PHYTASE MUTANT

Abstract

Disclosed is a thermostable phytase, in which at least one pair of introduced disulfide bonds is included in the amino acid sequence of wild-type Escherichia coli phytase or mutant Escherichia coli phytase, and after the introduction, the properties of the phytase can be improved, especially the heat stability, steam stability and granulation stability, which are superior to those of the existing wild-type or mutant phytase; compared to the engineered phytase in which disulfide bonds are introduced, the heat stability is also significantly improved.

Claims

1. A thermostable phytase, wherein at least one pair of introduced disulfide bonds is comprised in the amino acid sequence of wild-type Escherichia coli phytase or mutant Escherichia coli phytase, the amino acid sequence of the wild-type Escherichia coli phytase is as shown by SEQ ID NO: 1, and compared to the wild-type Escherichia coli phytase as shown by SEQ ID NO: 1, the mutant Escherichia coli phytase has a mutation on at least 1 position; and the introduced disulfide bonds are selected from: (A) a disulfide bond formed between an amino acid residue on a position corresponding to a 34.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 174.sup.th position of SEQ ID NO: 1; (B) a disulfide bond formed between an amino acid residue on a position corresponding to a 56.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 103.sup.rd position of SEQ ID NO: 1; (C) a disulfide bond formed between an amino acid residue on a position corresponding to a 57.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 366.sup.th position of SEQ ID NO: 1; (D) a disulfide bond formed between an amino acid residue on a position corresponding to a 61.sup.st position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 366.sup.th position of SEQ ID NO: 1; (E) a disulfide bond formed between an amino acid residue on a position corresponding to an 82.sup.nd position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 296.sup.th position of SEQ ID NO: 1; (F) a disulfide bond formed between an amino acid residue on a position corresponding to a 128.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 203.sup.rd position of SEQ ID NO: 1; (G) a disulfide bond formed between an amino acid residue on a position corresponding to a 140.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 262.sup.nd position of SEQ ID NO: 1; (H) a disulfide bond formed between an amino acid residue on a position corresponding to a 156.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 191.sup.st position of SEQ ID NO: 1; (I) a disulfide bond formed between an amino acid residue on a position corresponding to a 165.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 245.sup.th position of SEQ ID NO: 1; (J) a disulfide bond formed between an amino acid residue on a position corresponding to a 191.sup.st position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 210.sup.th position of SEQ ID NO: 1; (K) a disulfide bond formed between an amino acid residue on a position corresponding to a 196.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 211.sup.th position of SEQ ID NO: 1; (L) a disulfide bond formed between an amino acid residue on a position corresponding to a 264.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 312.sup.th position of SEQ ID NO: 1; (M) a disulfide bond formed between an amino acid residue on a position corresponding to a 315.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 380.sup.th position of SEQ ID NO: 1; (N) a disulfide bond formed between an amino acid residue on a position corresponding to a 322.sup.nd position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 356.sup.th position of SEQ ID NO: 1; (O) a disulfide bond formed between an amino acid residue on a position corresponding to a 346.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 393.sup.rd position of SEQ ID NO: 1; and (P) a disulfide bond formed between an amino acid residue on a position corresponding to a 349.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 390.sup.th position of SEQ ID NO: 1; and the conditions are as follows: the (C) item and the (D) item are not met at the same time; and the (H) item and the (J) item are not met at the same time.

2. The thermostable phytase according to claim 1, wherein compared to the wild-type Escherichia coli phytase as shown by SEQ ID NO: 1, the mutant Escherichia coli phytase has a mutation on at least one of the following positions: 1, 25, 30, 36, 37, 38, 39, 46, 55, 60, 62, 65, 69, 70, 73, 74, 75, 76, 77, 79, 80, 85, 101, 108, 109, 111, 114, 116, 118, 120, 123, 126, 127, 133, 137, 138, 139, 141, 142, 146, 151, 157, 159, 161, 173, 176, 178, 180, 183, 184, 185, 186, 187, 188, 189, 204, 211, 233, 235, 245, 253, 255, 267, 276, 282, 283, 284, 286, 287, 288, 291, 295, 297, 311, 315, 317, 318, 327, 341, 354, 363, 367, 369, 370, 380, 382, 383, 385, 391, 402 and 408.

