method of knowing or classical purification process.
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 being used to.The Needle program is carried out at Needleman S.B. and Wunsch, C.D. (1970) J.Mol.Biol.48, the overall comparison algorithm of describing among the 443-453 (globalalignment algorithm).Used substitution matrix is BLOSUM62, and it is 10 that breach is opened point penalty (gapopening penalty), and breach extension point penalty (gap extension penalty) is 0.5.
Identity degree between the aminoacid sequence (SEQID NO:2) that relates in aminoacid sequence of the present invention (" invention sequence ") and the claim, be by the accurate number of coupling in two sequence alignments, the shortest one is calculated in the length divided by the length of " invention sequence " or SEQ ID NO:2.The result represents with per-cent identity.
When " invention sequence " with SEQ ID NO:2 (representing this situation with " | " in the comparison example hereinafter) takes place when the eclipsed same position has identical amino-acid residue accurately to mate.The length of sequence is the total number of atnino acid (for example the length of the amino acid/11 of SEQ ID NO:2-411 is 411) in the sequence.
Embodiment 13 is comparison examples of the phytase of the phytase of SEQ ID NO:2 and SEQ ID NO:9, and this example has illustrated the identity per-cent that how to calculate these two main chains.
In the comparison example of another pure supposition, overlapping is the aminoacid sequence " HTWGER-NL " of sequence 1 below; Or the aminoacid sequence of sequence 2 " HGWGEDANL ".Breach is represented with "-" in this example.
The comparison example of supposing:
Sequence 1:ACMSHTWGER-NL
| ||| ||
Sequence 2:HGWGEDANLAMNPS
In an embodiment, amino acid sequence of polypeptide with or with respect to the following mensuration of identity per-cent of SEQ ID NO:2: i) use the Needle program, use the BLOSUM62 substitution matrix, breach opens point penalty 10 and breach extends point penalty 0.5, compares two aminoacid sequences; Ii) accurately mate number in the counting comparison; Iii) remove accurately coupling number with the length of the shortest sequence in two aminoacid sequences, iv) will iii) remove the result convert per-cent to.
In the superincumbent supposition example, accurately mating number and be in 6, two aminoacid sequences, the length of short sequence 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, the 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 selectable embodiment, the identity degree is at least 70%, 71%, 72% or at least 73%.
In embodiment further, phytase of the present invention is no more than 1,2 as comparing to have with SEQ IDNO:2, and 3,4,5,6,7,8,9 or be no more than 10 variations; Be no more than 11,12 as comparing to have, 13,14,15,16,17,18,19 or be no more than 20 variations with SEQ ID NO:2; Be no more than 21,22 as comparing to have, 23,24,25,26,27,28,29 or be no more than 30 variations with SEQ ID NO:2; Be no more than 31,32 as comparing to have, 33,34,35,36,37,38,39 or be no more than 40 variations with SEQ ID NO:2; Be no more than 41,42 as comparing to have, 43,44,45,46,47,48,49 or be no more than 50 variations with SEQ ID NO:2; Be no more than 51,52 as comparing to have, 53,54,55,56,57,58,59 or be no more than 60 variations with SEQ ID NO:2; Be no more than 61,62 as comparing to have, 63,64,65,66,67,68,69 or be no more than 70 variations with SEQ ID NO:2; Be no more than 71,72 as comparing to have, 73,74,75,76,77,78,79 or be no more than 80 variations with SEQ ID NO:2; Be no more than 81,82 as comparing to have, 83,84,85,86,87,88,89 or be no more than 90 variations with SEQ IDNO:2; Be no more than 91,92 as comparing to have, 93,94,95,96,97,98,99 or be no more than 100 variations with SEQ ID NO:2; Be no more than 101,102 as comparing to have, 103,104,105,106,107,108,109 or be no more than 110 variations with SEQ ID NO:2; Be no more than 111,112 as comparing to have, 113,114,115,116,117,118,119 or be no more than 120 variations with SEQ ID NO:2; Be no more than 121,122 as comparing to have, 123 or 124 variations with SEQ ID NO:2.
Position Number
The nomenclature of definition amino acid position used herein is based on the phytinic acid enzyme amino acid sequence that is 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 that is used for numbered positions is SEQ ID NO:2, begins to stop to E411 from E1.
Term when being used for this paper " maturation " partly (or sequence) is meant by the polypeptide portion of the polynucleotide that contain this polypeptide of encoding as the emiocytosis of its part heredity parts (genetic equipment).In other words, mature polypeptide partly is meant and cuts away the signal peptide part, and remaining polypeptide portion after the preceding peptide moiety (if exist any before peptide moiety).The signal peptide part can be predicted (for example SignalP) by program software known in the art.The SEQ ID NO:2 signal peptide part of expection is included in the sequence table of the present invention as SEQ ID NO:8, and 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 be determined by the N-end sequencing to purifying enzyme.The then inevitable existence of any difference between signal peptide part and the maturing part owing to propetide.
Change, as replacing, disappearance is inserted
The variant of phytase can comprise various types of variations with respect to template (i.e. reference or aminoacid sequence such as SEQ IDNO:2 relatively): an amino acid can replace with another amino acid; Can lack an amino acid; Can insert an amino acid; And the arbitrary combination of these variations of arbitrary number.Term " insertion " is intended to also comprise the extension of N-end and/or C-end in this article.
This paper is as follows to the used general nomenclature of independent variation: 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 or the like), it is present in the insertion.With following table 1 as a reference, it has described the example of supposition fully that this nomenclature is applied to various types of variations.
Chart 1
Type Describe Example
Replace Amino acid in the position c vacancy Y=variant in the amino acid D=template in the X=template G80A 80 AALNNSIGVLGVAPSAELYAVKVLGASGSG |||||||:|||||||||||||||||||||| AALNNSIAVLGVAPSAELYAVKVLGASGSG
Insert Position c=＂ a ＂ in the X=＂ * ＂ D=template before inserting is illustrated in first insertion of this position, and ＂ b ＂ represents the next one, or the like ＊80aT ＊80bY ＊85aS 8085 AALNNSIG..VLGVA.PSAELYAVKVLGASG |||||||| ||||| ||||||||||||||| AALNNSIGTYVLGVASPSAELYAVKVLGASG
Deletion Position c vacancy Y=＂ * ＂ in the amino acid D=template in the X=template V81 ＊80 AALNNSIGVLGVAPSAELYAVKVLGASGSG |||||||| ||||||||||||||||||||| AALNNSIG.LGVAPSAELYAVKVLGASGSG
N-is terminal to be extended Insertion at position ＂ 0 ＂ ＊0aA ＊0bT ＊0cG 1 ...AQSVPWGISRVQ |||||||||||| ATGAQSVPWGISRVQ
C-is terminal to be extended Insertion behind-terminal amino acid ＊275aS ＊275bT 270275 ATSLGSTNLYGSGLVNAEAATR.. |||||||||||||||||||||| ATSLGSTNLYGSGLVNAEAATRST
As explained above, positional number (" D ") is that first amino-acid residue from SEQ ID NO:2 begins counting.
Several variations in same sequence are separated with "/" (oblique line), for example sign " 1 */ 2 */ 3 *" be illustrated in location number 1,2 With3 amino acid all lacks, and the amino acid of sign " 104A/105F " expression location number 104 is replaced by A, and the amino acid of location number 105 is replaced by F.
Selectable variation is separated with ", " (comma), and for example, the amino acid on sign " 119R, K " the expression position 119 is replaced by R or K.
This paper used comma () in various other possibilities too numerous to enumerate is represented them usually in phraseological effect, promptly normally and/or.For example, (a V represented to select in two in first comma in the tabulation " 53V, Q, 121D and/or 167Q " OrAnd two commas of back are interpreted as Q), And/orSelection: 53V or Q, and/or 121D, and/or 167Q.
In this article, " at least one " (for example change) expression is one or more, and for example 1,2,3,4,5,6,7,8,9 or 10 variations; Or 12,14,15,16,18,20,22,24,25,28 or 30 variations; Or the like, until maximum variable number 125,130,140,150,160,170,180,190 or 200.Yet inositol six-phosphatase variants of the present invention also must have at least 74% identity with SEQID NO:2, and this per-cent is determined as mentioned above.
To with what replacement or extend and not carry out any specified replacement or extension and be meant and insert any naturally that or non-natural amino acid is in occupying template the amino acid of this position.
Embodiment 13 for how using this nomenclature provides further and has explained.
Identify corresponding location number
As top explanation, use the standard of the ripe phytase of Bu Shi citric acid bacillus ATCC 51113 (SEQ ID NO:2) as Position Number, also be the standard of nomenclature therefore.
For other phytases, especially inositol six-phosphatase variants of the present invention, position corresponding to position D among the SEQ ID NO:2 finds by two sequences of method comparison that described in detail in " phytase polypeptides, identity per-cent " by name joint.From comparison, can clearly and expressly identify (mutual those two positions in comparison) with the corresponding position of position D of SEQ ID NO:2 in the sequence of the present invention at the other side top.
Embodiment 13 is comparison examples of the phytase of the phytase of SEQ ID NO:2 and SEQ ID NO:9, this examples show the corresponding position in these two main chains how to identify.
Some other fully in the example of hypothesis is also included within, these example source are from above table 1, and table 1 comprises the comparison of a collection of two sequences in the 3rd row:
First the 3rd lattice of going with reference to table 1: top sequence is a template, and following is variant.Location number 80 is meant the amino-acid residue G in the template.Amino acid A has occupied the corresponding position in the variant.Therefore, this replacement is expressed as G80A.
Referring now to table 1 second the row the 3rd lattice: top sequence still be template and below be variant.Location number 80 is still the amino-acid residue G in the finger print plate.Variant has two insertions, that is, TY is behind the G80 of template and before the V81.And T and Y have certainly they own in the variant aminoacid sequence location number of " reality ", we are always with reference to the location number in the template, so T and Y are called respectively at location number 80a and 80b with regard to present case.
At last, with reference to last column the 3rd lattice of table 1: location number 275 is meant last amino acid of template.The extension ST of C-end is called at location number 275a and 275b, although same they also have the location number in the variant aminoacid sequence of they oneself " reality " certainly.
The characteristic of revising is with reference to phytase
In an embodiment, phytase of the present invention has modification, preferably improved characteristic.Term " modification " and " improvement " mean with another kind of phytase and compare.These other be used for reference to or phytase example relatively 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.Further example with reference to phytase can be SEQ ID NO:9, and in Fig. 1 disclosed its variant.
Revise, the non-limiting example that is preferably improved characteristic is as follows: thermostability, pH curve, specific activity, the performance in animal-feed, proteolytic enzyme susceptibility and/or glycosylation pattern.Phytase of the present invention can also have modification, advantageous embodiment, temperature curve, and/or it can mix the variation in potential proteolytic enzyme cutting site.
Thermostability
Thermostability, or temperature stability can be measured 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, the following mensuration of remaining activity wherein: the supernatant separated into two parts will ferment, part incubation 30 minutes under the temperature that expection improves, another part is 5 ℃ of incubations 30 minutes, measures the two activity at p-nitrophenyl phosphoric acid in 37 ℃ and pH5.5 then, and the activity that is used in the sample of incubation under the temperature of raising is divided by the activity in the same sample of 5 ℃ of incubations.The preferred temperature that improves is 50 ℃, 55 ℃, and 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃ or 85 ℃.If necessary, the sample that contains enzyme can dilute in 0.1M NaAc pH5.5.The remaining activity of phytase of the present invention is preferably with reference at least 105% of phytase remaining activity, or at least 110%, 120%, 130%, 140%, 150%.In embodiment further, the remaining activity of phytase of the present invention is with reference at least 200% of phytase remaining activity, or at least 250%, 300%, 400% or at least 500%.In embodiment further, the remaining activity of phytase of the present invention is at least 2 times with reference to the phytase remaining activity, 3 times, and 4 times, 5 times, 6 times, 7 times, 10 times, 15 times, 20 times or at least 25 times.
Thermostability can also be measured described in embodiment 5.Therefore, in a preferred embodiment, phytase of the present invention has the remaining activity higher than reference phytase, the following mensuration of remaining activity wherein: the supernatant separated into two parts will ferment, a part was 50 ℃ of incubations 30 minutes, another part is then measured the two activity at p-nitrophenyl phosphoric acid at 37 ℃ of pH5.5 5 ℃ of incubations 30 minutes, and the activity that is used in the sample of incubation under the temperature of raising is divided by the activity in the same sample of 5 ℃ of incubations.If desired, the sample that contains enzyme can dilute in 0.1M NaAc pH5.5.The remaining activity of phytase of the present invention is at least 2 times of reference phytase remaining activity of SEQ ID NO:3 preferably, and 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 preferably SEQ ID NO:2 reference phytase remaining activity at least 105%, 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 compare improve to some extent (seeing Table 3) 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 be measured as described in example 8 above.Therefore, one preferred embodiment in, phytase of the present invention has the remaining activity higher than reference phytase, the following mensuration of remaining activity wherein: the supernatant separated into two parts will ferment, a part was 60 ℃ of incubations 30 minutes, another part is then measured the two activity at p-nitrophenyl phosphoric acid at 37 ℃ of pH5.5 5 ℃ of incubations 30 minutes, and the activity that is used in the sample of incubation under the temperature of raising is divided by the activity at the sample of 5 ℃ of incubations.If desired, the sample that contains enzyme can dilute in choosing the 0.1M NaAcpH5.5 that comprises 0.005% Tween-20 wantonly.Phytase of the present invention and can in subtilis (Bacillus subtilis) host strain, express with reference to phytase.Host strain can shake 100ml PS1 substratum (100g/L sucrose, 40g/L soybean flakes (Soy flakes), 10g/LNa in the bottle at 500ml 2HPO 412H 2O, 0.1ml/L Dowfax 63N10 (Dow)) cultivated four days at 30 ℃ with 300rpm in.The remaining activity of phytase of the present invention preferably the reference phytase of SEQ ID NO:2 remaining activity at least 32%, or at least 34%, 36%, 38% or at least 40%.More preferably, the remaining activity of phytase of the present invention be SEQ ID NO:2 the reference phytase remaining activity at least 50%, or at least 60%, 70%, 80%, 90% or at least 100%.Also more preferably the remaining activity of phytase of the present invention be SEQ ID NO:2 the reference phytase remaining activity at least 120%, 140%, 160%, 180% or at least 200%.Most preferably, the remaining activity of phytase of the present invention is at least 2 times of 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 is preferred (seeing Table 5) especially:
(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 be measured as described in example 9 above, promptly uses the proteic denaturation temperature Td of phytase that the dsc measurement method is measured purifying.Td has indicative for proteic thermostability: Td is high more, and then thermostability is high more.Therefore, in a preferred embodiment, phytase of the present invention has the Td higher than reference phytase, wherein at the phytase sample of purifying use dsc from the 20-90 ℃ of scan rate with 90 ℃/h at the 20mM sodium acetate buffer, measure Td among the pH4.0 and (preferably be at least 95% with purity, measure by SDS-PAGE), dialysis (step of preferably then spending the night after 2-3 hour step) in 20mM sodium acetate pH4.0 before this measures is carried out that 0.45um filters again and with dialysis buffer liquid protein concentration is diluted to being equivalent to about 2 absorbance unit (A 280).In a preferred embodiment, the Td of phytase of the present invention is more preferably its at least 101% than the Td height of the phytase of SEQID NO:4, 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 be SEQ ID NO:4 phytase Td at least 120%, 130%, 140%, 150%, 160%, 170%, 180% or at least 190%.Following replacement is preferred (seeing Table 6) especially: 362K, 362R, 111P and/or 273L.In specific embodiment further, as use the dsc described in the embodiment 2 (DSC) (promptly at the 20mM sodium acetate, among the pH4.0) measure, heat-staple phytase of the present invention have at least 50 ℃ melting temperature(Tm) Tm (or denaturation temperature, Td).In embodiment further, 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 ℃.The dsc measurement method can also be carried out described in embodiment 1 (" dsc measurement method "), or carries out described in embodiment 2 (" measuring thermostability by DSC ").
