CN114164193A - Phytase mutant with improved thermal stability and protease resistance as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a phytase mutant with improved thermal stability and protease resistance, a preparation method and application thereof, belonging to the field of genetic engineering and enzyme engineering. In the invention, both the alanine at the 108 th site and the serine at the 322 th site in the phytase YiAPPA are mutated into cysteine, and a disulfide bond C108-C322 is introduced into the phytase YiAPPA to obtain a mutant YiAPPA-A108C/S322C. The half-life of the phytase mutant YIAPPA-A108C/S322C provided by the invention at 85 ℃ is improved to 15min from 6min of a control (before mutation), and is improved by 1.5 times, and the pepsin resistance and the trypsin resistance of the YIAPPA-A108C/S322C are respectively improved by 16% and 12%. The phytase mutant YIAPPA-A108C/S322C has obviously improved thermal stability and protease resistance, and has good application prospect in the field of animal feed additives.

Description

Phytase mutant with improved thermal stability and protease resistance as well as preparation method and application thereof

Technical Field

The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to a phytase mutant with improved thermal stability and protease resistance, and a preparation method and application thereof.

Background

Phytases, also known as phytases, are a class of phosphoesterases that decompose phytic acid into inositol and inorganic phosphorus, and are widely found in animals, plants, and microorganisms. The phytase has the greatest application in the feed industry, and has wide application prospect and great commercial value as a feed additive. The phytase serving as a feed additive can improve the utilization rate of phosphorus and reduce the pollution of phytate phosphorus to the environment; the anti-nutritional effect of the phytic acid in the feed is eliminated, the utilization rate of organisms on various trace elements and proteins is improved, the growth and development of animals are promoted, the production performance of the animals is improved, and the discharge of phosphorus in animal wastes is reduced.

Phytase is used as a feed additive for monogastric animals, and the feeding effect of phytase is confirmed worldwide. However, the popularization and application of phytase in feed in China are quite limited at present, and the reasons for this are mainly as follows: the phytase production cost is too high due to the low content of phytase in the natural material; the thermal stability, activity, pH action range, protease resistance and other enzymological properties of the current commercial phytase are difficult to completely meet the requirements of feed industry. In recent years, the improvement of the expression level of phytase by using a genetic engineering technology, and the modification of phytase genes on a molecular level by using the genetic engineering technology and a protein engineering technology along with the disclosure of the crystal structure of phytase have become hot spots for studying by scholars at home and abroad by changing the enzymological properties of phytase and improving the effectiveness of the phytase in feed.

The Yersinia Yersinia derived phytase has the outstanding properties of high stability and enzyme activity under acidic conditions, strong protease resistance, thermal stability and the like. Therefore, the phytase derived from Yersinia has a great potential for use in the feed industry, and the commercial production of the phytase derived from Yersinia is currently carried out by BASF corporation in Germany. The phytase YiAPPA derived from Yersinia intermedia is the phytase with the highest activity known at present, the enzyme activity of the phytase YiAPPA is as high as 3960U/mg (37 ℃, pH 4.5), and is 40 times of the enzyme activity of Aspergillus niger PhyA which is the most widely applied phytase at present. The enzymatic properties of YiAPPA make it have great potential for use in the feed industry, but the enzymatic properties of YiAPPA (especially thermostability and protease resistance) are difficult to fully meet the requirements of the feed industry. Taking the thermal stability of yiAPPA as an example, the feeding enzyme generally needs to be subjected to a short high temperature of 75-93 ℃ in the feed pelleting process, and the half-life of yiAPPA at 80 ℃ is only 15min, so that the requirement of the feed industry is difficult to achieve. The invention aims to carry out molecular modification on phytase YiAPPA so as to obtain the phytase mutant with improved heat stability and protease resistance.

Disclosure of Invention

In order to solve the problem that the stability (such as thermal stability and protease resistance) of the phytase YIAPPA can not completely meet the requirements of the feed industry, the invention provides a phytase mutant with improved thermal stability and protease resistance, which is characterized in that the amino acid sequence of the phytase mutant is shown as SEQ ID NO: 1 is shown.

The present invention provides a gene encoding the phytase mutant with improved thermostability and protease resistance according to claim 1; the nucleotide sequence of the gene is shown as SEQ ID NO: 2, respectively.

The invention also provides a vector capable of expressing and producing the phytase mutant YIAPPA-A108C/S322C with improved heat stability and protease resistance.

The invention also provides a genetically engineered bacterium capable of expressing and producing the phytase mutant YIAPPA-A108C/S322C with improved heat stability and protease resistance.

