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 variant having phytase activity that has at least 95% sequence identity to a polypeptide selected from the group consisting of the polypeptides of SEQ ID NO: 1, 2, 3, 79, and 99, wherein the phytase variant comprises a cysteine or a homocysteine residue at positions corresponding to positions 346 and 393 of the polypeptide of SEQ ID NO: 1, such that a disulfide bond is formed between the two residues, and optionally, the phytase variant further comprises one or more combinations of residues selected from (A) to (O), such that a disulfide bond is formed between the two residues of each combination: (A) a cysteine or a homocysteine residue at positions corresponding to positions 34 and 174 of the polypeptide of SEQ ID NO: 1; (B) a cysteine or a homocysteine residue at positions corresponding to positions 56 and 103 of the polypeptide of SEQ ID NO: 1; (C) a cysteine or a homocysteine residue at positions corresponding to positions 57 and 366 of the polypeptide of SEQ ID NO: 1; (D) a cysteine or a homocysteine residue at positions corresponding to positions 61 and 366 of the polypeptide of SEQ ID NO: 1; (E) a cysteine or a homocysteine residue at positions corresponding to positions 82 and 296 of the polypeptide of SEQ ID NO: 1; (F) a cysteine or a homocysteine residue at positions corresponding to positions 128 and 203 of the polypeptide of SEQ ID NO: 1; (G) a cysteine or a homocysteine residue at positions corresponding to positions 140 and 262 of the polypeptide of SEQ ID NO: 1; (H) a cysteine or a homocysteine residue at positions corresponding to positions 156 and 191 of the polypeptide of SEQ ID NO: 1; (I) a cysteine or a homocysteine residue at positions corresponding to positions 165 and 245 of the polypeptide of SEQ ID NO: 1; (J) a cysteine or a homocysteine residue at positions corresponding to positions 191 and 210 of the polypeptide of SEQ ID NO: 1; (K) a cysteine or a homocysteine residue at positions corresponding to positions 196 and 211 of the polypeptide of SEQ ID NO: 1; (L) a cysteine or a homocysteine residue at positions corresponding to positions 264 and 312 of the polypeptide of SEQ ID NO: 1; (M) a cysteine or a homocysteine residue at positions corresponding to positions 315 and 380 of the polypeptide of SEQ ID NO: 1; (N) a cysteine or a homocysteine residue at positions corresponding to positions 322 and 356 of the polypeptide of SEQ ID NO: 1; and (O) a cysteine or a homocysteine residue at positions corresponding to positions 349 and 390 of the polypeptide of SEQ ID NO: 1; wherein the thermostable phytase variant does not comprise the combinations of residues of (C) and (D) at the same time; or does not comprise the combinations of residues (H) and (J) at the same time.

2. The thermostable phytase variant according to claim 1, wherein the thermostable phytase variant comprises at least one combination of residues selected from (A), (B), (C), (D), (E), (J), or (M), but not both (C) and (D) at the same time.

3. The thermostable phytase variant according to claim 2, wherein the thermostable phytase variant comprises one, two, or more combinations of residues selected from (A), (B), (C), (D), (E), (J), and (M), but not both (C) and (D) at the same time.

4. The thermostable phytase variant according to claim 2, wherein the thermostable phytase variant comprises the combination of residues of (A), (B), (C), (E), (J) or (M).

5. The thermostable phytase variant according to claim 2, wherein the thermostable phytase variant further comprises the combination of residues of (D).

6. The thermostable phytase variant according to claim 2, wherein the thermostable phytase variant comprises the combinations of residues of (B); (C); (D); (M); (B) and (D); or (D) and (M).

7. The thermostable phytase variant according to claim 6, wherein the thermostable phytase variant comprises the combination of residues of (B).

8. The thermostable phytase variant according to claim 6, wherein the thermostable phytase variant comprises the combination of residues of (C).

9. The thermostable phytase variant according to claim 6, wherein the thermostable phytase variant comprises the combination of residues of (M).

10. The thermostable phytase variant according to claim 6, wherein the thermostable phytase variant comprises the combinations of residues of (B) and (D).

11. The thermostable phytase variant according to claim 6, wherein the thermostable phytase variant comprises the combinations of residues of (D) and (M).

12. The thermostable phytase variant according to claim 1, wherein the phytase variant comprises at least one combination of residues selected from (i) a cysteine or homocysteine residue at the positions corresponding to positions 31 and 176 of the polypeptide of SEQ ID NO: 1; (ii) a cysteine or homocysteine residue at the positions corresponding to positions 31 and 177 of the polypeptide of SEQ ID NO: 1; (iii) a cysteine or homocysteine residue at the positions corresponding to positions 52 and 99 of the polypeptide of SEQ ID NO: 1; (iv) a cysteine or homocysteine residue at the positions corresponding to positions 59 and 100 of the polypeptide of SEQ ID NO: 1; (v) a cysteine or homocysteine residue at the positions corresponding to positions 91 and 46 of the polypeptide of SEQ ID NO: 1; (vi) a cysteine or homocysteine residue at the positions corresponding to positions 141 and 200 of the polypeptide of SEQ ID NO: 1; (vii) a cysteine or homocysteine residue at the positions corresponding to positions 162 and 248 of the polypeptide of SEQ ID NO: 1; and (viii) a cysteine or homocysteine residue at the positions corresponding to positions 205 and 257 of the polypeptide of SEQ ID NO: 1; wherein the thermostable phytase variant does not comprise the combinations of residues of (i) and (ii) at the same time.