Thermostability can also be measured as described in example 12 above.Therefore, one preferred embodiment in, incubation is after 60 minutes under 70 ℃ and pH4.0 condition, phytase of the present invention is compared with the reference phytase of handling according to the same manner has improved remaining activity, and described remaining activity is to calculate with respect to the activity that (at the 0th minute) before the incubation exists for every kind of phytase.The remaining activity preferred pin is measured when pH5.5 and 37 ℃ sodium phytate.Incubation is preferably at the 0.1M sodium acetate, among the pH4.0.Phytase is purifying preferably, more preferably is purified to and is determined as at least 95% purity by SDS-PAGE.Preferred phytase activity assay buffer is 0.25M sodium acetate pH5.5.Use present method, the remaining activity of phytase of the present invention is preferably with reference at least 105% of the remaining activity of phytase, and more preferably at least 110%, 115%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or at least 200%.Perhaps, the active remaining activity with respect to the 0th minute preferably at least 31% or at least 32%.The following replacement that improved thermostability is provided is preferred (seeing Table 9): 273L, 46E, 362R and/or 53V.
In an embodiment, inositol six-phosphatase variants of the present invention is with to compare heat with reference to phytase more stable, and wherein thermostability is measured (based on example 1,5,8,9 or 12) by any test of using in above-mentioned four kinds of tests.
In embodiment, the following variant of the phytase of expection SEQ ID NO:2 has improved thermostability (in each grouping, with preferred order):
(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) replace the ring that comprises between C178 and the C187 with shorter ring, described shorter ring for example is selected from, 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 for example being selected from, HQEKMGTMDPT, HQQDIKQVDSL, HQPEIGKMDPV, TQADTSSPDPL, HQQDIKQADPL, TQTDTSSPDPL, the ring of NQADLKKTDPL replace between the residue 114 and 124 ring (YQKDEEKNDPL) in the face of avtive spot;
(x)R339D。
Temperature curve
Phytase of the present invention is compared the temperature that whether has modification and can be come as described in example 10 above to determine with the reference phytase.Therefore, phytase of the present invention is compared the temperature curve with modification with the reference phytase in an embodiment, and phytase is at the activity of the sodium phytate function with respect to temperature when wherein temperature curve being determined as in 20-90 ℃ of scope (is one-level with 10 ℃) pH5.5.Preferred damping fluid is at the 0.25M sodium acetate buffer, among the pH5.5.The activity of each temperature preferably is expressed as with respect to the relative reactivity (representing with %) after the value stdn of optimum temps.Optimum temps is the temperature of temperature (promptly with 10 ℃ of those temperature for once crossing over) when middle activity is the highest of test.
In a preferred embodiment, phytase of the present invention has at least 18% in the time of 70 ℃, or at least 19%, 20%, 21%, 22%, 23%, 24%, or at least 25% relative reactivity.As top explanation, this is with respect to the activity when the optimum temps.More preferably, phytase of the present invention has at least 26%, 27%, 28%, 29%, 30%, 31% in the time of 70 ℃, or at least 32% relative reactivity.It is (seeing Table 7) that the preferred replacement (with the form that has higher relative reactivity at 70 ℃) of the temperature curve of modification is provided: 57Y, 76G, 107G, 273L, 362K, 46E, 362R, 53V and/or 241Q.It is higher that their relative reactivities in the time of 70 ℃ and SEQ ID NO:3 compare with 4 reference phytase, and (57Y, 76G in some cases, 107G, 273L, 362K, 362R and/or 53V) to compare with the reference phytase of SEQ ID NO:2 also be like this.
The pH curve
Phytase of the present invention is compared the pH curve that whether has modification with the reference phytase can be as measuring described in embodiment 11.Therefore, phytase of the present invention is compared the pH curve with modification with the reference phytase in an embodiment, wherein the pH curve is defined as in the time of 37 ℃ in pH2.0 to 7.5 scope (is one-level with 0.5 pH unit) at the phytase activity of the sodium phytate function as pH.Preferred damping fluid is the 50mM glycine, the mixed solution (cocktail) of 50mM acetate and 50mM Bis-Tris.Another preferred damping fluid is the 0.25M sodium acetate.Activity at each pH preferably is expressed as with respect to the relative reactivity (representing with %) after the value stdn at best pH.
The example of the pH curve of revising is that pH curve (relative reactivity is as the function of pH) is to higher or lower pH skew.With SEQ ID NO:2,3 or 4 reference phytase is compared, and it is (seeing Table 8) that the preferred replacement to higher pH skew 0.5pH unit is provided: 46E and/or 218Q.
Other examples of the pH curve of revising are that wherein best pH changes upward or downwards.With SEQID NO:2,3 or 4 compare, and it is (seeing Table 8) that the preferred replacement of lower best pH is provided: 46E, 121D and/or 200K.With SEQ ID NO:2,3 or 4 compare, and it is (seeing Table 8) that the preferred replacement of higher best pH is provided: 218Q and/or 241Q.
The pH curve of revising can also be measured as embodiment 1 (" the pH curve of modification: the mensuration of pH3.5/5.5 specific activity ") described in, that is, and and by the phosphatase activity of comparison pH3.5 and 5.5 o'clock.Alternatively, the activity during pH3.5 can with pH4.0,4.5 or 5.0 o'clock specific activity.In alternative embodiment further, relatively be phytase activity but not phosphatase activity.
In an embodiment, phytase of the present invention is compared the pH curve with modification with the reference phytase.More specifically, the pH curve is revised in pH 3.5-5.5 scope.Further more specifically, at pH4.0,4.5,5.0 and/or 5.5 o'clock activity be active at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% when best pH (pH3.5), 90% or at least 95% level.
The pH curve of polypeptide, and best pH can measure by this polypeptide of incubation under various pH values, described incubation is used the substrate of determining good concentration in advance and is also carried out under the fixed heated culture temperature.The pH curve is the diagram of phytase activity with respect to pH, and best pH determines from this pH curve.In an embodiment, used Phosphoric acid esterase or the phytinic acid enzyme assay of embodiment 1, for example substrate is the 5mM sodium phytate, 37 ℃ of temperature of reaction, and in each pH pH-value determination pH activity, for example pH2-12 replaces the pH5.5 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 (pH4.0-5.0), 0.1MBis-Tris/HCl (pH5.5-6.5), 0.1M Tris/HCl (pH7.0).Other examples of damping fluid are: be adjusted to pH value 2.0,2.5,3.0,3.5 with HCl or NaOH, 4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0 and 12.0 100mM succsinic acid, 100m MHEPES, 100mM CHES, 100mMCABS.
In specific embodiment, the following variant of the phytase of expection SEQ ID NO:2 has the pH curve (in each grouping, with preferred order) of modification:
(i)E218Q，D324N，T200R，K，N121D，E196Q，D202N，E406A，E167Q，E53V，Q，E241Q，D314N，G，E239Q，E285N；
(ii) with being selected from for example HQEKMGTMDPT, HQQDIKQVDSL, HQPEIGKMDPV, TQADTSSPDPL, HQQDIKQADPL, TQTDTSSPDPL, the ring of NQADLKKTDPL replace between the residue 114 and 124 ring (YQKDEEKNDPL) in the face of avtive spot.
Specific activity
In one embodiment, phytase of the present invention has improved specific activity with respect to the reference phytase.More specifically, the specific activity of phytase of the present invention is with respect at least 105% of the specific activity of the reference phytase of measuring by same steps as.In embodiment further, 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 measuring by same steps as.
Under alternative situation, the specific activity that term is high refers to the specific activity of 200FYT/mg zymoprotein (EP) at least.In 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 at highly purified sample (sds page should show to have only 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 the concrete inositol six-phosphatase variants discussed, and it is calculated as the phytase activity of measuring with following unit, i.e. the FYT units of every milligram of inositol six-phosphatase variants zymoprotein.Further details is referring to embodiment 7.
In embodiment, the following variant of the phytase of expection SEQ ID NO:2 has the specific activity of modification, wherein with preferred order, the ring (YQKDEEKNDPL) in the face of avtive spot between residue 114 and 124 is replaced with being selected from following ring, for example, HQEKMGTMDPT, HQQDIKQVDSL, HQPEIGKMDPV, TQADTSSPDPL, HQQDIKQADPL, TQTDTSSPDPL, NQADLKKTDPL.
Performance in animal-feed
Phytase of the present invention is compared with the reference phytase in animal-feed and is had improved performance in a specific implementations.Performance in animal-feed can be measured by the external model among the embodiment 6.Therefore, phytase of the present invention in a preferred embodiment has improved performance in animal-feed, wherein said performance is measured in external model, feed sample by preparation is made up of 30% soyflour (soybean meal) and 70% Semen Maydis powder (maize meal) adds CaCl to this feed sample 2Concentration to every kilogram of feed 5 gram calcium; Under 40 ℃ and pH3.0 condition preincubation they 30 minutes, then add stomach en-(3000U/g feed) and phytase; Preincubation sample 60 minutes is then under the pH4.0 condition 30 minutes under 40 ℃ and pH3.0 condition; Termination reaction; By adding HCl, then carry out freeze-thaw cycle and extracted phytinic acid and phosphoinositide in 1 hour at 40 ℃ of incubations to final concentration 0.5M and 40 ℃ of incubations 2 hours; (high performance ion chromatography) separates phytinic acid and phosphoinositide by the high-effect ionic chromatography; Measure the amount of remaining phytinic acid phosphorus (IP6-P); Calculating through phytase treatment and without the blank sample of phytase treatment between the difference (this difference be degraded IP6-P) of remaining IP6-P; The IP6-P that represents the degraded of phytase of the present invention with respect to the IP6-P of the degraded of reference phytase (phytase that for example has SEQ ID NO:3 and 4).
Certain phytase of the present invention be with identical amount with reference to phytase, be preferably based on phytase activity unit (FYT) and come administration.Preferred dosage is the 125FYT/ kilogram of feed.Another preferred dosage is the 250FYT/ kilogram of feed.Phytase can be with the phytase form of purifying, or with the form administration of fermentation supernatant.The phytase of purifying preferably has at least 95% purity, as being measured by SDS-PAGE.
In preferred embodiment, the IP6-P value of the degraded of purifying phytase of the present invention with respect to the IP6-P value of the degraded of reference phytase, is at least 101%, or at least 102%, 103%, 104%, 105%, 110%, 115%, or at least 120%.In preferred implementation further, the IP6-P value of the degraded of purifying phytase of the present invention with respect to the IP6-P value of the degraded of reference 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 with respect 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%.
Compare with the phytase of SEQ ID NO:3, following replacement provides the improved or same at least outstanding performance in external animal-feed (seeing Table 4A): 4P, 5P, 111P, 1 *, 1 */ 2 *, 1 */ 2 */ 3 *, 273L, 286Q.
Compare with the phytase of SEQ ID NO:3, following replacement also provides the improved or same at least outstanding performance in external animal-feed (seeing Table 4A): 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 that is more preferably about the 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 recently be calculated as the percentage of the phosphorus that is discharged by the reference phytase.
In embodiment further, the relative performance of phytase of the present invention can be used as the phosphorus that discharged by phytase of the present invention and recently calculates with respect to the percentage of the phosphorus amount that is discharged by the reference phytase.
In embodiment further, 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 proteolytic enzyme susceptibility that reduces
In an embodiment, phytase of the present invention has the proteolytic enzyme susceptibility of reduction.More specifically, it has the susceptibility of reduction to Kex2 proteolytic enzyme, means the trend reduction that takes place by the cutting of this proteolytic enzyme.
Variant 339D, preferred R339D is the example of phytase of the present invention with proteolytic enzyme susceptibility of reduction.
Glycosylation pattern
Glycosylation is observed phenomenon during expressing protein in eukaryotic cell such as fungi and transgenic plant only, but does not observe in prokaryotic cell prokaryocyte such as bacterium.Various types of glycosylations are arranged, but maximally related in this article be the N-glycosylation, i.e. the glycosylation that connects of l-asparagine, it is initial wherein to attach to l-asparagine from the N-acetyl-glucosamine molecule, makes sugar attach to albumen.Have been found that the N-glycosylation only betides in sequence the l-asparagine of a part that is following tripeptides: 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), compare during with expression in subtilis, found lower thermostability, see embodiment 2.
This discovery has caused proposal of the present invention, promptly by changing the 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 modification, the inositol six-phosphatase variants of the N-glycosylation site of preferred modification.Expectation is when expressing in fungi, and the glycosylation meeting of modification gives inositol six-phosphatase variants improved thermostability.