3. The thermostable phytase according to claim 2, wherein compared to the wild-type Escherichia coli phytase as shown by SEQ ID NO: 1, the mutant Escherichia coli phytase has at least one of the following mutations: Q1S, Q1V, Q1N, A25F, Q30K, A36K, W37F, P38Y, T39D, W46E, I55V, H60S, H60Q, Q62W, R65H, D69N, G70E, A73P, A73D, A73E, K74D, K74P, K74L, K74N, K75C, K75Q, G76T, C77A, Q79L, Q79R, Q79A, Q79G, Q79F, S80P, I85V, A101L, C108A, A109D, A109E, A109G, A109F, A109P, THIS, T111D, T111Q, T114H, T116A, T118R, T118S, S120R, P123E, N126Y, P127V, P127L, C133A, N137V, N137E, N137S, N137P, A138V, A138H, A138D, A138P, N139P, N139A, N139H, T141R, T141E, T141G, T141A, D142R, S146E, S146R, S151P, G157R, G157Q, G157N, G157L, G157A, R159Y, T161P, P173Y, P173S, N176P, N176K, C178A, K183R, Q184S, D185N, D185L, E186V, E186A, S187P, C188A, S189T, N204C, V211W, G233E, G235Y, T245E, Q253V, Y255D, R267A, H282N, P283G, P284T, K286F, Q287Y, A288E, A288R, A288V, V291I, T295I, V297T, G311S, E315G, E315S, N317L, W318Y, T327Y, L341Y, L341V, F354Y, K363A, K363L, S367F, N369P, T370P, A380P, A380R, A380T, C382A, E383S, R385S, R385V, R385T, C391A, E402R, E402T, E402D, E402P, E402N and C408A.

4. The thermostable phytase according to claim 3, wherein compared to the wild-type Escherichia coli phytase as shown by SEQ ID NO: 1, the mutant Escherichia coli phytase has any combination of mutations selected from the following group: W46E, Q62W, A73P, K75C, S146E, R159Y, N204C and Y255D; A25F, W46E, Q62W, G70E, A73P, K75C, T114H, N137V, D142R, S146E, R159Y and Y255D; W46E, Q62W, G70E, A73P, K74N, K75Q, G76T, S146E, R159Y, P173S, Y255D, H282N, P283G and P284T; and A25F, W46E, Q62W, G70E, A73P, K74N, K75Q, T114H, N137V, D142R, S146E, R159Y, P173S, Y255D, H282N, P283G and P284T.

5. The thermostable phytase according to claim 4, wherein compared to the wild-type Escherichia coli phytase as shown by SEQ ID NO: 1, the mutant Escherichia coli phytase has any combination of mutations selected from the following group: W46E, Q62W, A73P, K75C, S146E, R159Y, N204C and Y255D; A25F, W46E, Q62W, G70E, A73P, K75C, T114H, N137V, D142R, S146E, R159Y and Y255D; and W46E, Q62W, G70E, A73P, K74N, K75Q, G76T, S146E, R159Y, P173S, Y255D, H282N, P283G and P284T.

6. The thermostable phytase according to claim 3, wherein the amino acid sequence of the mutant Escherichia coli phytase is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 79 or SEQ ID NO: 99.

7. The thermostable phytase according to claim 1, wherein the disulfide bonds are selected from at least one item of (A), (B), (C), (D), (E), (J), (M) or (O), and the condition is that the (C) item and the (D) item are not met at the same time.

8. The thermostable phytase according to claim 7, wherein the amino acid sequence meets any two, three or more items in the follow group: the (A) item, the (B) item, the (C) item, the (D) item, the (E) item, the (J) item, the (M) item and the (O) item, and the condition is that the (C) item and the (D) item are not met at the same time.

9. The thermostable phytase according to claim 7, wherein the amino acid sequence meets the (A) item, the (B) item, the (C) item, the (E) item, the (J) item or the (M) item.

10. The thermostable phytase according to claim 7, wherein the amino acid sequence at least meets the (D) item or the (O) item.

11. The thermostable phytase according to claim 10, wherein the amino acid sequence meets the (D) item.

12. The thermostable phytase according to claim 10, wherein the amino acid sequence meets the (O) item.

13. The thermostable phytase according to claim 7, wherein the amino acid sequence meets the (B) item and the (O) item; the (C) item and the (O) item; the (D) item and the (O) item; the (M) item and the (O) item; the (B) item, the (D) item and the (O) item; or the (D) item, the (M) item and the (O) item.