13. The thermostable phytase variant according to claim 1, wherein the thermostable phytase variant comprises the amino acid sequence of SEQ ID NO: 18.

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

15. The thermostable phytase variant according to claim 1, wherein the disulfide bond is formed between two cysteine residues or two homocysteine residues.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a pPIC9K-WT plasmid profile.

(2) FIG. 2 is a diagram showing a heat stability test result of a wild-type and disulfide bond mutant.

(3) FIG. 3 is a diagram of a heat stability test result of an APPA-M1 and mutant.

(4) FIG. 4 is a diagram of a heat stability test result of an APPAan-WT and mutant.

(5) FIG. 5 is a diagram of a heat stability test result of an APPA-M2 and mutant.

DETAILED DESCRIPTION

(6) Embodiment 1 Disulfide bond mutant building, and wild-type and mutant expression in Pichia

(7) 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.

(8) 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

(9) 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.

(10) 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.

(11) TABLE-US-00003 Disulfide bond Disulfidebond name site Mutationprimer A P34C/Q174C A-F1GATGTCACCtgtGACGCTTGGCCAACCTGGA- F2AACTTCCCAtgtTCAAACTTGTGCTTGAAG A-R1CCAAGCGTCacaGGTGACATCTTGCATAAG A-R2CAAGTTTGAacaTGGGAAGTTAAGAACTCT B A56C/G1030 B-F1GAGCTCATTtgtTACTTGGGTCACTACCAA B-F2TTCGCCGCCtgtCTTGCTCCTGACTGTGCC B-R1ACCCAAGTAacaAATGAGCTCACCACCTCT B-R2AGGAGCAAGacaGGCGGCGAAGGCTTCACC C T57C/L366C C-F1CTCATTGCTtgtTTGGGTCACTACCAAAGAC- F2AAGACTCCAtgtTCTTTGAACACGCCTCCA C-R1GTGACCCAAacaAGCAATGAGCTCACCACC C-R2GTTCAAAGAacaTGGAGTCTTGTCTCTCAT D T61C/L366C D-F1TTGGGTCACtgtCAAAGACAGCGTCTTGTT D-F2AAGACTCCAtgtTCTTTGAACACGCCTCCA D-R1CTGTCTttgacaGTGACCCAACTAAGCAAT D-R2GTTCAAAGAacaTGGAGTCTTGTCTCTCAT E Q82C/S296C E-F1CAATCTGGTtgtGTAGCTATTATTGCTGAC E-F2TTGCCCACTtgtGTCTTGTTCATTGCCGGT E-R1AATAGCTACacaACCAGATTGTGGACAACC E-R2GAACAAGACacaAGTGGGCAAGGTAACACC F L128C/D203C F-F1TTCAACCCTtgtAAGACTGGTGTTTGCCA F-F2 GTCTCCGCCtgtAACGTCTCTTTGACCGGTF-R1 ACCAGTCTTacaAGGGTTGAACAATGGATC F-R2AGAGACGTTacaGGCGGAGACCTTCAACTC G V140C/E262C G-F1 aacGCTAACtgtACTGACGCTATCTTGTCC G-F2AGAACTCCAtgtGTTGCTAGATCCAGAGCC G-R1AGCGTCAGTacaGTTAGCGTTGTCCAATTG G-R2TCTAGCAAcacATGGAGTTCTCTGCAGCAA H T156C/T191C H-F1GCTGACTTCtgtGGTCACAGACAGACTGCC H-F2TGTTCCTTGtgtCAAGCATTACCATCTGAGH- R1tctGTGACCacaGAAGTCAGCAATGGATCC H-R2TAATGCTTGacaCAAGGAACAGGATTCGTC I E165C/T245C I-F1GCCTTCAGAtgtTTGGAAAGAGTTCTTAAC I-F2CAATGGAACtgtTTGTTGTCCTTGCACAAC I-R1TCTTTCCAAacaTCTGAAGGCAGTCTGTCT I-R2 GGACAACAAacaGTTCCATTGGTGAGAGTC T191C/A210C J-F1TGTTCCTTGtgtCAAGCATTACCATCTGAG J-F2TTGACCGGTtgtGTCAGCTTGGCTTCCATG J-RI TAATGCTTGacaCAAGGAACAGGATTCGTC J-R2 CAAGCTGACacaACCGGTCAAAGAGACGT K S196C/V211C