The example of phytase is a bacterial phytases, gram-feminine gender (Gram-negative) phytase for example, 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, SEQ ID NO:4, the phytase of 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 concrete embodiment, following phytase of the present invention (for example variant of SEQ ID NO:2) expectation has the glycosylation pattern of modification, with preferred order: N31T, N74A, N171T, N203T, N281H, N316D, N308A.Following is to replace N-X-T pattern formula: N31T, N74A, N281H.Following is to replace N-X-S pattern formula: N171T, N203T, N308A, N316D.
Hypoallergenic variant
In an embodiment, phytase of the present invention (also) is hypoallergenic variant, and plan is worked as and animal, comprises when the people contacts, and can cause the immunne response of reduction.The term immunne response should be understood to any reaction by the immunity system generation of the animal of contact inositol six-phosphatase variants.The immunne response of a type is an allergic response, and the IgE level increases in its animal body that causes being touched.Hypoallergenic variant can use technology known in the art to prepare.For example the epi-position of the inositol six-phosphatase variants inositol six-phosphatase variants that can relate to polymeric groups masked segment (polymer moieties shielding portions) or immunne response combines.Can relate to the external chemical coupling of polymkeric substance to inositol six-phosphatase variants with combining of polymkeric substance, for example, as at WO 96/17929, WO 98/30682, described in WO98/35026 and/or the WO 99/00489.In conjunction with can be additionally or selectively relate to polymkeric substance and inositol six-phosphatase variants coupling in vivo.This combination can realize by following method: genetically engineered is handled the nucleotide sequence of coding inositol six-phosphatase variants, in inositol six-phosphatase variants, insert the consensus sequence of other glycosylation sites of coding, with in can making the glycosylated host of inositol six-phosphatase variants, express inositol six-phosphatase variants, referring to for example WO 00/26354.Thereby the another kind of approach that hypoallergenic variant is provided be with the nucleotide sequence that genetically engineered is handled the coding inositol six-phosphatase variants cause inositol six-phosphatase variants from oligomerization, cause the described monomeric epi-position of other inositol six-phosphatase variants of inositol six-phosphatase variants monomer maskable and reduce the antigenicity of oligomer thus.These products and their preparation are for example being described among the WO 96/16177.Can be by the whole bag of tricks such as WO 00/26230 and WO 01/83559 described phage display method, or the random device described in the EP 561907 is identified the epi-position that participates in immunne response.In case identified epi-position, can pass through known gene manipulation techniques such as directed mutagenesis (referring to for example WO 00/26230, WO 00/26354 and/or WO 00/22103) change the immune property of its aminoacid sequence with mutagenic inositol six-phosphatase variants, and/or can enough carry out the combination of polymkeric substance near the epi-position place, so that the described epi-position of polymer shield.
Nucleotide sequence and construct
The invention still further relates to the nucleotide sequence of the nucleotide sequence that comprises code book invention inositol six-phosphatase variants.
Term " isolated nucleic acid sequences " is meant that basically (essentially) do not contain the nucleotide sequence of other nucleotide sequences, for example as measuring by agarose gel electrophoresis, pure at least about 20%, preferably pure at least about 40%, more preferably pure at least about 60%, even it is more preferably pure, most preferably pure at least about 90% at least about 80%.For example, isolated nucleic acid sequences can obtain by standard cloning process used in genetically engineered, so that nucleotide sequence is repositioned onto the different positions that it will duplicate from its natural place.Cloning process can relate to excision and separate the expectation nucleic acid fragment that comprises the nucleic acid encoding sequence, fragment is inserted carrier molecule, and incorporate recombinant vectors into host cell, will duplicate a plurality of copies or the clone of this nucleotide sequence in this host cell.Nucleotide sequence can be genomic, cDNA, and RNA, semisynthetic, synthetic source, or their arbitrary combination.
Nucleotide sequence of the present invention can suddenly change and prepares in template phytase encoding sequence or its subsequence by introducing at least one, wherein Tu Bian nucleic acid sequence encoding variant phytase.In nucleotide sequence, introduce sudden change, to replace another nucleic acid with a nucleic acid, this can finish by any known method in this area, for example pass through directed mutagenesis, pass through random mutagenesis, or (doped) by mixing, (spiked) of wedging, or partial random mutagenesis (localized random mutagenesis).
Random mutagenesis is suitable for carrying out at least three parts of the gene of translating into described aminoacid sequence as the random mutagenesis of partial or regiospecificity, or carries out in complete genome.When using oligonucleotide to carry out mutagenesis, can be in the building-up process of oligonucleotide described three non-parents (non-parent) Nucleotide be mixed (doped) or wedge (spiked) this oligonucleotide in the position that will change.Thereby mix or wedge and avoid undesired amino acid whose codon.Oligonucleotide that mix or wedging can be incorporated among the DNA of the phytase of encoding by any technology, for example, and PCR, LCR or any archaeal dna polymerase and the ligase enzyme that see fit.
Preferably, mixing usefulness " constant mix at random (constant random doping) " and carry out, is predefined in the wild-type of each position and the per-cent of sudden change wherein.In addition, can make to mix and be devoted to preferably to introduce specific nucleotide, and therefore preferred one or more specified amino acid residues of introducing.Mix and for example to make, make it allow to introduce 90% wild-type and 10% and suddenly change in each position.Be based on restriction heredity and protein structure in another consideration aspect the selection of mixing design.
Random mutagenesis can advantageously be confined to the part of parent's phytase of being discussed.For example, when some zone of having identified enzyme was even more important for the particular characteristics of this enzyme, this mode may be favourable.
Provide alternative method of variant of the present invention to comprise gene reorganization (shuffling), for example described in WO95/22625 or WO 96/00343, and the consensus sequence deriving method described in EP 897985 (consensus derivation process).
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 sequences, and described regulating and controlling sequence instructs the expression of encoding sequence under the condition compatible with this regulating and controlling sequence in proper host cell.Expression should be interpreted as to comprise any step that relates in the polypeptide generation, include but not limited to, transcribe, post transcriptional modificaiton, translation, posttranslational modification and secretion.
Terminology used here " nucleic acid construct " is meant strand or double-stranded nucleic acid molecule, described nucleic acid molecule separates from naturally occurring gene, perhaps modifies described nucleic acid molecule so that it contains the fragment of nucleic acid in the mode that can not be present in occurring in nature (would not otherwise exist in nature) originally.When nucleic acid construct comprises when expressing the required regulating and controlling sequence of 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 at this paper and comprises that expressing for the polynucleotide of coding polypeptide of the present invention is essential or favourable all the components.Each regulating and controlling sequence can be natural or external source for the nucleic acid encoding sequence.Such regulating and controlling sequence includes, but not limited to leader sequence, polyadenylation sequence, propeptide sequence, promotor, signal peptide sequence and transcription terminator.Minimum situation, regulating and controlling sequence comprise promotor and transcribe with the translation termination signal.Regulating and controlling sequence can provide together with the joint that is used to introduce the specificity restriction site, and described specificity restriction site promotes being connected of nucleotide sequence coded district of regulating and controlling sequence and coded polypeptide.
Term " is operably connected " and represents such configuration at this paper, wherein regulating and controlling sequence is placed the appropriate position with respect to the encoding sequence of polynucleotide sequence, thereby makes regulating and controlling sequence instruct the expression of polypeptid coding sequence.
Term when being used for this paper " encoding sequence " (CDS) represents directly to specify the nucleotide sequence of the aminoacid sequence of its protein product.The border of encoding sequence is generally by opening frame decision, begins with ATG initiator codon or other initiator codon such as GTG and TTG usually.Encoding sequence can be DNA, cDNA or recombinant nucleotide sequence.
Expression vector
Term " expression " comprises any step that relates in the polypeptide generation, but is not limited to, and transcribes post transcriptional modificaiton, translation, posttranslational modification and secretion.
Term " expression vector " is defined as linear or Circular DNA molecular structure at this paper, and it comprises the polynucleotide of the polypeptide of the present invention of encoding, and described polynucleotide are operably connected with the extra Nucleotide that is provided for its expression.
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 promotor of encoding, operon, ribosome bind site, translation initiation signal and randomly suppressor gene or various activating gene.
The recombinant expression vector that carries the dna sequence dna of coding inositol six-phosphatase variants of the present invention can be any carrier, and it can carry out recombinant DNA method easily, and the selection of carrier often depends on that it is with the host cell that imports.Carrier can be a kind of like this carrier, and when it was imported host cell, this vector integration advanced the host cell gene group and duplicates with the karyomit(e) of having integrated it.
Inositol six-phosphatase variants can also be with at interested at least a other enzyme coexpression aspect the animal-feed, described other enzyme phytase for example, Phosphoric acid esterase, zytase, Galactanase, alpha-galactosidase, proteolytic enzyme, Phospholipid hydrolase, amylase, and/or beta-glucanase.Enzyme can be from different carrier coexpressions, from a carrier coexpression, or the combination of using 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 be from one or more promoter expressions.If clone (two or polycistronic) under the regulation and control of a promotor, the order of gene clone may influence proteic expression level.Inositol six-phosphatase variants can also be expressed as fusion rotein, the gene of the inositol six-phosphatase variants of promptly will having encoded meets frame ground (in frame) and is fused in another proteic gene of coding.This albumen can be other enzymes or from the functional domain of other enzymes.
Host cell
Term " host cell " as used herein, comprises the conversion that comprises the nucleic acid construct of polynucleotide of the present invention for use, transfection, any cell type of susceptibles such as transduction.
The invention still further relates to the recombinant host cell of the recombinant production that is suitable for polypeptide, it comprises polynucleotide of the present invention.The carrier that will comprise polynucleotide of the present invention imports to host cell, thereby makes this carrier as the chromosomal integration body or as keeping as the outer carrier of previous described self replication karyomit(e).Term " host cell " comprises any filial generation of parental cell, and it is incomplete same with parental cell owing to the sudden change that takes place between replicative phase.The selection of host cell will depend on the gene and the source thereof of coded polypeptide to a great extent.
Host cell can be a unicellular microorganism, prokaryotic cell prokaryocyte for example, or non-unicellular microorganism, for example eukaryotic cell.
Useful unicellular microorganism is a bacterial cell, gram positive bacterium for example, comprise, but be not limited to, bacillus (Bacillus) cell, for example, Alkaliphilic bacillus (Bacillus alkalophilus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus brevis (Bacillus brevis), Bacillus circulans (Bacillus circulans), gram Lloyd's genus bacillus (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, for example, shallow Streptomyces glaucoviolaceus (Streptomyces lividans) and mouse ash streptomycete (Streptomycesmurinus); Or gram negative bacterium, for example intestinal bacteria (E.coli) and Rhodopseudomonas bacterial classification (Pseudomonas sp).Aspect preferred, bacterial host cell is a bacillus lentus, Bacillus licheniformis, bacstearothermophilus or bacillus subtilis mycetocyte.Another preferred aspect, bacillus cell is Alkaliphilic bacillus (alkalophilic Bacillus).
Importing carrier to bacterial host cell can finish by the following method: for example protoplast transformation (referring to, for example, Chang and Cohen, 1979, Molecular General Genetics 168:111-115), utilize experience polypeptide cell (referring to, for example, Young and Spizizin, 1961, Journal of Bacteriology81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology56:209-221), electroporation (referring to, for example, Shigekawa and Dower, 1988, Biotechniques6:742-751), or joint (conjugation) (referring to, Koehler and Thorne, 1987, Journal of Bacteriology 169:5771-5278).
Host cell can also be an eukaryote, Mammals for example, insect, plant or fungal cell.
Aspect preferred, host cell is the fungal cell." fungi " of using here comprises with the 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, define among the UK) and oomycetes door (Oomycota) (as at Hawksworth etc., 1995, the same, quote in 171 pages), with all mitospore fungies (mitosporic fungi) (Hawksworth etc., 1995, the same).
Aspect preferred, the host cell of fungi is a yeast cell." yeast " ascosporogenous yeast (ascosporogenous yeast) (Endomycetale (Endomycetales)) that comprises as used herein, produce load yeast (basidiosporogenous yeast) and belong to the yeast of imperfect fungi (Fungi Imperfecti) (gemma guiding principle (Blastomyetes)).Because being sorted in, zymic may change in the future, for the present invention, yeast should be defined as at biology of yeast and activity (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 aspect 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 aspect 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 aspect another is most preferred, yeast host cell is Kluyveromyces lactis (Kluyveromyces lactis) cell.Another most preferably aspect in, yeast host cell is to separate fat the West alpine yarrow mould (Yarrowia lipolytica) cell.
In aspect another is preferred, fungal host cells is a filamentous fungal cells." filamentous fungus " comprises the whole thread form (as Hawksworth etc., 1995, the same definition) in fungi (Eumycota) and oomycetes (Oomycota) subphylum.The feature of filamentous fungus is usually by chitin, Mierocrystalline cellulose, dextran, chitosan, mannosans, and the mycelia body wall formed of other complicated polysaccharide.Nourish and grow is to prolong by mycelia to realize and carbon katabolism is obligate aerobic.On the contrary, yeast for example yeast saccharomyces cerevisiae nourish and grow be realize by sprouting of unicellular thalline and carbon katabolism can ferment.
In aspect preferred, filamentous fungal host cell is a mould genus of 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), the curved mould genus of neck (Tolypocladium), trametes (Trametes) or Trichoderma (Trichoderma) cell.
In aspect most preferred, filamentous fungal host cell is an 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 aspect another is most preferred, filamentous fungal host cell is a 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) is intended silk spore sickle spore (Fusarium trichothecioides) or empiecement sickle spore (Fusariumvenenatum) cell.Another most preferred aspect, filamentous fungal host cell is black thorn smoke pipe bacterium (Bj erkandera adusta), do and intend wax bacterium (Ceriporiopsis aneirina), do and intend the wax bacterium, Ceriporiopsiscaregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa or worm are intended wax bacterium (Ceriporiopsis subvermispora), Coprinus cinereus (Coprinus cinereus), hairy fungus (Coriolus hirsutus), special humicola lanuginosa (Humicolainsolens), dredge cotton shape humicola lanuginosa (Humicola lanuginose), rice black wool mould (Mucor miehei), the thermophilic silk mould (Myceliophthora thermophila) of ruining, Neuraspora crassa (Neurospora crassa), penicillium purpurogenum (Penicilliumpur purogenum), Phanerochaete chrysosporium (Phanerochaetechrysosporium), arteries and veins bacterium (Phlebia radiata) is penetrated in radiation, eryngo pick up the ears (Pleurotus eryngii), autochthonal shuttle spore mould (Thielavia terrestris), long wool hair bolt bacterium (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.