14. The thermostable phytase according to claim 13, wherein the amino acid sequence meets the (B) item and the (O) item.

15. The thermostable phytase according to claim 13, wherein the amino acid sequence meets the (C) item and the (O) item.

16. The thermostable phytase according to claim 13, wherein the amino acid sequence meets the (M) item and the (O) item.

17. The thermostable phytase according to claim 13, wherein the amino acid sequence meets the (B) item, the (D) item and the (O) item.

18. The thermostable phytase according to claim 13, wherein the amino acid sequence meets the (D) item, the (M) item and the (O) item.

19. The thermostable phytase according to claim 1, further comprising at least one pair of introduced disulfide bonds, wherein the disulfide bonds are selected from: (i) a disulfide bond formed between an amino acid residue on a position corresponding to a 31.sup.st position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 176.sup.th position of SEQ ID NO: 1; (ii) a disulfide bond formed between an amino acid residue on a position corresponding to a 31.sup.st position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 177.sup.th position of SEQ ID NO: 1; (iii) a disulfide bond formed between an amino acid residue on a position corresponding to a 52.sup.nd position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 99.sup.th position of SEQ ID NO: 1; (iv) a disulfide bond formed between an amino acid residue on a position corresponding to a 59.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 100.sup.th position of SEQ ID NO: 1; (v) a disulfide bond formed between an amino acid residue on a position corresponding to a 91.sup.st position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 46.sup.th position of SEQ ID NO: 1; (vi) a disulfide bond formed between an amino acid residue on a position corresponding to a 141.sup.st position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 200.sup.th position of SEQ ID NO: 1; (vii) a disulfide bond formed between an amino acid residue on a position corresponding to a 162.sup.nd position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 248.sup.th position of SEQ ID NO: 1; and (viii) a disulfide bond formed between an amino acid residue on a position corresponding to a 205.sup.th position of SEQ ID NO: 1 and an amino acid residue on a position corresponding to a 257.sup.th position of SEQ ID NO: 1; and the condition is that the (i) item and the (ii) item are not met at the same time.

20. The thermostable phytase according to claim 1, wherein the thermostable phytase has any one amino acid sequence selected from the following group: SEQ ID NOs: 4-40, SEQ ID NOs: 80-88 and SEQ ID NOs: 100-108.

21. The thermostable phytase according to claim 1, wherein the thermostable phytase is obtained through heterologous expression in a Pichia or Aspergillus niger host.

22. The thermostable phytase according to claim 1, wherein the amino acid residue capable of forming the disulfide bonds is a cysteine residue or a homocysteine residue.

23. A polynucleotide, coding the thermostable phytase according to claim 1.

24. The polynucleotide according to claim 23, wherein a coding sequence of the polynucleotide is subjected to codon optimization so as to realize expression in Pichia or Aspergillus niger.

25. The polynucleotide according to claim 23, having a nucleotide sequence as shown by any one of SEQ ID NOs: 41-77, SEQ ID NOs: 90-98 and SEQ ID NOs: 100-118.

26. A host cell, comprising the polynucleotide according to claim 23.

27. The host cell according to claim 26, wherein the host cell is a fungus cell, a bacterium cell or a plant cell.

28. The host cell according to claim 27, being a yeast cell or a filamentous fungus cell.

29. The host cell according to claim 28, being a Pichia cell or an Aspergillus niger cell.

30.-48. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0129] FIG. 1 is a pPIC9K-WT plasmid profile.

[0130] FIG. 2 is a diagram showing a heat stability test result of a wild-type and disulfide bond mutant.

[0131] FIG. 3 is a diagram of a heat stability test result of an APPA-M1 and mutant.

[0132] FIG. 4 is a diagram of a heat stability test result of an APPAan-WT and mutant.

[0133] FIG. 5 is a diagram of a heat stability test result of an APPA-M2 and mutant.

DETAILED DESCRIPTION

[0134] Embodiment 1 Disulfide bond mutant building, and wild-type and mutant expression in Pichia

[0135] A 3D structure of an Escherichia coli phytase has been published (referring to Lim D et al, Nat Struct Biol. 2000, 7(2):108-13), and with reference to a 3D structure document PDB ID 1DKO, disulfide bonds as described in the table below were designed and introduced.