K-F1GCATTACCAtgtGAGTTGAAGGTCTCCGCC K-F2ACCGGTGCTtgtAGCTTGGCTTCCATGTTG K-R1CTTCAACTCacaTGGTAATGCTTGAGTCAA K-R2AGCCAAGCTacaAGCACCGGTCAAAGAGAC L A264C/G312C L-F1CCAGAGgTTtgtAGATCCAGAGCCACCCCA L-F2 AATCTCGGCtgtGCTTTGGAGTTGAACTGG L-R1TCTGGATCTacaAAcCTCTGGAGTTCTCTG L-R2CTCCAAAGCacaGCCGAGATTTGCCAAGTT M E315C/A380C M-F1GGTGCTTTGtgtTTGAACTGGACTCTTCCT M-F2TTGACCTTGtgtGGATGTGAAGAGAGAAAT M-R1CCAGTTCAAacaCAAAGCACCGCCGAGATT M-R2TTCACATCCacaCAAGGTCAATTTGACTTC N G322C/T356C N-F1ACTCTTCCTtgtCAACCTGATAACACTCCA N-F2GTCTTCCAAtgtTTGCAGCAGATGAGAGAC N-R1ATCAGGTTGacaAGGAAGAGTCCAGTTCAA N-R2CTGCTGCAAacaTTGGAAGACCAACGAAAC 0 Q346C/L393C O-F1GATAACTCTtgtTGGATTCAGGTTTCGTTG O-F2ATGTGTTCCtgtGCTGGTTTCACTCAAATC O-R1CTGAATCCAacaAGAGTTATCAGATAGTCT O-R2GAAACCAGCacaGGAACACATACCCTGAGC P Q349C/M390C P-F1CAATGGATTtgtGTTTCGTTGGTCTTCCAA P-F2GCTCAGGGTtgtTGTTCCTTGGCTGGTTTC P-R1CAACGAAACacaAATCCATTGAGAGTTATC P-R2CAAGGAACAacaACCCTGAGCATTTCTCTC Q T33C/L170C Q-F1CAAGATGTCtgtCCAGACGCTTGGCCAACC Q-F2GAAAGAGTTtgtAACTTCCCACAAtcaAAC Q-R1AGCGTCTGGacaGACATCTTGCATAAGTTG Q-R2TGGGAAGTTacaAACTCTTTCCAACTCTCT R 155C/A99C R-F1GGTGAGCTCtgtGCTTACTTGGGTCACTAC R-F2ACAGGTGAAtgtTTCGCCGCCGGTCTTGCT R-R1CAAGTAAGCacaGAGCTCACCACCTCTAGG R-R2GGCGGCGAAacaTTCACCTGTCTTACGGGT S A268C/N309C S-F1AGATCCAGAtgtACCCCATTGTTGGACTTG S-F2 AACTTGGCAtgtCTCGGCGGTGCTTTGGAG S-R1CAATGGGGTacaTCTGGATCTAGCAAcCTC S-R2ACCGCCGAGacaTGCCAAGTTAGTATCGTG T 185C/G97C T-F1 CAAGTAGCTtgtATTGCTGACGTCGACGAA T-F2CGTAAGACAtgtGAAGCCTTCGCCGCCGGT T-R1GTCAGCAATacaAGCTACTTGACCAGATTG T-R2GAAGGCTTCACaTGTCTTACGGGTTCTTTC U P123C/T130C U-F1TCTCCAGATtgtTTGTTCAACCCTTTGAAG U-F2CCTTTGAAGtgtGGTGTTTGCCAATTGGAC U-R1GTTGAACAAacaATCTGGAGAAGAAGTGTC U-R2GCAAACACCacaCTTCAAAGGGTTGAACAA V A226C/M360C V-F1CTGCAACAAtgtCAAGGTATGCCTGAGCCA V-F2TTGCAGCAGtgtAGAGACAAGACTCCACTG V-R1CATACCTTGAcaTTGTTGCAGAAGAAAGAT V-R2CTTGTCTCTacaCTGCTGCAAAGTTTGGAA W W243C/P324C W-F1TCTCACCAATGtAACACCTTGTTGTCCTTG W-F2CCTGGTCAAtgtGATAACACTCCACCAGGT W-R1CAAGGTGTTacaTTGGTGAGAGTCGGTGAT W-R2AGTGTTATCacaTTGACCAGGAAGAGTCCA X W347C/M390C X-F1AACTCTCAAtgtATTCAGGTTTCGTTGGTC X-F2GCTCAGGGTigtTGTTCCTTGGCTGGTTTC X-R1AACCTGAATaCATTGAGAGTTATCAGATAG X-R2CAAGGAACAacaACCCTGAGCATTTCTCTC Y I348C/F396C Y-F1TCTCAATGGtgtCAGGTTTCGTTGGTCTTC Y-F2TTGGCTGGTtgtACTCAAATCGTTAACGAA Y-R1CGAAACCTGacaCCATTGAGAGTTATCAGA Y-R2GATTTGAGTacaACCAGCCAAGGAACACAT

(12) 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.

(13) 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

(14) 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

(15) 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

(16) 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

(17) 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.

(18) 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.

(19) 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.