The fungal cell can form by relating to protoplastis, and protoplast transformation and cell walls regenerated method transform in a manner known way.The appropriate method that transforms Aspergillus and Trichoderma host cell is at EP 238 023 and Yelton etc., and 1984, among the Proceedings of the National Academy of SciencesUSA 81:1470-1474 description is arranged.The appropriate method that transforms the fusarium bacterial classification is by Malardier etc., and 1989, Gene78:147-156 and WO 96/00787 describe.Can utilize Becker and Guarente, at Abelson, the Guide to Yeast Genetics and MolecularBiology that J.N. and Simon, M.I. edit, Methods in Enzymology, volume 194,182-187 page or leaf, Academic Press, Inc., NewYork; Ito etc., 1983, Journal of Bacteriology153:163; With Hinnen etc., 1978, the method described in the Proceedings of the National Academy of Sciences USA 75:1920 is with yeast conversion.
The preparation method
The invention still further relates to the method that is used to prepare phytase of the present invention, it comprises that (a) cultivates host cell under the condition that is of value to the phytase generation; And (b) reclaim phytase.
In preparation method of the present invention, use method known in the art culturing cell in the nutritional medium that is fit to the polypeptide generation.For example, can pass through shake-flask culture, and small-scale or large scale fermentation in laboratory or industrial fermentation jar (comprise successive; in batches; fed-batch (fed-batch), or solid state fermentation), in suitable medium and under described expression of polypeptides of permission and/or isolating condition, carry out.Utilize methods known in the art, cultivate in suitable nutritional medium, described substratum comprises carbon source and nitrogenous source and inorganic salt.Suitable medium can obtain from suppliers, or can be according to disclosed composition preparation (for example, in the catalogue of American type culture collection (American Type CultureCollection)).If polypeptide is secreted in the nutritional medium, then can directly reclaim polypeptide from substratum.If polypeptide is secretion not, then can from cell lysate, reclaim.
The polypeptide that obtains can reclaim with means known in the art.For example, can include, but not limited to centrifugally, filter, extract by the method for routine, spraying drying, evaporation or precipitation reclaim polypeptide from nutritional medium.
Polypeptide of the present invention can come purifying by multiple methods known in the art, includes but not limited to chromatography (for example, ion-exchange, affine, hydrophobic, chromatofocusing, and size exclusion), electrophoresis method (for example, the isoelectrofocusing of preparation type), differential solubleness is (for example, ammonium sulfate precipitation), SDS-PAGE or extraction (referring to, for example, 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 that has used coding to have the polypeptide of phytase activity of the present invention transforms, thereby reaches and produce described polypeptide with callable scale.Polypeptide can reclaim from plant or plant part.Alternatively, comprise the plant of recombinant polypeptide or plant part can as application, in order to improve food or quality of the fodder, for example, improve nutritive value, palatability and rheological property, or destroy anti-nutrition (antinutritive) factor.
In embodiment, the endosperm storage bubble (endospermstoragevacuole) of polypeptide target in seed.This can have the appropriate signals propeptide and realize by it is synthesized, referring to Horvath etc., and at PNAS, on February 15th, 2000,97 volumes, No. 4, in the 1914-1919 page or leaf.
Transgenic plant can be dicots (dicotyledons) or monocotyledonous (monocotyledons) or their genetically engineered variants.Monocotyledonous example is a grass, English grass (meadowgrass) (bluegrass (blue grass) for example, Gramineae (Poa)), herbage (forage grass) is festuca (Festuca) for example, lolium (Lolium), the temperate zone grass is Agrostis (Agrostis) for example, and cereal, for example, 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 a tobacco, beans, for example Sunflower Receptacle (Helianthus (Helianthus)), cotton (Gossypium (Gossypium)), lupine, potato, sugar beet (sugar beet), pea, Kidney bean (bean) and soybean, and cress (Cruciferae (family Brassicaceae)), for example Cauliflower, Semen Brassicae campestris (rape seed), and the model animals Arabidopis thaliana (Arabidopsisthaliana) of tight association.Low-phytate salt plant is as for example United States Patent (USP) no.5,689,054 and United States Patent (USP) no.6,111,168 described be the example of genetically engineered plant.
The example of plant part is a stem, callus, leaf, root, fruit, seed and stem tuber, and the independent body that comprises these parts, epidermis for example, mesophyll, parenchyma, vascular tissue, meristematic tissue.Specific vegetable cell compartment, chloroplast(id) for example, apoplast (apoplast), plastosome, vacuole, peroxysome, and tenuigenin also is considered to plant part.In addition, no matter any vegetable cell how tissue-derived, is all thought plant part.Equally, plant part, for example for promoting isolating particular organization of application of the present invention and cell also to be considered to plant part, embryo for example, endosperm, aleuron and plant skin (seed coats).
Also comprise these plants within the scope of the present invention, the filial generation of plant part and vegetable cell.
Can be according to the transgenic plant or the vegetable cell of methods known in the art construction expression polypeptide of the present invention.Briefly, following structure transgenic plant or vegetable cell by one or more code book invention polypeptide expression constructs are mixed the plant host genome, and make the modified plant or the vegetable cell propagation that obtain become transgenic plant or vegetable cell.
Easily, expression construct is a nucleic acid construct, and it comprises the nucleotide sequence of the polypeptide of the present invention of encoding, and the described nucleotide sequence suitable regulating and controlling sequence required with express this nucleotide sequence in selected plant or plant part is operably connected.In addition, expression construct can comprise that selective marker is used to identify the host cell of having integrated expression construct and for construct being imported the necessary dna sequence dna of described plant (latter depends on applied DNA introduction method).
Regulating and controlling sequence, for example promotor and terminator sequence and the randomly selection of signal or transit sequence, based on expectation for example when, where and how express polypeptide decides.For example, the expression of gene of the polypeptide of the present invention of encoding can be composing type or induction type, maybe can be to grow, and the stage or tissue-specific, and gene product can target specific cells compartment, tissue or plant part such as seed or leaf.Regulating and controlling sequence is, for example, by Tague etc., 1988, Plant Physiology 86:506 is described.
For constitutive expression, can use following promotor: 35S-CaMV promotor (Franck etc., 1980, Cell 21:285-294), corn 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 R 1991, Analysis of rice Act1 5 ' region activity in transgenicrice plants.Plant Cell 3,1155-1165).Organ specific promotor can be that for example, depots tissue (storage sink tissue) is seed for example, the promotor (Edwards﹠amp of potato tuber and fruit; Coruzzi, 1990, Ann.Rev.Genet.24:275-303), or from for example merismatic promotor (Ito etc. of metabolic pool tissue (metabolicsink tissue), 1994, Plant Mol.Biol.24:863-878), the promotor of seed-specific is for example from the gluten of rice, prolamine, sphaeroprotein or albuminous promotor (Wu etc., 1998, Plant and Cell Physiology 39:885-889), from legumin B4 with from the broad bean promotor (Conrad etc. of the unknown seed protein gene of broad bean (Vicia faba), 1998, Journal of Plant Physiology 152:708-711), promotor (Chen etc. from seed oil body protein (seed oil bodyprotein), 1998, Plant and Cell Physiology 39:935-941), storage protein napA promotor from colea (Brassica napus), or the promotor of other any seed-specifics known in the art, for example, described in WO 91/14772.In addition, promotor can be that the leaf specific promoter is for example from the rbcs promotor (Kyozuka etc. of rice or tomato, 1993, PlantPhysiology 102:991-1000), chlorella virus VITAMIN B4 methyl transferase gene promotor (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 damage for example potato pin2 promotor (Xu etc. of inducible promoter, 1993, Plant Molecular Biology22:573-588).Equally, promotor can be handled for example temperature by abiotic (abiotic), and arid or salinity change induces, maybe the material of the activation promotor that can use by external source is induced, and described material is ethanol for example, oestrogenic hormon, plant hormone such as ethene, dormin, gibberic acid and/or heavy metal.
Promotor strengthens element and can also be used for obtaining the higher expression of polypeptide plant.For example, it can be intron that promotor strengthens element, its place promotor and the nucleotide sequence of the polypeptide of the present invention of encoding between.For example, above-mentioned Xu etc., 1993, disclose and utilized first intron of rice Actin muscle 1 gene to strengthen expression.
Further, can select to express to improve with respect to the floristics optimizing codon of research (referring to Horvath above-mentioned etc.).
Any other part of selectable marker gene and expression construct can be from this area those obtainable choosing.
Incorporate nucleic acid construct into into Plant Genome according to routine techniques known in the art, these technology comprise the conversion of Agrobacterium (Agrobacterium) mediation, virus-mediated conversion, microinjection, particle bombardment, biology launches and transforms and electroporation (Gasser etc., 1990, Science 244:1293; Potrykus, 1990, Bio/Technology 8:535; Shimamoto etc., 1989, Nature 338:274).
At present, the transgenosis of agrobacterium tumefaciens (Agrobacterium tumefaciens) mediation be used to prepare the transgenosis dicotyledons preferred method (referring to 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, the monocotyledonous preferred method of preparation transgenosis except the Agrobacterium method, is particle bombardment (with the meticulous gold (microscopic gold) or the tungsten particle of transfering DNA the coating) (Christou of embryo Yu injured tissue 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 monocotyledons method for transformation is based on protoplast transformation, as Omirulleh etc., and 1993, Plant Molecular Biology21:415-428 is described.
After the conversion, transformant and the regeneration selecting to have incorporated expression construct into according to method known in the art become whole plant.Usually method for transformation is designed to can optionally remove the selection gene in regenerative process or in ensuing offspring, it is by utilizing the cotransformation that for example uses two isolating T-DNA constructs or by specific recombinase the selection gene being carried out locus specificity and excise.
The invention still further relates to the method for preparation polypeptide of the present invention, it is included under the condition that is of value to described polypeptide generation and cultivates transgenic plant or vegetable cell, 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 for example breast and blood, organ, meat and a zooblast of body fluid.The technology that is used for expressing protein, the technology of expressing protein in mammalian cell for example, be known in the art, referring to for example 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 translation post-treatment.As a rule, in order to prepare transgenic animal, the nucleotide sequence that has the polypeptide of phytase activity of the present invention with coding transforms the selected cell of selected animal, thereby expresses and the preparation polypeptide.Can from animal body, reclaim polypeptide, for example from the Ruzhong of jenny, maybe can be with expression of polypeptides for being of value to animal itself, for example auxiliary animal digestion.The example of animal title below is to mention in the part of animal-feed.
In order to prepare to reclaim polypeptide from the animal Ruzhong is the transgenic animal of purpose, the gene of coded polypeptide can be inserted in the zygote of the animal of being discussed, for example comprise the transgene expression vector of the gene of suitable milk-protein promotor and coding said polypeptide by use.Zygote is advanced in the transgene expression vector microinjection, preferably forever be integrated into karyomit(e).In case ovum begins growth and division, the parent (surrogate mother) that potential embryo implantation is substituted just, and identify and carry this genetically modified animal.The animal that obtains can be raised by routine immediately and breed.Polypeptide can be from animal Ruzhong purifying, referring to for example Meade, H.M. etc. (1999): Expression of recombinant proteins in the milk oftransgenic animals, Gene expression systems:Using nature for the art ofexpression. is (at transgenic animal Ruzhong express recombinant protein, 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 prepare the non-human transgenic animal who in the genome of its somatocyte and/or stem cell, carries the nucleotide sequence that comprises the heterologous transgene construct, wherein said heterologous transgene construct comprises the transgenosis of coded polypeptide, can be operably connected by first regulating and controlling sequence that this transgenosis and the sialisterium that is used for polypeptide is specific expressed, disclosed as WO 00/064247.
Composition and purposes
In a still further aspect, the present invention relates to comprise the composition of polypeptide of the present invention, and use these method for compositions.
Peptide composition can prepare according to methods known in the art, and can be with the form of liquid or dry composition.For example, peptide composition can exist with particle or microparticle form.The polypeptide that will be included in the composition can come stabilization according to methods known in the art.
Phytase of the present invention can be used for degraded in any industrial environment, for example, and phytate, phytinic acid and/or list, two, three, four and/or five phosphoinositides.Know for example metal ion of the phosphoric acid part chelating divalence of these compounds and tervalent positively charged ion, include but not limited to the essential ionized calcium of (i.a.) nutrition, iron, zinc and magnesium, and trace-metal manganese, copper and molybdenum.In addition, phytinic acid is also conjugated protein by electrostatic interaction to a certain extent.
Therefore, the preferable use of polypeptide of the present invention is the additive that (comprises human foods) or be used for these prepared products in the animal-feed prepared product.
In an embodiment, polypeptide of the present invention can be used to improve the nutritive value of animal-feed.The non-limiting example that improves the nutritive value of animal-feed (comprising human foods) is: improve the feed digestibility; Promote growth of animal; Improve feed applications; Improve proteic bioavailability; Raising can digest phosphatic level; Improve the release and/or the degraded of phytate; Improve the bioavailability of trace minerals; Improve the bioavailability of big mineral substance; Eliminate the phosphoric acid salt that adds complementarity, the needs of trace minerals and/or big mineral substance; And/or improvement 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 (promptly the feed weight of She Ruing increases with respect to weight) of animal.
And then polypeptide of the present invention can be used for reducing the phytinic acid salt level of ight soil.
Animal, animal-feed and animal feedstuff additive
Term animals comprises all animals, comprises the mankind.The example of animal is a non-ruminant animal, and ruminating animal.Ruminating animal comprises, sheep for example, and goat and ox (cattle), for example milk cow (cow) is as beef cattle (beefcattle) and milk cow (dair ycow).In an embodiment, animal is the non-animal that ruminates.Non-ruminant animal comprises the animal of simple stomach, for example pig (pig) or pig (swine) (including but not limited to piggy (piglet), pig in the growth (growing pig) and sow (sow)); Poultry is turkey for example, 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)); And crustaceans (including but not limited to river prawn (shrimp) and prawn (prawn)).
Term feed or feed composition are meant any compound, prepared product, mixture or the composition that is suitable for the animal absorption or is intended to be taken in by animal.
In according to purposes of the present invention, can be before the meal, after the meal or with have meal simultaneously to the feeding animal polypeptide.The latter is preferred.