TABLE-US-00002 Disulfide bond name Disulfide bond site A P34C/Q174C B A56C/G103C C Y57C/L366C D Y61C/L366C E Q82C/S296C F L128C/D203C G V140C/E262C H T156C/T191C I E165C/T245C J T191C/A210C K S196C/V211C L A264C/G312C M E315C/A380C N G322C/T356C O Q346C/L393C P Q349C/M390C Q T33C/L170C R I55C/A99C S A268C/N309C T I85C/G97C U P123C/T130C V A226C/M360C W W243C/P324C X W347C/M390C Y I348C/F396C

[0136] The amino acid sequence of a wild-type phytase was as shown by SEQ ID NO: 1. A nucleotide sequence thereof expressed in the Pichia was as shown by SEQ ID NO: 78. An expression vector was pPIC9K. A Saccharomyces cerevisiae Alpha factor was used as a signal peptide. An expression plasmid pPIC9K-WT of the wild-type phytase was as shown in FIG. 1.

[0137] In order to build the mutant in the above table, primers were respectively designed for Polymerase Chain Reaction (PCR). The primers were as shown by the table below.

TABLE-US-00003 Disul- Disul- fide fide bond bond name site Mutationprimer A P34/ A-F1GATGTCACCtgtGACGCTTGGCCAACCTGGA- Q174C F2AACTTCCCAtgtTCAAACTTGTGCTTGAAG A-R1CCAAGCGTCacaGGTGACATCTTGCATAAG A-R2CAAGTTTGAacaTGGGAAGTTAAGAACTCT B A56/ B-F1GAGCTCATTtgtTACTTGGGTCACTACCAA G103C B-F2TTCGCCGCCtgtCTTGCTCCTGACTGTGCC B-R1ACCCAAGTAacaAATGAGCTCACCACCTCT B-R2AGGAGCAAGacaGGCGGCGAAGGCTTCACC C T57C/ C-F1CTCATTGCTtgtTTGGGTCACTACCAAAGAC- L366C F2AAGACTCCAtgtTCTTTGAACACGCCTCCA C-R1GTGACCCAAacaAGCAATGAGCTCACCACC C-R2GTTCAAAGAacaTGGAGTCTTTGTCTCTCAT D T61C/ D-F1TTGGGTCAtgtCAAAGACAGCGTCTTGTT L36C D-F2AAGACTCCAtgtTCTTTGAACACGCCTCCA D-R1CTGTCTttgacaGTGACCCAAGTAAGCAAT D-R2GTTCAAAGAacaTGGAGTCTTGTCTCTCAT E Q82C/ E-F1CAATCTGGTtgtGTAGCTATTATTGCTGAC S296C E-F2TTGCCCACTtgtGTCTTGTTCATTGCCGGT E-R1AATAGCTACacaACCAGATTGTGGACAACC E-R2GAACAAGACacaAGTGGGCAAGGTAACACC F L128C/ F-F1TTCAACCCTtgtAAGACTGGTGTTTGCCA D203C F-F2GTCTCCGCCtgtAACGTCTCTTTGACCGGT F-R1ACCAGTCTTacaAGGGTTGAACAATGGATC F-R2AGAGACGTTacaGGCGGAGACCTTCAACTC G V140C/ G-F1aacGCTAACtgtACTGACGCTATCTTGTCC E262C G-F2AGAACTCCAtgtGTTGCTAGATCCAGAGCC G-R1AGCGTCAGTacaGTTAGCGTTGTCCAATTG G-R2TCTAGCAAcacATGGAGTTCTCTGCAGCAA H T156C/ H-F1GCTGACTTCtgtGGTCACAGACAGACTGCC T191C H-F2TGTTCCTTGtgtCAAGCATTACCATCTGAGH- R1tctGTGACCacaGAAGTCAGCAATGGATCC H-R2TAATGCTTGacaCAAGGAACAGGATTCGTC I E165C/ I-F1GCCTTCAGAtgtTTGGAAAGAGTTCTTAAC T245C I-F2CAATGGAACtgtTTGTTGTCCTTGCACAAC I-R1TCTTTCCAAacaTCTGAAGGCAGTCTGTCT I-R2GGACAACAAacaGTTCCATTGGTGAGAGTC J T191C/ J-F1TGTTCCTTGtgtCAAGCATTACCATCTGAG