In an embodiment, polypeptide adds form in the feed to it, or when it is included in the fodder additives, is that (substantially) is pure basically.It is (well-defined) that determines in an embodiment.Term " definite " means that the phytase prepared product is at least 50% pure, as measured (referring to the embodiment 12 of WO 01/58275) by size exclusion chromatography.In other concrete embodiments, as measuring by this method, the phytase prepared product is at least 60,70,80,85,88,90, and 92,94 or at least 95% is pure.
Basically pure, and/or the polypeptide prepared product of determining is favourable.For example, there is not the polypeptide of other polypeptide interference or pollution much easier basically with the correct dose administration.Term specifically is meant the purpose that obtains constant (consistent) and constant (constant) result with the correct dose administration, and based on the ability of desired effects optimization dosage.
Yet for the application in animal-feed, phytase polypeptides of the present invention does not need so pure; For example it can comprise other polypeptide, can be called the phytase prepared product in the case.
The phytase prepared product can (a) directly add feed (or being directly used in proteic treatment process) to, or (b) can be used for the production of one or more intermediate component, fodder additives or the premix (premix) of described intermediate component as adding (or in treatment process, using) in the feed subsequently to.The degree of above-mentioned purity is meant the purity of original polypeptide prepared product, no matter uses according to top (a) or (b).
Polypeptide prepared product with purity of this order of magnitude especially can be used recombinant method for production and obtain, and is not to be easy to and to have a much higher difference between batch (batch-to-batch variation) otherwise obtain them by the traditional zymotic method.
Such polypeptide prepared product certainly mixes with other polypeptide.
Polypeptide can add in the feed in any form, as pure relatively polypeptide (be it as arelatively pure polypeptide), or mix with other components that are intended to add in the animal-feed, promptly with the form of animal feedstuff additive, the premix of so-called animal-feed (pre-mixes) for example.
The present invention relates to the composition of use aspect animal-feed further, for example animal-feed, and animal feedstuff additive, for example premix.
Except that polypeptide of the present invention, animal additive of the present invention contains at least a liposoluble vitamin, and/or at least a water-soluble vitamins, and/or at least a trace minerals.The animal additive can also comprise at least a big mineral substance (macro mineral).
In addition, optionally, the fodder additives composition is a tinting material, for example carotenoid such as β-Hu Luobusu, astaxanthin and xenthophylls; Aromatic compound; Stablizer; Antimicrobial peptide; Polyunsaturated fatty acid; Active oxygen resultant (reactive oxygen generating species); And/or be selected from least a other polypeptide among following: 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-Dian Fenmei (EC 3.2.1.1) for example; And/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).
These other polypeptide is (as above defined in the face of the phytase prepared product) determined in an embodiment.
Phytase of the present invention can also make up with other phytase, for example sac fungi (ascomycete) phytase such as Aspergillus phytase, for example be derived from Fructus Fici aspergillus (Aspergillus ficuum), aspergillus niger or Aspergillus awamori; Or club fungi (basidiomycete) phytase, for example be derived from Peniophora lycii, flat the edge of a field mushroom (Agrocybe pediades), fine hair bolt bacterium (Trametes pubescens) or involute paxillus (Paxillus involutus); Or have their derivative, fragment or a variant of phytase activity.
Thereby, 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, with phytase of the present invention and the combination of these phytases.
The example of antimicrobial peptide (AMP ' s) is CAP18, LeucocinA, Tritrpticin, Protegrin-1, Thanatin, defensin (Defensin), lactoferrin (Lactoferrin), Lactoferricin and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins and statins (Statins) are included in disclosed compound and polypeptide among WO 03/044049 and the WO 03/048148, and above-mentioned every variant or the fragment that keep antimicrobial acivity.
The example of anti-fungus polypeptide (AFP ' s) is huge aspergillus (Aspergillus giganteus) and aspergillus niger peptide, and their variant and the fragment that keeps 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, and as arachidonic acid, docosahexenoic acid (docosohexaenoic acid), eicosapentaenoic acid and gamma-linoleic acid.
The example of active oxygen resultant is a for example perborate of chemical reagent, persulphate or antihypo; And polypeptide for example oxydase, oxygenase or synthetic enzyme.
Usually fat-soluble and water-soluble vitamins, and trace minerals forms the part that is intended to the so-called premix of feed interpolation, and big mineral substance then adds in the feed separately usually.In these types of compositions any just becomes animal feedstuff additive of the present invention when adding has polypeptide of the present invention.
In an embodiment, be intended to animal feedstuff additive of the present invention is contained in (or regulation must be contained in) animal diet followed or the feed with 0.01 to 10.0% level; More specifically with 0.05 to 5.0% level; Or 0.2 to 1.0% level (% represents the gram number of additive in per 100 gram feeds).In premix especially like this.
Following is the nonexcludability example list of these components:
The example of liposoluble vitamin is a vitamin A, Vitamin D3 500,000 I.U/GM, vitamin-E and vitamin K, for example vitamin K3.
The example of water-soluble vitamins is a vitamin B12, vitamin H and choline, VITMAIN B1, Wei ShengsuB2, vitamin B6, nicotinic acid, folic acid and pantothenate (panthothenate), for example D-calcium pantothenate (Ca-D-panthothenate).
The example of trace minerals is a manganese, zinc, iron, copper, iodine, selenium and cobalt.
The example of big mineral substance is a calcium, phosphorus and sodium.
Nutritional requirement (is example with poultry and piggy/pig) to these components is listed in the Table A of WO 01/58275.Nutritional requirement means that these components should provide with the concentration of regulation in diet.
On the other hand, animal feedstuff additive of the present invention comprise at least a in the Table A of WO 01/58275 specified individual components.At least one means, and is 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, this at least a individual components is included in the additive of the present invention with such amount, and this amount is provided at the 4th row of Table A, or the 5th row, or concentration (in-feed-concentration) in the feed in the scope of explanation in the 6th row.
The invention still further relates to the composition of animal-feed.Animal feedstuff compositions or diet have high relatively protein content.The feature of the diet of poultry and pig can be described in WO 01/58275 table B 2-3 row.The feature of the diet of fish can as this show B the 4th row described in.So in addition fish diet has the crude fat content of 200-310g/kg usually.
WO 01/58275 incorporates it into by reference corresponding to US 09/779334.
Animal feedstuff compositions of the present invention has the crude protein content of 50-800g/kg, and comprises the claimed polypeptide of at least a this paper in addition.
In addition, or under alternative situation (for above-mentioned crude protein content), but animal feedstuff compositions of the present invention has the metabolisable energy that content is 10-30MJ/kg; 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(Met) content of 0.1-100g/kg; And/or the methionine(Met) of 0.1-150g/kg adds cysteine content; And/or the lysine content of 0.5-50g/kg.
In embodiment, but metabolisable energy, crude protein, calcium, phosphorus, methionine(Met), methionine(Met) adds halfcystine, and/or the content of Methionin is within arbitrary in scope 2,3,4 in the table B of WO 01/58275 or 5 (R.2-5).
Crude protein multiply by the factor 6.25 with nitrogen (N) and calculates, i.e. crude protein (g/kg)=N (g/kg) x6.25.Nitrogen content is measured (A.O.A.C., 1984, Official Methods of Analysis14th ed., Association of Official Analytical Chemists, Washington DC) by Kjeldahl determination.
But the calculating of metabolisable energy can be 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.The country academic press, the Washington D.C., 2-6 page or leaf (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, research of Spelderholt poultry and expansion center, 7361 DA Beekbergen, Holland.Grafisch bedrij fPonsen & 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 the intact animal diet, the calculating of available phosphorus and amino acid whose diet content is based on the feed table, for example 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 a albumen.Described albumen can be animal proteinum, for example meat and bone meal, and/or fish meal; Or it can be a vegetable-protein.Term vegetable-protein used herein is meant any compound, composition, and prepared product or mixture, it comprises at least a albumen from plant derivation or origin, comprises modified albumen and protein derivatives.In embodiment, the protein content of vegetable-protein is at least 10,20,30,40,50 or 60% (w/w).
Vegetable-protein can plant-derived protein source, for example beans and cereal, for example from pulse family (Fabaceae) (pulse family (Leguminosae)), Cruciferae (Cruciferaceae), the material of the plant of Chenopodiaceae (Chenopodiaceae) and Gramineae (Poaceae), for example soyflour, feather fan bean powder (lupin meal) and canola (rapeseed meal).
In an embodiment, plant protein source is the material from one or more plants of pulse family, for example soybean, lupine, pea or Kidney bean.
In another embodiment, plant protein source is the material from one or more plants of Chenopodiaceae, beet for example, 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 for example barleys of cereal, wheat, rye, oat, corn (corn), rice, triticale and Chinese sorghum.
In embodiment further, animal feedstuff compositions of the present invention comprises the 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 for example can be produced and be mass (mash feed) (non-particulate (nonpelleted)) or granulated feed (pelleted feed).Normally, the ground feed is mixed and add the essential vitamin and the mineral substance of capacity according to standard (specification) to the species discussed.Polypeptide can be used as the form of solid or liquid polypeptide formulations to be added.For example, the solid polypeptide preparation usually before the mixing step or during add; Liquid polypeptide formulations adds after granulation step usually.Polypeptide also can be blended in fodder additives or the premix.
The final concentration of polypeptide is in every kilogram of diet 0.01-200mg polypeptide protein scope in the diet, for example the scope of every kilogram of animal diet followed 5-30mg polypeptide protein.
Phytase of the present invention should be used with effective amount, promptly dissolves and/or improve the amount of feed nutritive value with enough improvement.The consideration polypeptide is used with one or more following amounts (dosage range) at present: 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 proteic milligram of the phytase polypeptides number (ppm) in every kilogram of feed.
For the proteic milligram of the phytase polypeptides of measuring every kilogram of feed number, phytase purifying from feed composition is come out, the specific activity of the phytase of purifying is used the related assays method and is measured.The phytase activity of feed composition like this is also used identical assay method and is determined, and based on these two kinds of mensuration, calculates the proteic milligram of every kilogram of feed mysoinositol six-phosphatase dosage.
Identical mechanism is applicable to the proteic milligram of the phytase polypeptides of measuring in fodder additives number.Certainly, if can obtain to be used to prepare the sample of the phytase of fodder additives or feed, then from this sample, measure specific activity (need not purifying phytase from feed composition or additive).
Embodiment
The invention still further relates to following embodiment:
I. phytase, itself and SEQ ID NO:2 have at least 74% identity, and it is compared with SEQ ID NO:2 to be included in and is selected from least one following locational at least one variation: 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 that 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 compared with SEQ ID NO:2 to be included in and is selected from least one following locational at least one variation: 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 compared with SEQ ID NO:2 to be included in and is selected from least one following locational at least one variation: 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 being selected from least one following optimum seeking site: 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 that this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, is not SEQID NO:6, and is not the variant of listed it among SEQ ID NO:9 and Fig. 1.
IV. phytase, itself and SEQ ID NO:2 have at least 74% identity, and it is compared with SEQ ID NO:2 to be included in and is selected from least one following locational at least one variation: 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 that this phytase is not the variant of listed it among SEQ ID NO:9 and Fig. 1.
V. phytase, itself and SEQ ID NO:2 have at least 74% identity, and it is compared with SEQ ID NO:2 to be included in and is selected from least one following locational at least one variation: 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 variation: 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 wherein position 179,180,181,182,183,184,185 and 186 amino acid by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
Preferred at least one following variation: 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 in the position 179,180,181,182,183,184,185 and 186 amino acid by KEKHQ, KEKQQ, KEKKV or KTDKL replace;
Condition is that 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 variation: 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 in the position 179,180,181,182,183,184,185 and 186 amino acid by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
Preferred at least one following variation: 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 in the position 179,180,181,182,183,184,185 and 186 amino acid by KEKHQ, KEKQQ, KEKKV or KTDKL replace.
IIX. phytase, itself and SEQ ID NO:2 have at least 74% identity and it comprises at least one following variation: 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 in the position 179,180,181,182,183,184,185 and 186 amino acid by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
Preferred at least one following variation: 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 in the position 179,180,181,182,183,184,185 and 186 amino acid by KEKHQ, KEKQQ, KEKKV or KTDKL replace.
Condition is that this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, is not SEQID NO:6, and is not the variant of listed it of 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 variation: 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 in the position 179,180,181,182,183,184,185 and 186 amino acid by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
Preferred at least one following variation: 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, 200K 202N, 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 in the position 179,180,181,182,183,184,185 and 186 amino acid by KEKHQ, KEKQQ, KEKKV or KTDKL replace.
Condition is that this phytase is not the variant of listed it of 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 variation: 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 in the position 179,180,181,182,183,184,185 and 186 amino acid by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
Preferred at least one following variation: 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 in the position 179,180,181,182,183,184,185 and 186 amino acid by KEKHQ, KEKQQ, KEKKV or KTDKL replace.
XI. phytase, itself and SEQ ID NO:2 have at least 74% identity and it comprises at least one following variation: 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 that this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, is not SEQID NO:6, and is not its variant of listing among 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 variation: 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 variation: 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. each phytase among the embodiment 1-13, it is the variant of the phytase of SEQ ID NO:2.
XV. each phytase among the embodiment 1-13, it is the variant of the phytase of SEQ ID NO:3.
XVI. each phytase among the embodiment 1-13, it is the variant of the phytase of SEQ ID NO:4.
XVII. each phytase among the embodiment 1-13, it is the variant of the phytase of SEQ ID NO:6.
IIXX. each phytase among the embodiment 1-13, it is the variant of the phytase of SEQ ID NO:9.
IXX. each phytase among the embodiment 1-13, its be relevant with SEQ ID NO:9 and the inositol six-phosphatase variants in Fig. 1, listed in any variant.
XX. each phytase among the embodiment 1-19, itself so that comprise and be selected from the replacement that every ranks go out among Fig. 1 and replace the replacement in the combination or replace combination.
XXI. each phytase among the embodiment 1-20, it has improved thermostability, improved pH curve, improved specific activity, the glycosylation pattern of revising, improved temperature curve, improved performance in animal-feed, and/or it contains the change in (incorporate) potential proteolytic enzyme cutting site.
XXII. isolated nucleic acid sequences, it comprises the nucleotide sequence of each phytase among the coding embodiment I-XXI.
XXIII. nucleic acid construct, it comprises the nucleotide sequence of embodiment XXII, and this nucleotide sequence is operably connected to guidance produces phytase in appropriate expressive host one or more regulating and controlling sequences.
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 each phytase among the embodiment I-XXI, it comprises
(a) host cell of cultivation embodiment XXV comprises the supernatant liquor of phytase with generation; (b) reclaim phytase.
XXVII. transgenic plant, or plant part, it can express among the embodiment I-XXI each phytase.
IIXXX. genetically modified, non-human animal, or its product, or component (element), it can express among the embodiment I-XXI each phytase.
IXXX. composition, its comprise among the embodiment I-XXI each at least a phytase and
(a) at least a liposoluble vitamin;
(b) at least a water-soluble vitamins; And/or
(c) at least a trace minerals.
XXX. the composition of embodiment IXX, it further comprises at least a enzyme in the enzyme that is selected from down group: amylase, phytase, Phosphoric acid esterase, zytase, Galactanase, alpha-galactosidase, proteolytic enzyme, Phospholipid hydrolase and/or beta-glucanase.
XXXI. each composition among the embodiment IXX-XXX, it is an animal feedstuff additive.
XXXII. animal feedstuff compositions, its have 50 to 800g/kg crude protein content and comprise among the embodiment I-XXI each phytase or embodiment IXXX-XXXI in each composition.
XXXIII. be used to improve the method for animal-feed nutritive value, wherein with among the embodiment I-XXI each phytase or embodiment IXXX-XXXI in each composition add in the feed.
XXXIV. be used to reduce the method for animal excrement mysoinositol six phosphoric acid salt levels, it comprises the forage feed animal with the embodiment XXXII of significant quantity.
XXXV. be used to handle the method for vegetable-protein, it comprise at least a vegetable-protein or protein source add among the embodiment I-XXI each phytase or embodiment IXXX-XXXI in each the step of composition.
XXXVI. each the purposes of composition in animal-feed among each phytase or the embodiment IXXX-XXXI among the embodiment I-XXI; Purposes in the preparation of animal-feed; Be used to improve the purposes of the nutritive value of animal-feed; Be used for reducing the purposes of the phytinic acid salt level of animal excrement; The purposes that is used for the processing of vegetable-protein; Or be used for from the purposes of phytinic acid enzyme substrates release phosphorus.
A). phytase, it has at least 70% identity and its with SEQ ID NO:2 and compares with SEQ ID NO:2 to comprise and be selected from least one following variation: 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 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 and its with SEQ ID NO:2 and compares with SEQ ID NO:2 to comprise and be selected from least one following variation: 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 and its with SEQ ID NO:2 and compares with SEQ ID NO:2 to comprise and be selected from least one following variation: 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 variation: 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 in the position 179,180,181,182,183,184,185 and 186 amino acid by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace.
A4). embodiment a2)-a3) in each phytase, it comprises at least one following variation:
(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) K179 wherein, T180, T181, E182, K183, S184, T185 and K186 be by QADKP, GEDKP, and NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
(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) in each phytase, it comprises at least one following variation:
(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) K179 wherein, T180, T181, E182, K183, S184, T185 and K186 be by QADKP, GEDKP, and NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
(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 variation: 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 in the position 179,180,181,182,183,184,185 and 186 amino acid by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace.
C). each phytase in the top embodiment, it comprises at least one following variation:
(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) K179 wherein, T180, T181, E182, K183, S184, T185 and K186 be by QADKP, GEDKP, and NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
(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). each phytase in the above-mentioned embodiment, it comprises at least one following variation:
(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) K179 wherein, T180, T181, E182, K183, S184, T185 and K186 be by QADKP, GEDKP, and NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL, 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, the preferred 46E of E, 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, the preferred 114T115Q of V, 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). each phytase in the top embodiment, it has improved characteristic.
E). embodiment c) or phytase c1), it comprises the characteristic (i) of embodiment 3, (ii), and (iii), (iv), (v), (vi), (vii), (viii), (x), at least one (xi) and/or in one or more variations (xii), and have improved thermostability.
F). embodiment c) or phytase c1), it comprises embodiment c) characteristic (ix) and/or at least one in one or more variations (x), and have improved pH curve.
G). embodiment c) or phytase c1), it comprises embodiment c) one or more variations of characteristic (x) at least one, and have improved specific activity.
H). embodiment c) or phytase c1), it comprises embodiment c) one or more variations of characteristic (xi) at least one, and have the glycosylation pattern of modification.
I). embodiment c) or phytase c1), it comprises embodiment c) the variation of characteristic (xii), it has changed potential proteolytic enzyme cutting site.
J). comprise a1)-a5) and c1) embodiment a)-d) in each phytase, it comprises at least one following variation:
(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) K179 wherein, T180, T181, E182, K183, S184, T185 and K186 be by QADKP, GEDKP, and NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
(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 c1) embodiment a)-d) in each phytase, comprise at least one following variation:
(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) K179 wherein, T180, T181, E182, K183, S184, T185 and K186 be by QADKP, GEDKP, and NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
(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。
L). embodiment k) phytase, it is the variant of SEQ ID NO:2.
M). comprise a1)-a5) and c) embodiment a)-d) in each phytase, it comprises at least one following variation:
(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) K179 wherein, T180, T181, E182, K183, S184, T185 and K186 be 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 c1) embodiment a)-d) in each phytase, it comprises at least one following variation:
(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) K179 wherein, T180, T181, E182, K183, S184, T185 and K186 be by QADKP, GEDKP, and NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
(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 c1) embodiment a)-d) in each phytase, it comprises at least one following variation:
(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) K179 wherein, T180, T181, E182, K183, S184, T185 and K186 be by QADKP, GEDKP, and NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
(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 c1) embodiment a)-d) in each phytase, it comprises at least one following variation:
(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) K179 wherein, T180, T181, E182, K183, S184, T185 and K186 be by QADKP, GEDKP, and NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace;
(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). isolated nucleic acid sequences, it comprises the coding embodiment and a)-t) comprises a1)-a5) and c1) in each the nucleotide sequence of phytase.
V). nucleic acid construct, it comprises embodiment u) nucleotide sequence, this nucleotide sequence is operably connected to guidance produces phytase in suitable expressive host one or more regulating and controlling sequences.
W). recombinant expression vector, it comprises embodiment nucleic acid construct v).
v).
X). recombinant host cell, it comprises embodiment nucleic acid construct and/or embodiment w v)) expression vector.
Y). be used for producing embodiment and a)-t) comprise a1)-a5) and c1) each the method for phytase, it comprises
(a) host cell cultivation embodiment x) comprises the supernatant liquor of phytase with generation; (b) reclaim phytase.
Z). transgenic plant, or plant part, it can be expressed embodiment and a)-t) comprise a1)-a5) and c1) in each phytase.
Ae). genetically modified, non-human animal, or its product, or component, it can be expressed embodiment and a)-t) comprise a1)-a5) and c1) in each phytase.
Oe). composition, it comprises embodiment and a)-t) comprises a1)-a5) and c1) in each at least a phytase and
(a) at least a liposoluble vitamin;
(b) at least a water-soluble vitamins; And/or
(c) at least a trace minerals.
Aa). embodiment oe) composition, its further comprise be selected from down the group enzyme at least a enzyme: amylase, phytase, Phosphoric acid esterase, zytase, Galactanase, alpha-galactosidase, proteolytic enzyme, Phospholipid hydrolase, and/or beta-glucanase.
Bb). embodiment oe)-aa) in each composition, it is an animal feedstuff additive.
Cc). animal feedstuff compositions, its have 50 to 800g/kg crude protein content and comprise embodiment a)-t) comprise a1)-a5) and c1) in each phytase or embodiment oe)-aa) in each composition.
Dd). be used to improve the method for animal-feed nutritive value, wherein embodiment a)-t) comprised a1)-a5) and c1) in each phytase or embodiment oe)-aa) in each composition be added in the feed.
Ee). be used to reduce the method for animal excrement mysoinositol six phosphoric acid salt levels, it comprises the embodiment cc with significant quantity) the forage feed animal.
Ff). be used to handle the method for vegetable-protein, it comprise embodiment a)-t) is comprised a1)-a5) and c1) in each phytase or embodiment oe)-aa) in each composition add step at least a vegetable-protein or the protein source to.
Gg). embodiment a)-t) comprises a1)-a5) and c1) in each phytase or embodiment oe)-aa) in each the purposes of composition in animal-feed; Purposes in the preparation animal-feed; Be used to improve the purposes of the nutritive value of animal-feed; Be used for reducing the purposes of the phytinic acid salt level of animal excrement; Be used to handle the purposes of vegetable-protein; Or be used for from the purposes of phytinic acid enzyme substrates release phosphorus.
This paper describes and the scope of claimed invention is not restricted to embodiment disclosed herein, because these embodiments are intended to the explanation as several aspects of the present invention.Any embodiment that is equal to should be within the scope of the invention.Really, show except that this paper and describe will be conspicuous to those skilled in the art to various modifications of the present invention.These modifications also should be within the scope of the appended claims.Having under the situation of conflict, be as the criterion to comprise the disclosure in being defined in.
The various reference papers that this paper quotes are incorporated its disclosure into this paper by reference.
Embodiment
Used chemical agent is the SILVER REAGENT commodity at least.
Embodiment 1: the detection of the preparation of variant and temperature stability and pH curve
The preparation of inositol six-phosphatase variants
The DNA of inositol six-phosphatase variants that coding has the aminoacid sequence of SEQ ID NO:2 produces by methods known in the art, and construct is blended in by Takami etc. at the DNA of the coded signal peptide described in the Biosci.Biotechnol.Biochem.56:1455 (1992) and be integrated into by homologous recombination in the genome of Bacillus subtillis host cell (referring to (1990) such as Diderichsen by PCR with standard technique, J.Bacteriol., 172,4315-4321).The phytinic acid zymoprotein that gene is expressed under the control of three promoter systems (as described in WO 99/43835) and obtained with the ordinary method purifying.
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) have about 10 every milliliter in microgram albumen and be dissolved in the 0.1M sodium acetate buffer, among the pH5.5, with this protein solution separated into two parts, a part is in (for example 50 ℃ of the temperature of the rising of expectation, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃ or 85 ℃) incubation in plastic containers, another part is stored in 5 ℃.Incubation was transferred to ice bath with protein solution after 30 minutes at elevated temperatures, and measured the activity of the sample of (deduction damping fluid blank (buffer blind subtracted)) refrigerative sample and heating by following phosphatase assay.Residual activity is defined as activity after the thermal treatment divided by the activity (with the % form) of refrigerative sample.If the residual activity in Phosphoric acid esterase or phytinic acid enzyme assay, thinks then that variant is to temperature stable more (thermally-stabilised) with higher with reference to comparing.
The mensuration of phosphatase activity
The enzyme solution that 75 microlitres are contained phytase is assigned to micro titer plate well (microtiter platewell) for example among the NUNC 269620, and add 75 microlitre substrates (for the preparation substrate, with two 5mg p-nitrophenyl phosphoric acid tablet (Sigma, catalog number (Cat.No.) N-9389) is dissolved in 10ml 0.1M sodium acetate buffer, pH5.5).Flat board was sealed then incubation 15 minutes, at 37 ℃ with the 750rpm wave and culture.After incubation period, add the absorbancy that 75 microlitres stop reagent (termination reagent is the 0.1M disodium tetraborate in the water) and be determined at 405nm on the microtiter plate spectrophotometer.A phosphatase unit is defined as the enzymic activity (deduction damping fluid blank) that under given reaction conditions per minute discharges 1 micromole's phosphate radical.The absorbancy of measuring 1 micromole's p-nitrophenol under test condition is 56AU (an AU=absorbance units).
Dsc measurement
Differential scan calorimetry (DSC) can be carried out by the VP-DSC with Micro Cal under various pH-values.Scanning is carried out under 1.5 ℃/min of constant scan rate, scope 20-90 ℃.Before beginning DSC, phytase is used in suitable damping fluid (0.1M glycine-HCl for example, pH2.5 or 3.0; 20mM sodium acetate pH4.0; 0.1M sodium acetate, pH5.5; 0.1M Tris-HCl, pH7.0) middle equilibrated NAP-5 post (Pharmacia) desalination.Data processing is to use MicroCal Origin software (MicroCal Originsoftware) (edition 4 .10) to carry out, and denaturation temperature Td (is also referred to as melting temperature(Tm), Tm) is defined as the tip temperature at peak in the thermolysis curve (thermogram).
The pH curve of revising: the mensuration of pH3.5/5.5 activity ratio
The correction of the pH curve of inositol six-phosphatase variants can followingly be determined: (the 0.1M acetate buffer, (the 0.1M acetate buffer pH5.5) is measured activity, all deducts the damping fluid blank under two kinds of situations during pH3.5) with pH5.5 when pH3.5.The activity that the activity of measuring when being used in pH3.5 is measured during divided by pH5.5 is about to two absorption values be divided by (face as follows).For measuring activity, with the suitably dilution (for example 1:5000) in damping fluid separately of the supernatant liquor of variant and reference.75 microlitres enzyme solution separately is assigned to micro titer plate well for example among the NUNC269620, and add the substrate that 75 microlitres have corresponding pH (by respectively at the 10ml0.1M sodium acetate buffer, pH5.5 and 10ml0.1M acetate buffer, two 5mg p-nitrophenyl phosphoric acid tablets of dissolving (Sigma, catalog number (Cat.No.) N-9389) prepare substrate among the pH3.5).With flat board sealing and incubation 15 minutes, at 37 ℃ with the 750rpm wave and culture.After incubation period, add 75 microlitres and stop reagent (the 0.1M disodium tetraborate aqueous solution in the water), and in the microtiter plate spectrophotometer, measure absorbancy at 405nm.