A2I0C J-F2TTGACCGGTtgtGTCAGCTTGGCTTCCATG J-R1TAATGCTTGacaCAAGGAACAGGATTCGTC J-R2CAAGCTGACacaACCGGTCAAAGAGACGT K S196C/ K-F1GCATTACCAtgtGAGTTGAAGGTCTCCGCC V211C K-F2ACCGGTGCTtgtAGCTTGGCTTCCATGTTG K-R1CTTCAACTCacaTGGTAATGCTTGAGTCAA K-R2AGCCAAGCTacaAGCACCGGTCAAAGAGAC L A264C/ T-F1CCAGAGgTTtgtAGATCCAGAGCCACCCCA G312C T-F2AATCTCGGCtgtGCTTTGGAGTTGAACTGG T-R1TCTGGATCTacaAAcCTCTGGAGTTCTCTG T-R2CTCCAAAGCacaGCCGAGATTTGCCAAGTT M E315C/ M-F1GGTGCTTTGtgtTTGAACTGGACTCTTCCT A380C M-F2TTGACCTTGtgtGGATGTGAAGAGAGAAAT M-R1CCAGTTCAAacaCAAAGCACCGCCGAGATT M-R2TTCACATCCacaCAAGGTCAATTTGACTTC N G322C/ N-F1ACTCTTCCTtgtCAACCTGATAACACTCCA T356C N-F2GTCTTCCAAtgtTTGCAGCAGATGAGAGAC N-R1ATCAGGTTGacaAGGAAGAGTCCAGTTCAA N-R2CTGCTGCAAacaTTGGAAGACCAACGAAAC O Q346C/ O-F1GATAACTCTtgtTGGATTCAGGTTTCGTTG L393C O-F2ATGTGTTCCtgtGCTGGTTTCACTCAAATC O-R1CTGAATCCAacaAGAGTTATCAGATAGTCT O-R2GAAACCAGCacaGGAACACATACCCTGAGC P Q349C/ P-F1CAATGGATTIgtGTTTCGTTGOTCTTCCAA M390C P-F2GCTCAGGGTtgtTGTTCCTTGGCTGGTTTC P-R1CAACGAAACacaAATCCATTGAGAGTTATC P-R2CAAGGAACAacaACCCTGAGCATTTCTCTC Q T33C/ Q-F1CAAGATGTCtgtCCAGACGCTTGGCCAACC L170C Q-F2GAAAGAGTTtgtAACTTCCCACAAtcaAAC Q-R1AGCGTCTGGacaGACATCTTGCATAAGTTG Q-R2TGGGAAGTTacaAACTCTTTCCAACTCTCT R I55C/ R-F1GGTGAGCTCtgtGCTTACTTGGGTCACTAC A99C R-F2ACAGGTGAAtgtTTCGCCGCCGGTCTTGCT R-R1CAAGTAAGCacaGAGCTCACCACCTCTAGG R-R2GGCGGCGAAacaTTCACCTGTCTTACGGGT S A268C/ S-F1AGATCCAGAtgTACCCCATTGTTGGACTTG N309C S-F2AACTTGGCATgtCTCGGCGGTGCTTTGGAG S-R1CAATGGGGTacaTCTGGATCTAGCAAcCTC S-R2ACCGCCGAGacaTGCCAAGTTAGTATCGTG T I85C/ T-F1CAAGTAGCTtgtATTGCTGACGTCGACGAA G97C T-F2CGTAAGACACgtGAAGCCTTCGCCGCCGGT T-RTGTCAGCAATacaAGCTACTTGACCAGATTG T-R2GAAGGCTTCACaTGTCTTACGGGTTCTTTC U P123C/ U-F1TCTCCAGATtgtTTGTTCAACCCTTTGAAG T130C U-F2CCTTTGAAGtgtGGTGTTTGCCAATTGGAC U-RTGTTGAACAAacaATCTGGAGAAGAAGTGTC U-R2GCAAACACCacaCTTCAAAGGGTTGAACAA V A226C/ V-F1CTGCAACAAtgtCAAGGTATGCCTGAGCCA M360C V-F2TTGCAGCAGtgtAGAGACAAGACTCCACTG V-R1CATACCTTGAcaTTGTTGCAGAAGAAAGAT V-R2CTTGTCTCTacaCTGCTGCAAAGTTTGGAA W W243C/ W-F1TCTCACCAATGtAACACCTTGTTCJTCCTTG P324C W-F2CCTGGTCAAtgtGATAACACTCCACCAGGT W-R1CAAGGTGTTacaTTGGTGAGAGTCGGTGAT W-R2AGTGTTATCacaTTGACCAGGAAGAGTCCA X W347C/ X-F1AACTCTCAAtgTATTCAGGTTTCGTTGGTC M390C X-F2GCTCAGGGTgtTTGTTCCTTGGCTGGTTTC X-R1AACCTGAATaCATTGAGAGTTATCAGATAG X-R2CAAGGAACAacaACCCTGAGCATTTCTCTC Y I348C/ Y-F1TCTCAATGGtgtCAGGTTTCGTTGGTCTTC F396C Y-F2TTGGCTGGTtgtACTCAAATCGTTAACGAA Y-R1CGAAACCTGacaCCATTGAGAGTTATCAGA Y-R2GATTTGAGTacaACCAGCCAAGGAACACAT

[0138] In order to introduce 25 pairs of disulfide bonds of A-Y, plasmid pPIC9K-WT was used as a template. F1/R2 and F2/R1 were used as introduction pairs. Two PCR amplification reactions were respectively performed. The amplification reaction was completed by a Phusion High-Fidelity DNA polymerase (New England Biolabs, article number: M0530L). Setting was performed with reference to its manual. After the amplification was completed, a Dpnl endonuclease (New England Biolabs) was added for digesting the template. Then, Gibson Assembly Master Mix Kit (New England Biolabs, article number: E2611) was used for fragment recombination. Through sequencing, successful building of the mutant plasmid was confirmed. The mutant plasmid was respectively named as pPIC9K-A to pPIC9K-Y according to the disulfide bond name in the above table.

[0139] In order to express the phytase and the mutant, the Pichia GS115 and the plasmid were operated with reference to Pichia expression kit (Invitrogen) manual. Specifically, after a Pichia GS115 strain was subjected to plate culture at 30 C. for 48 h by a YPD culture medium (1% of yeast extracts, 2% of protein, 2% of glucose and 1.5% of agar), monoclone was selected into a 4 mL YPD liquid culture medium (1% of yeast extracts, 2% of protein, and 2% of glucose), cultured at 30 C. and 200 rpm for 12 h, transferred to an Erlenmeyer flask with 30 mL YPD liquid culture medium, and cultured at 30 C. and 220 rpm for 4-5 h. After an OD600 value was detected to be in a range of 1.1-1.3, culture solution was centrifuged at 4 C. and 9,000 rpm for 2 min. 4 mL of thalli were respectively collected into sterile EP tubes. Supernatants were slightly abandoned. After residual supernatants were thoroughly absorbed by sterile filter paper, 1 mL of precooled sterile water was used for resuspending the thalli. Centrifugation at 4 C. and 9,000 rpm was performed for 2 min. Supernatants were abandoned. The above steps were repeated. The thalli were resuspended by 1 mL of precooled sorbitol (1 mol/L). Centrifugation at 4 C. and 9,000 rpm was performed for 2 min. Supernatants were abandoned. The thalli were resuspended by 100-150 l of precooled sorbitol (1 mol/L). Hereto, preparation of competent cells was completed. The expression plasmid pPIC9K-WT and the other 25 disulfide bond mutants were linearized by BglII. Linearized fragments were purified and recovered and were then converted into the Pichia GS115 competent cells by an electroporation method. A mixture was uniformly coated onto an MD plate to be subjected to 30 C. reverse culture for 2-3 d. All bacterial colonies on the plate were washed down by sterile water and were then coated onto a YPD (0.5 mg/mL-8 mg/mL) plate containing different concentrations of geneticin for screening multicopy converters. Pichia recombination strains were obtained through screening on the MD plate, and were named as APPA-WT and APPA-A, APPA-B, APPA-C, APPA-D, APPA-E, APPA-F, APPA-G, APPA-H, APPA-I, APPA-J, APPA-K, APPA-L, APPA-M, APPA-N, APPA-O, APPA-P, APPA-Q, APPA-R, APPA-S, APPA-T, APPA-U, APPA-V, APPA-W, and APPA-X and APPA-Y The above clones obtained through screening were respectively transferred into a BMGY culture medium to be cultured for 24 h in an oscillation table concentrator at 30 C. and 250 rpm, and were then transferred into a BMMY culture medium. Conditions of 30 C. and 250 rpm were maintained. 0.5% of methanol was added every day. After 120 h of induction expression, 9000-12000 rpm centrifugation was performed for 10 min so as to remove thalli. A fermentation supernatant containing a phytase APPA-WT and its 25 mutants was obtained. An SD S-PAGE result showed that three mutants of APPA-S, APPA-X and APPA-Y were not expressed, and the other 22 mutants were expressed.