The mensuration of phytase activity
To suitably dilute (for example at the 0.25M sodium acetate, among 0.005% (w/v) Tween-20.pH5.5) the 75 microlitres enzyme solution that contains phytase be assigned to and littlely come that titration plate hole for example among the NUNC 269620, and add 75 microlitre substrates (by at 10ml 0.25M sodium acetate buffer, dissolving 100mg prepares from the sodium phytate (Aldrich catalog number (Cat.No.) 274321) of rice among the pH5.5).With flat board sealing and incubation 15 minutes, at 37 ℃ with the 750rpm wave and culture.Add 75 microlitres termination reagent after the incubation period and (stop reagent by mixing 10ml molybdate solution (10% (w/v), seven-ammonium molybdate is in 0.25% (w/v) ammonia solution); 10ml ammonium vanadate (0.24%Bie﹠amp; The commerical prod of Berntsen, catalog number (Cat.No.) LAB17650) and 21.7% (w/v) nitric acid prepare), on the microtiter plate spectrophotometer, measure the absorbancy of 405nm.Phytase activity represents with FYT unit, and FYT is that per minute discharges the enzyme amount of the inorganic ortho-phosphoric acid root of 1 micromole under these conditions.(such standard enzyme prepared product with known activity can be from Novozymes A/S from the typical curve of the suitable diluent preparation of inorganic phosphate or with reference to the phytase prepared product from known activity by reference, Krogshoejvej 36, and DK-2880 Bagsvaerd asks for acquisition) the absolute value of the typical curve made of the diluent phytase activity that obtains to measure.
Embodiment 2: expressive host/glycosylation is to the influence of thermostability
Expression in the bacillus
The phytase of SEQ ID NO:2 is expressed in subtilis as described in embodiment 1, and use the ordinary method purifying: centrifugal, microbe filter (germ filtration), ammonium sulfate precipitation (80% ammonium sulfate saturated solution), centrifugal, at buffer A (50mM sodium acetate, 1.5M resuspended precipitation ammonium sulfate pH4.5), filter, hydrophobic interaction chromatography (hydrophobic interaction chromatography) (Phenyl Toyopearl, with sample on the buffer A, with buffer B (50mM sodium acetate pH4.5) wash-out) and cation-exchange chromatography (SP-agarose, go up sample with 10mM Trisodium Citrate pH4.0, 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.1,2000, general description among the pp.105-112.From the supernatant of fermentation culture as following purifying phytase: with ammonium sulfate (80% saturated solution) precipitation, dissolving again in 10ml 25mM sodium acetate buffer pH4.5 with respect to the same buffer dialysis, and is filtered by the 0.45mm filter.This solution of 150ml is added to same buffer pH4.5 equilibrated 40ml SP Sepharose FF post (Pharmacia), with albumen LINEAR N aCl gradient (0-0.5M) wash-out.Analysis is from the phytase activity of the fraction of post.Have the fraction of phytase activity by the SDS-PAGE detection, and compile (pool) pure fraction.Measure protein concentration by using BCA test kit (Pierce).
Thermostability by DSC mensuration
The phytase of the SEQ ID NO:2 that Pichia and bacillus are expressed carries out the measurement by the thermostability of differential scan calorimetry (DSC).
Specimen preparation:
Sample (volume is less than 3ml) (about 5 degrees centigrade) in the cold house was dialysed minimum 1 hour with respect to the 20mM sodium acetate buffer pH4.0 of 500ml.With sample transfer to fresh, cold buffer formulation of 500ml and dialysed overnight.Sample is filtered with 0.45 micron syringe filter, with dialysis buffer liquid with volume-adjustment to about 1.5ml, and write down A 280(absorbancy) at the 280nm place.In DSC scanning, use dialysis buffer liquid as a reference.With vacuum take-off and stir about 10 minutes was the sample exhaust.
During the specimen preparation of the phytase that Pichia is expressed, (dialyse) and formed precipitation with respect to 20mM sodium acetate (NaAc) pH4.0.Supernatant liquor is used for first experiment.Then with the remainder of the liquid storage of purifying with respect to 20mM NaAc pH4.0 dialysis, make like this under some low Mw contamination precipitations that in this batch, exist.This batch is used for second experiment, and this experiment has disclosed and the closely similar Tm (54 couples 55 ℃) of first experiment.
The DSC experiment:
Use MicroCal TMThe experiment setting of VP-DSC equipment: scanning speed: 90K/h.Scanning interval: 20-90 degree centigrade.Feedback model: do not have.The filtration phase (filtering period): 16 seconds.
The enzyme concn of sample is about 1-1.5mg/ml, as passes through A 280With estimate at the Theoretical Calculation optical extinction coefficient (Vector NTI version 9.0.0) of 280nm.Temperature (Td is folded in pyrolysis; Thermal unfoldingtemperature) assesses with MicroCal Origin software (edition 4 .10), and denaturation temperature is defined as the summit temperature of thermolysis curve.
Sum up in result's table 2 below.
Table 2
Table 2 clearly illustrates that the phytase of Pichia expression is more very different than the thermostability of the phytase of bacillus expression.
The phytase that Pichia is expressed has the severe glycosylation, and being seen by wide in range molecular weight ranges as using mass spectroscopy (Maldi-TOF), the phytase that bacillus is expressed does not then have glycosylation.
Embodiment 3: inositol six-phosphatase variants R339D
Preparation has the phytase protein engineering variant of the SEQ ID NO:2 that replaces R339D, and expresses with methods known in the art in aspergillus oryzae.Use as embodiment 2 described its denaturation temperature Td of DSC mensuration be 62.5 ℃ (the 20mM sodium acetate, pH4.0).
In addition, the R339D replacement is used for removing and expresses potential relevant Kex2 proteolytic enzyme cutting 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 inositol six-phosphatase variants R339D that will comprise 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 Wei ShengsuB2s
1500 mg vitamin B6s
7666 mcg vitamin B12
12333 mg nicotinic acid
33333 mcg vitamin Hs
300 mg folic acid
3000 mg D-calcium pantothenate
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 the 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
The 0.2%DL-methionine(Met)
0.22%DCP (Lin Suanergai)
0.76%CaCO 3(lime carbonate)
0.32% sand
0.15%NaCl (sodium-chlor)
1% above-mentioned pre-composition
Each composition is mixed, and feed is made particle in desired temperatures (for example 60,65,75,80,85,90 or even 95 ℃).
Embodiment 5: the mensuration of temperature stability
Eight variants (variation of comparing with SEQ ID NO:2 shows in the following Table 3) that prepare SEQ ID NO:2 as described in example 1 above.Two with SEQ ID NO:2 and SEQ ID NO:3 prepare in the same manner with reference to phytase.
The following mensuration of temperature stability:
With variant and reference protein 200 microlitre supernatant liquor separated into two parts separately, a part is at 50 ℃ of incubations in plastic containers, and another part is kept at 5 ℃.After 30 minutes protein solution is transferred to ice bath at 50 ℃ of incubations.At the 0.1M sodium acetate buffer, dilution after, by phosphatase assay in embodiment 1 (" mensuration of phosphatase activity ") measure the activity of the sample of refrigerative and heating among the pH5.5 with 1:100, deduction damping fluid blank.The result is shown as in the following Table 3 respectively 5 ℃ and the enzymic activity (with absorbance units (AU) expression) of 50 ℃ of incubations after 30 minutes, and residual activity (RA) is calculated as with the activity of heat treated sample (the 50 ℃ of incubations) activity divided by cooling sample (5 ℃ of incubations), represents with %.
Table 3: inositol six-phosphatase variants with improved thermostability
Figure A200780020771D00731
Embodiment 6: the performance in animal-feed in external model
The performance of inositol six-phosphatase variants in animal-feed compared described reference protein such as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:6 with the performance of reference protein in external model.The gastrointestinal conditions of external model simulation monogastric animal and with body in result that animal experiment obtained fully related.Relatively be performed as follows:
Measure described in phytase activity in the variant sample such as the embodiment 1 " mensuration of phytase activity ".
Prepare the feed sample of forming by 30% soyflour and 70% Semen Maydis powder then, add CaCl 2To concentration be every kilogram of feed 5g calcium, and 40 ℃ and pH3.0 preincubation 30 minutes, next add the phytase (using same dosage so that comparison) of stomach en-(3000U/g feed) and suitable dosage, for example 0.25 to 0.75 phytase unit FYT/g feed for all phytases that will detect.The blank that also comprises no phytase activity as a reference.Thereafter with sample 40 ℃ and pH3.0 incubation 60 minutes, then pH4.0 incubation 30 minutes.
By adding HCl, then carried out a freeze-thaw cycle and 40 ℃ of incubations 1 hour, thus with reaction terminating and extract phytinic acid and phosphoinositide to final concentration 0.5M and 40 ℃ of incubations 2 hours.
By high-effect ionic chromatography such as Chen etc. at Journal of Chromatography A (2003) vol.1018, described in the pp.41-52 phytinic acid is separated with phosphoinositide, and as Skoglund etc. in J.Agric.Food Chem. (1997), vol.45 carries out described in the pp.431-436 quantitatively.
Then the phosphorus that discharges is calculated as phytase treatment and untreated sample between the difference of phosphoinositide bonded phosphorus (IP-P).The relative performance of variant is calculated as the per-cent of the phosphorus that discharges with respect to the contrast phytase.
As mentioned above with the external performance of a plurality of 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 shows respectively to show among 4A and the 4B below.Remaining IP6-P refers to the amount of remaining IP6-P after external incubation (phytinic acid phosphorus), represents with mg/gDM (dry-matter).The IP6-P of degraded is determined as the difference between the remaining IP6-P of the remaining IP6-P of barren and each sample.At last, in the end one be listed as, represent the IP6-P that degrades with respect to phytase with SEQ ID NO:2.In table 4A empty with reference to (SEQ ID NO:2) value is a plurality of independent averages of measuring, and other values are based on unitary determination.In table 4B empty value is a plurality of independent averages of measuring, and other values are based on unitary determination.
The external performance of table 4A. inositol six-phosphatase variants supernatant liquor
Figure A200780020771D00741
Figure A200780020771D00751
Variant 015,016,018,040,041,042,044 and 050 looks to have that to compare with 3 phytase with SEQ IDNO:2 be the same good or better external performance at least.
Table 4B: the external performance of the inositol six-phosphatase variants of purifying
Figure A200780020771D00752
Figure A200780020771D00761
Variant 030,031,037,056,072,085,087,089,090,095,098 seems that in external performance the phytase with SEQ ID NO:3 is the same good at least with 125.
Embodiment 7: specific activity
Based on respect to the 250mM sodium acetate, the highly purified sample of pH5.5 dialysis is measured the specific activity of inositol six-phosphatase variants.On sds page, detect purity in advance, show only to have a component.
Protein concentration is measured by following amino acid analysis: with the aliquots containig of sample in the Glass tubing of finding time at 6N HCl, in 0.1% phenol 110 ℃ of hydrolysis 16 hours.Come 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, can calculate proteic total mass and concentration in the aliquots containig of hydrolysis.
Described in embodiment 1 (" mensuration of phytase activity "), measure phytase activity with FYT unit, and specific activity is calculated as phytase activity, described phytase activity is measured with FYT unit's every mg inositol six-phosphatase variants zymoprotein.
The specific activity such as the above-mentioned mensuration of the phytase of SEQ ID NO:2 and variant 072 (I362R of SEQ ID NO:2).The specific activity of variant 072 be SEQ ID NO:2 the phytinic acid specific enzyme activity 86%.Uncertain (standard deviation) estimated to be about 10%, mainly is owing to the substrate of phytase activity assay method based on complexity.
Embodiment 8: temperature stability
The a plurality of variants that prepare SEQ ID NO:2 as described in example 1 above, and the subtilis host strain shaken 100ml PS1 substratum (100g/L sucrose, 40g/L soybean flakes, 10g/L Na in the bottle at 500ml 2HPO 4.12H 2O, 0.1ml/L Dowfax 63N10 (Dow)) cultivated four days with 300rpm at 30 ℃ in.
Two prepare in the same manner with reference to phytase, promptly have the phytase (corresponding to the variant N31D/Q139K/L197F/N316K of SEQ ID NO:2) of SEQ ID NO:3, and the phytase (corresponding to the variant N31D/N121T/K132T/Q139K of SEQ ID NO:2) with SEQ ID NO:4.
Comprise that also the phytase with SEQ ID NO:9 compares (corresponding to the variant Q3P/N31D/N121T/K132T/Q139K of SEQ IDNO:2).
Variant and following definite with reference to the temperature stability of phytase:
By adding 20ul (microlitre) supernatant liquor to 180ul 0.1M sodium acetate buffer, pH5.5+0.005% Tween-20 is with ten times of supernatant liquor dilutions.With the enzyme separated into two parts of dilution, a part is at 60 ℃ of incubations in plastic containers, and another part is kept at 5 ℃.Protein solution is transferred to ice bath at 60 ℃ of incubations after 30 minutes.At the 0.1M sodium acetate buffer, with after the 1:10 dilution, measure the activity of the sample of refrigerative and heating, deduction damping fluid blank among pH5.5 and the 0.005%Tween-20 by the phosphatase assay of (determining of phosphatase activity) among the embodiment 1.
Table 5 is to compare the list of variants with improved temperature stability with the reference phytase.For each variant, this table has also been described the variation of comparing with SEQ ID NO:2.Measured respectively 5 ℃ and the enzymic activity (with absorbance units (AU) expression) of 60 ℃ of incubations after 30 minutes, and residual activity (RA) is calculated as with the activity of heat treated sample (60 ℃ of incubations) to come divided by the activity of cooling sample (5 ℃ of incubations).Next, with the result of residual activity residual activity stdn with respect to the phytase of the SEQ ID NO:2 that expresses in the same manner and handle.The improvement factor (IF) that draws is displayed in Table 5.Phytase for SEQ ID NO:2, IF is 1.0, SEQ ID NO:3 with the phytase of SEQ ID NO:2 compares with reference to phytase with 4 two that then thermostability is relatively poor, and this IF from these two phytases only is respectively to be conspicuous 0.1 and 0.3 the fact.
Table 5: inositol six-phosphatase variants with improved thermostability
Figure A200780020771D00771
Figure A200780020771D00781
Figure A200780020771D00791
Embodiment 9: according to the thermostability of DSC
A plurality of purifying variants as preparation SEQ ID NO:2 as described in summarizing among the embodiment 1.Prepare two in the same manner with reference to phytase, promptly have the phytase (corresponding to the variant N31D/Q139K/L197F/N316K of SEQ ID NO:2) of SEQ ID NO:3 and have the phytase (corresponding to the variant N31D/N121T/K132T/Q139K of SEQ ID NO:2) of SEQ ID NO:4.Comprise that also the phytase with SEQ ID NO:9 is used for comparison (corresponding to the variant Q3P/N31D/N121T/K132T/Q139K of SEQ ID NO:2).