Embodiment 2 Heat Stability Determination

[0140] Phytase activity determination conforms to GB/T 18634-2009. 23 samples in Embodiment 1 were diluted to 100 U/mL. 9 mL of water was taken in a 25 mL colorimetric tube to be respectively preheated in an 80 C. constant-temperature water bath. 1 mL of each enzyme sample was sucked by a pipette and were added fast into each corresponding test tube. Fast mixing and blending was performed by a mixing and blending device, followed by standing for 3 min. The temperature was fast cooled to a room temperature. Dilution was performed by water. Residual activity of each sample was determined. Therefore, the enzyme activity residue rates (the enzyme activity before heat treatment was set to be 100%) at different treatment temperatures were calculated. The heat stability data was as shown in FIG. 2. Some mutants showed good heat stability. APPA-B, APPA-C, APPA-D, APPA-M, APPA-O and APPA-P had the best performance, with the residual activity improved by about 20-25%, or about 2-3 times, compared with that of APPA. The above results showed that the introduction of the disulfide bonds had significant influence on the mutants. Some mutations even caused normal expression incapability, such as APPA-S, APPA-X and APPA-Y. Additionally, the introduction of some specified disulfide bonds may cause stability reduction. For example, the two mutants, such as APPA-Q and APPA-V caused heat stability significantly lower than that of the wild type. There were also some introduced disulfide bonds beneficial to the enzyme structure stability, such as APPA-A to APPA-P which can improve the heat resistance capability of the wild type.

Embodiment 3 Introduction of Disulfide Bonds on Basis of Mutant and Determination of its Stability

[0141] Nov9X is a mutant with good heat resistance obtained through mutation screening by the wild-type phytase (as described in U.S. Pat. No. 7,432,098). 8 mutations were introduced on the basis of the wild type. A specific sequence was as shown by SEQ ID NO: 2. Other mutations were continuously introduced on the basis of the Nov9X sequence. The sequence became one as shown in SEQ ID NO: 3, and the mutant was named as APPA-M1. Its heat stability can be further improved. In order to test whether the disulfide bond mutant described in Embodiment 1 can achieve a function on the phytase mutant or not, the stability was further improved. The disulfide bonds D and O and disulfide bond combinations B+O, C+O, D+O, M+O, B+D+O and D+M+O were introduced on the basis of APPA-M1 according to a method in Embodiment 1. Each mutant was respectively named as APPA-M1-D, APPA-M1-O, APPA-M1-BO, APPA-M1-CO, APPA-M1-DO, APPA-M1-MO, APPA-M1-BDO and APPA-M1-DMO. Meanwhile, according to description in US20170240872 and US20130017185, two best mutant disulfide bonds in each embodiment were introduced on the basis of APPA-M1, and the mutants were respectively named as US20170240872-A, US20170240872-B, US20130017185-B and US20130017185-C. Each mutant was expressed in Pichia. Then, heat stability was determined according to a method in Embodiment 2. A different parameter was incubation for 3 min at 85 C. The results were as shown in FIG. 3. The disulfide bonds D and O both further improved the heat stability of the mutant, and the improvement brought by the disulfide bond 0 on the stability of the APPA-M1 mutant was greater than the improvement brought on the wild-type APPA-WT. The disulfide bond O improved the stability of the APPA-M1 mutant by up to 35.5%. Disulfide bond combined introduced mutants APPA-M1-CO and APPA-M1-DO showed stability similar to APPA-M1-O. Other combination mutants, such as APPA-M1-BO, APPA-M1-MO, APPA-M1-BDO and APPA-M1-DMO had higher stability. The residual activity of APPA-M1-BO with the best stability can reach 77.2%, and was improved by about 1-1.5 times compared with that of APPA-M1, the significant heat resistance characteristic was realized, and APPA-M1-BO was predicted to have good performance in feed granulation. The above results showed that the proper combination can create more stable mutants. The stability of US20170240872-A and US20170240872-B determined by this method was improved by 1.1-8.7% compared with that of APPA-M1. The stability of US20130017185-B and US20130017185-C was improved by 5.0-14.8%. It can be seen that the introduction of the disulfide bonds provided by the inventor showed more effective results.

Embodiment 4 Expression of Escherichia coli Wild-Type Phytase in Aspergillus niger and Disulfide Bond Introduced Mutant Phytase

[0142] According to description of patent application CN107353327, the Escherichia coli phytase wild type (SEQ ID NO: 1) and mutants (A to P) were expressed. The wild-type phytase was named as APPAan-WT. Each mutant was named according to APPAan-A to APPAan-P. After shake flask supernatant was obtained, heat stability determination was performed according to description in Embodiment 2. A different parameter was incubation for 3 min at 85 C. The experiment results were as shown in FIG. 4. It is found that the wild-type phytase expressed in Aspergillus niger showed more significant stability than that expressed in Pichia probably because they had different glycosylation states. Through experiments, it is also found that the mutant APPAan-P cannot be expressed, APPAan-G expressed stability similar to WT, and the stability of APPAan-H was significantly reduced. The other 13 mutants all expressed significant stability performance improvement, and all expressed improvement of 5% to 20.5%, which was different from that of mutants expressed in Pichia. The above results showed that 16 mutants expressed in at least one host cell, and showed more excellent stability than the wild type. Through the proper introduction of the disulfide bond combination, mutants with higher stability can be obtained.

Embodiment 5 Stability Test Results after Introduction of Disulfide Bonds to Phytase Mutants

[0143] Nov9X is a mutant with good heat resistance obtained through mutation screening by the wild-type phytase (as described in U.S. Pat. No. 7,432,098). 8 mutations were introduced on the basis of the wild type. A specific sequence was as shown by SEQ ID NO: 2. On the basis of a Nov9X sequence, a glycosylation site is continuously introduced according to a literature report (Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design). The sequence became one as shown in SEQ ID NO: 79, and the mutant was named as APPA-M2. Its heat stability can be further improved. In order to test whether the disulfide bond mutant described in Embodiment 1 can achieve a function on the phytase mutant APPA-M2 or not, the stability was further improved. The disulfide bonds B, C, D, M and O and disulfide bond combinations B+O, D+O, M+O and C+O were introduced on the basis of APPA-M2 according to a method in Embodiment 1. Each mutant was respectively named as APPA-M2-B, APPA-M2-C, APPA-M2-D, APPA-M2-M, APPA-M2-O, APPA-M2-BO, APPA-M2-DO, APPA-M2-MO and APPA-M2-CCX. The amino acid sequence of each mutant was as shown by SEQ ID Nos: 80-88. Corresponding nucleotide sequences were as shown by SEQ ID NOs: 90-98. Each mutant was expressed in Aspergillus niger. Then, heat stability was determined according to a method in Embodiment 2. The results were as shown in FIG. 5. The disulfide bonds adopted in the experiment all significantly improved the heat stability of the mutant. The effect of the combined disulfide bonds C+O was best. The residual activity after heat resistance treatment can reach 84.5%. The significant heat resistance characteristic was realized. It was predicted to have good performance in feed granulation. The above results showed that the proper combination can create more stable mutants. The above results showed that the introduction of the disulfide bonds provided by the inventor still showed very effective results on the phytase mutant sequence.

[0144] The inventor also tried continuously introducing a glycosylation site on the basis of the Nov9X sequence. Its sequence was as shown by SEQ ID NO: 99. The mutant was named as APPA-M3. Its heat stability can also be improved. Disulfide bonds C, C, D, M and O and disulfide bond combinations B+O, D+O, M+O, and C+O were introduced on the basis of APPA-M3 according to the method in Embodiment 1. Each mutant was respectively named as APPA-M3-B, APPA-M3-C, APPA-M3-D, APPA-M3-M, APPA-M3-O, APPA-M3-BO, APPA-M3-DO, APPA-M3-MO and APPA-M3-CCX. The amino acid sequence of each mutant was as shown by SEQ ID Nos: 100-108. Corresponding nucleotide sequences were as shown by SEQ ID NOs: 110-118. Each mutant was expressed in Aspergillus niger. Then, heat stability was determined according to the method in Embodiment 2. It was found that the disulfide bond introduced mutants achieve a good heat resistance characteristic like the disulfide bond introduced AMMA-M2 mutants. Identically, it was predicted to have good performance in feed granulation.

[0145] The modifications and variations of the method according to the present invention are obvious to a person skilled in the art, and do not deviate from the scope of the invention. Although the present invention is described with reference to particular embodiments, it should be understood that the invention requiring protection is not improperly limited to these particular exemplary embodiments. In fact, the various mutational modifications of wild-type phytase that are apparent to a person skilled in the art to achieve the technical effects of the present invention are covered by the claims.