The aliquots containig of protein sample is dialysed in 2-3 hour step at 4 ℃ with respect to 2 x 500ml 20mM sodium acetate pH4.0, then dialysed overnight.Each sample is diluted to about 2 A through the 0.45um filtration and with damping fluid 280Unit.The definite absorption value that records is providing in the table as a result.On MicroCalVP-DSC, carry out DSC,, among the pH4.0, in 20-90 ℃ of scope, carry out with 90 ℃/h scanning speed at the 20mM sodium acetate buffer.
The denaturation temperature that obtains (Td) shows in the following Table 6, has summed up the result of three different experiments.
Table 6: the Td by DSC measures
Figure A200780020771D00801
Embodiment 10: purifying and temperature curve
Inositol six-phosphatase variants used herein and reference and the comparative following purifying that carries out of phytase: the fermented supernatant fluid that contains phytase is earlier centrifugal one hour with 7200rpm and 5 ℃, then through four layers of Whatman glass microfiber filter paper (2.7,1.6,1.2 and 0.7 microns) multi-layered devices (sandwich) filter.Then solution is carried out sterile filtration (, perhaps utilizing pressure) through Seitz-EKS degree of depth filter (Seitz-EKS depth filter) perhaps through having the quick PES bottle top filter (Fast PES Bottle top filter) of 0.22 μ m cutoff value (cut-off).In solution, add solid-state ammonium sulfate, obtain final concentration 1.5M, use 6M HCl pH regulator to 6.0.
The solution that will contain phytase is added on butyl-agarose column, about 50ml in the 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 (referring to embodiment 1, " mensuration of phosphatase activity ") to analyze activity, and compile and have active fraction from the fraction of pillar.The fraction that to compile is dialysed with respect to 10mM sodium acetate pH4.5 abundant (extensively).Then, the solution that will contain phytase on the S agarose by chromatography purification, about 75ml in the XK26 post, usefulness 50mM sodium acetate pH4.5 as buffer A and 50mM sodium acetate+1MNaCl pH4.5 as buffer B.Equally, analyze from the active of the fraction of pillar and compile activated fraction.The solution concentration that will contain at last, the phytase of purifying with the Amicon ultra-15 filtration unit of the film that has the 10kDa cutoff value.
In all cases, the molecular weight of all phytases (as being estimated by SDS-PAGE) is about 40kDa, and purity is greater than 95%.
The temperature curve of variant (phytase activity is as the function of temperature) is determined 20-90 ℃ temperature range as (" mensuration of phytase activity ") as described in the embodiment 1 basically, yet enzyme reaction (100 microlitres contain the enzyme solution+100 microlitre substrates of phytase) is not on the microtiter plate and carry out in the PCR pipe.After 15 minute reaction period of desired temperature finishes, pipe is cooled to 20 ℃ continues 20 seconds, 150 microlitre reaction mixtures are transferred on the microtiter plate.Add that 75 microlitres stop reagent and in the microtiter plate spectrophotometer, measure absorbancy at 405nm.The result sums up in the following Table 7.The numeral that provides for each temperature (20-90 ℃ is one-level with 10 ℃) is with respect to the standardized relative reactivity of optimum value (representing with %).
Table 7: temperature curve
Figure A200780020771D00811
Figure A200780020771D00821
Variant 030,031,037,044,056,062,072,083 and 093 compares with 102 with reference phytase 026 70 ℃ the time and has higher relative reactivity.
Embodiment 11:pH curve
The pH curve (phytase activity is as the function of pH) of a plurality of variants used among the embodiment in front and same reference and comparative phytase is determined as (" mensuration of phytase activity ") as described in the embodiment 1 in 2.0 to 7.5 pH scope (is one-level with 0.5 pH unit) at 37 ℃, only be to use damping fluid mixed solution (cocktail) (50mM glycine, 50mM acetate and 50mMBis-Tris) to replace 0.25M sodium acetate pH5.5 damping fluid.The result sums up in the following Table 8.The numeral that provides for each pH (2.0-7.5) is with respect to the standardized relative reactivity of optimum value (representing with %).
Table 8:pH curve
Figure A200780020771D00831
To YC062 and YC091, pH curve (relative reactivity is as the function of pH) seems to be offset 0.5pH unit to higher pH.
And then, when optimum value is at pH3.5-pH4.0 for the majority of the phytase (comprising with reference to phytase 026 and 102) of table 8, the optimal pH of observing for numbering 062,085 and 089 is 3.5, and the optimal pH of observing for numbering 091 and 093 is 4.0.
Embodiment 12: temperature stability
By 70 ℃ with pH4.0 (0.1M sodium acetate) incubation after measure remaining phytase the activity variant of measuring a plurality of purifying as in the previous examples and identical reference and the temperature stability of comparative phytase.With the phytase incubation, sample is 0,10, withdraws from after 30 and 60 minutes and in cooled on ice.The method of (" mensuration of phytase activity ") is measured the residual activity of pH5.5 described in the use embodiment 1.The result with respect to the active stdn 0 minute the time, is shown in the following Table 9.
Table 9: temperature stability
Figure A200780020771D00841
Top result shows numbering 044,062,072 and 083, and (70 ℃ and pH4) may be more stable than reference phytase under these conditions (although observing big deviation for numbering 000 in this experiment).
Embodiment 13: calculate identity per-cent and identify the corresponding position
Needle program with EMBOSS software package 2.8.0 version is compared SEQ ID NO:9 and SEQ IDNO:2.Used substitution matrix is BLOSUM62, and it is 10.0 that breach is opened point penalty, and it is 0.5 that breach extends point penalty.
The comparison result that obtains is represented in Fig. 2.
The following calculating of identity degree between SEQ ID NO:9 and the SEQ ID NO:2: accurately the number of coupling is 406 (all these are with vertically drawing (vertical stroke) expression).The length of short sequence is 411 (SEQ ID NO:2).The per-cent of identity is 406/411 x 100%=98.8%.
The corresponding position that also comparison result of Fig. 2 is used to derive, as follows: the amino acid at the top is in corresponding position mutually in this comparison.For example the amino acid Q on SEQ ID NO:2 position 3 is corresponding 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 that comprises the SEQ ID NO:2 that replaces Q3P.
Other difference that exists with the replacement form in the lap of comparison result is in position 31,121,132 and 139, i.e. N31D, N121T, K132T and Q139K.
Difference in addition is present in the N-end, compares SEQ ID NO:9 at the N-end with SEQ ID NO:2 and has 22 amino acid whose extensions.
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/ *0jF/ *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
<130>10945.204-WO
<160>9
<170>PatentIn version 3.4
<210>1
<211>1233
<212>DNA
＜213〉Bu Shi citric acid bacillus (Citrobacter braakii) ATCC 51113
<220>
＜221〉mature peptide
<222>(1)..(1233)
<220>
<221>CDS
<222>(1)..(1233)
<400>1
Figure A200780020771D00861
Figure A200780020771D00871
<210>2
<211>411
<212>PRT
＜213〉Bu Shi citric acid bacillus ATCC 51113
<400>2
Figure A200780020771D00872
<210>3
<211>411
<212>PRT
＜213〉Bu Shi citric acid bacillus YH-15
<220>
＜221〉mature peptide
<222>(1)..(411)
<400>3
Figure A200780020771D00882
Figure A200780020771D00891
<210>4
<211>411
<212>PRT
＜213〉citrobacter freundii (Citrobacter freundii)
<220>
＜221〉mature peptide
<222>(1)..(411)
<400>4
Figure A200780020771D00901
<210>5
<211>1236
<212>DNA
＜213〉variant of SEQ ID NO:2
<220>
<221>CDS
<222>(1)..(1233)
<220>
＜221〉mature peptide
<222>(1)..(1233)
<400>5
Figure A200780020771D00912
Figure A200780020771D00921
<210>6
<211>411
<212>PRT
＜213〉variant of 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
Figure A200780020771D00922
<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
<210>8
<211>22
<212>PRT
＜213〉Bu Shi citric acid bacillus ATCC 51113
<400>8
Figure A200780020771D00942
<210>9
<211>433
<212>PRT
＜213〉citrobacter freundii NCIMB 41247
<220>
＜221〉mature peptide
<222>(1)..(433)
<400>9
Figure A200780020771D00943
Figure A200780020771D00951

Claims (29)

1. phytase, it has at least 74% identity with SEQ ID NO:2 and at least one position of comparing with SEQ IDNO:2 in being selected from group down comprises at least one variation: 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 phytase is not SEQ ID NO:3, is not SEQ ID NO:4, and is not SEQ ID NO:6.
2. the phytase of claim 1 is not the variant of listing among SEQ ID NO:9 and its Fig. 1.
3. any one phytase in the claim 1 and 2, it comprises at least one following variation: 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.
4. the phytase of claim 3, wherein the position 179,180, and 181,182,183,184,185 and 186 amino acid is by QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ, KEKKV or KTDKL replace.
5. phytase, itself and SEQ ID NO:2 have at least 74% identity and comprise at least one following variation: 1H, K, R, 60P, 105E, 106A, G, 155F, 157F, 173P, 175L, 188P, 205P, 215M, 231P, 254Y, 280P, 330D and/or 371P; Condition is that this phytase is not SEQ ID NO:3, is not SEQ ID NO:4, is not SEQ ID NO:6, and is not SEQ IDNO:9 and the variant listed in Fig. 1 thereof.
6. each phytase among the claim 1-4, it comprises and is selected from following variation: 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。
7. each phytase among the claim 1-6, it is the variant of the phytase of SEQ ID NO:2.
8. each phytase among the claim 1-6, it is the variant of the phytase of SEQ ID NO:3.
9. each phytase among the claim 1-6, it is the variant of the phytase of SEQ ID NO:4.
10. each phytase among the claim 1-6, it is the variant of the phytase of SEQ ID NO:6.
11. each phytase among the claim 1-6, it is the variant of the phytase of SEQ ID NO:9.
12. each phytase among the claim 1-6, its for any one variant of SEQ ID NO:9 inositol six-phosphatase variants relevant and that in Fig. 1, list.
13. each phytase among the claim 1-12, it also comprises the replacement or the replacement that are selected from the replacement of listing and replace in the combination in every row of Fig. 1 and makes up.
14. each phytase among the claim 1-13, it has improved thermostability, improved pH curve, improved specific activity, the glycosylation pattern of revising, improved temperature curve, improved performance and/or its mix the variation in potential proteolytic enzyme cutting site in animal-feed.
15. isolated nucleic acid sequences, it comprises the nucleotide sequence of each phytase among the coding claim 1-14.
16. nucleic acid construct, it comprises the nucleotide sequence of claim 15, and described nucleotide sequence is operably connected to one or more regulating and controlling sequences that instruct phytase to produce in suitable expressive host.
17. comprise the recombinant expression vector of the nucleic acid construct of claim 16.
18. comprise the recombinant host cell of the expression vector of the nucleic acid construct of claim 16 and/or claim 17.
19. produce the method for each phytase among the claim 1-14, it comprises
(a) host cell of cultivation claim 18 comprises the supernatant liquor of described phytase with generation; With
(b) reclaim described phytase.
20. transgenic plant or plant part, it can express among the claim 1-14 each phytase.
21. can express the transgenic nonhuman animal of each phytase among the claim 1-14, or its integral part or product.
22. composition, its comprise among the claim 1-14 each at least a phytase and
(a) at least a liposoluble vitamin;
(b) at least a water-soluble vitamins; And/or
(c) at least a trace minerals.
23. the composition of claim 22, it further comprises at least a enzyme that is selected from down group: amylase, phytase, Phosphoric acid esterase, zytase, Galactanase, alpha-galactosidase, proteolytic enzyme, Phospholipid hydrolase and/or beta-glucanase.
24. each composition among the claim 22-23, it is an animal feedstuff additive.
25. animal feedstuff compositions, its have 50 to 800g/kg gross protein value and comprise among the claim 1-14 each phytase or claim 22-24 in each composition.
26. be used to improve the method for the nutritive value of animal-feed, wherein with among the claim 1-14 each phytase or claim 22-24 in each composition be added into feed.
27. reduce the method for animal excrement mysoinositol six phosphoric acid salt levels, it comprises the forage feed animal with the claim 25 of significant quantity.
28. the method for treatment of vegetable protein, it comprise with among the claim 1-14 each phytase or claim 22-24 in each the composition step of adding at least a plant protein or protein source to.
29. among the claim 1-14 among each phytase or the claim 22-24 each composition be used for following purposes: at animal-feed; In the preparation of animal-feed; Be used to improve the nutritive value of animal-feed; Be used for reducing the phytinic acid salt level of animal excrement; Be used for treatment of vegetable protein; Or be used for containing the phosphorous material from the release of phytinic acid enzyme substrates.
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CN102854158A (en) * 2011-07-01 2013-01-02 北京昕大洋科技发展有限公司 Method for rapidly determining heat resistance of phytase
CN102906255A (en) * 2010-03-26 2013-01-30 诺维信公司 Thermostable phytase variants
CN105358706A (en) * 2013-03-08 2016-02-24 生物语法公司 Yeast promoters for protein expression
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EP1546316B1 (en) * 2002-08-12 2010-12-01 Danisco US Inc. Mutant e. coli appa phytase enzymes and natural variants thereof, nucleic acids encoding such phytase enzymes, vectors and host cells incorporating same and methods of making and using same
WO2004085638A1 (en) * 2003-03-25 2004-10-07 Republic Of National Fisheries Research And Development Institute Phytase produced from citrobacter braakii
WO2006038128A2 (en) * 2004-10-04 2006-04-13 Danisco A/S Citrobacter freundii phytase and homologues
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CN102906255A (en) * 2010-03-26 2013-01-30 诺维信公司 Thermostable phytase variants
CN102854158A (en) * 2011-07-01 2013-01-02 北京昕大洋科技发展有限公司 Method for rapidly determining heat resistance of phytase
CN102854158B (en) * 2011-07-01 2016-01-20 北京昕大洋科技发展有限公司 A kind of method of Fast Measurement phytase heat resistance
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US10619164B2 (en) 2013-03-08 2020-04-14 Keck Graduate Institute Of Applied Life Sciences Yeast promoters from Pichia pastoris
US10676750B2 (en) 2013-03-08 2020-06-09 Biogrammatics, Inc. Yeast promoters for protein expression

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