The invention also provides a preparation method of the phytase mutant YIAPPA-A108C/S322C with improved heat stability and protease resistance, which is characterized by comprising the following specific steps:

1) according to the gene sequence of the phytase YiAPPA of Yersinia intermedia, the gene sequence is shown as SEQ ID NO: 4, synthesizing an optimized gene by adopting a chemical total synthesis method, cloning the optimized gene into a recombinant plasmid pSTOP1622, and constructing the recombinant plasmid pSTOP 1622-yiappah;

2) replacing a 322 th base G with a base T, a 323 rd base C with a base G, a 324 th base G with a base C, a 965 th base C with a base G and a 966 th base A with a base C in a phytase YIAPPA gene sequence by adopting a site-directed mutagenesis method to construct an expression vector pSTOP1622-yiappahA108C/S322C of a mutant YIAPPA-A108C/S322C;

3) transforming the expression vector pSTOP1622-yiappahA108C/S322C into Bacillus subtilis WB600 to obtain Bacillus subtilis genetic engineering bacteria, and performing induced expression to obtain the phytase mutant YIAPPA-A108C/S322C.

The phytase mutant YIAPPA-A108C/S322C is applied to feed additives.

The invention carries out site-directed mutagenesis on amino acid residues which can possibly form disulfide bonds in phytase YiAPPA, introduces new disulfide bonds into the YiAPPA and constructs the following mutants: YIAPPA-S49C/W68C, YIAPPA-V70C/Y74C, YIAPPA-T106C/V109C, YIAPPA-A108C/S322C, YIAPPA-P150C/A157C, YIAPPA-H153C/D156C, YIAPPA-Q311C/A314C, and YIAPPA-L350C/Q353C. By comparing the phytase activity and the thermal stability of each mutant, the phytase mutant YIAPPA-A108C/S322C with remarkably improved thermal stability and basically unchanged activity is screened.

The invention has the advantages that: the half-life of the phytase mutant YIAPPA-A108C/S322C provided by the invention at 85 ℃ is improved to 15min from 6min of a control (before mutation), and is improved by 1.5 times, and the pepsin resistance and the trypsin resistance of the YIAPPA-A108C/S322C are respectively improved by 16% and 12%. The phytase mutant YIAPPA-A108C/S322C has obviously improved thermal stability and protease resistance, and has good application prospect in the field of animal feed additives.

Drawings

FIG. 1 shows the optimum reaction pH for phytases YIAPPA and YIAPPA-A108C/S322C;

FIG. 2 is the pH stability of phytases YIAPPA and YIAPPA-A108C/S322C;

FIG. 3 shows the optimal reaction temperatures for phytases YIAPPA and YIAPPA-A108C/S322C;

FIG. 4 shows the thermostability of phytases YIAPPA and YIAPPA-A108C/S322C at 85 ℃;

FIG. 5 shows the protease resistance of phytases YIAPPA and YIAPPA-A108C/S322C.

Detailed Description

A phytase mutant with improved thermostability and protease resistance of the present invention, a method for preparing the same and use thereof are further described in detail with reference to the following examples.

The experimental conditions are as follows:

1. bacterial strains and vectors

Escherichia coli JM109 (stored in the laboratory), Bacillus subtilis WB600 (stored in the laboratory), and Bacillus subtilis expression vector pSTOP1622 (available from MoBiTec).

2. Enzymes and other biochemical reagents

KOD DNA polymerase and KOD-Plus-neo DNA polymerase were obtained from Toyobo Co., DNA restriction enzyme and T4 DNA ligase were obtained from Fermentase Co., DNA gel recovery kit and plasmid extraction kit E.Z.N.A. were obtained from Omega Bio-tek Co., QuickMutation^TMGene site-directed mutagenesis kit is purchased from Shanghai Binyan biotechnology, Inc., chemical Sepharose^TMFast Flow was purchased from GE Healthcare, USA, Bradford method protein concentration determination kit was purchased from Shanghai Biotechnology, Inc., gene synthesis was completed by Shanghai Bo Probiotics technology, Inc., polymerase chain reaction was introducedThe synthesis and sequencing of the compound are completed by Shanghai biological engineering Co., Ltd, and other chemical reagents are made in China or imported for analytical purification.

3. Culture medium

LB medium (g/L): tryptone 10, yeast extract 5, NaCl 10, pH 7.0. The screening medium was LB medium containing 50. mu.g/mL ampicillin.

The molecular cloning and protein detection techniques used in the present invention are conventional in the art. The techniques not described in detail in the following examples were performed in accordance with the relevant portions of the following experimental manuals. Green M R, Sambrook J.molecular cloning: a Laboratory Manual [ M ]. New York: Cold Spring Harbor Laboratory Press, 2012.

Example 1: construction and screening of phytase mutants

1) Synthesis of Gene yiappa

According to the gene ID 58046988 of the phytase YIAPPA from Yersinia intermedia, the gene sequence is obtained by searching, and is shown as SEQ ID NO: 4, the gene is handed to Shanghai Bo probiotic science and technology Limited company for the whole gene synthesis of the phytase YiAPPA.

2) Construction of expression vector pSTOP1622-yiappah

PCR primers P1 and P2 (Table 1) were designed based on the gene sequence of YIAPPA, and PCR amplification was carried out using the synthetic gene YiAPPA as a template and P1 and P2 as primers. The PCR amplification conditions were: 5min at 98 ℃; 20sec at 98 ℃, 40sec at 60 ℃ and 2min at 74 ℃ for 30 cycles; 74 ℃ for 10 min. The amplified product was digested with Spe I and Sac I, ligated to vector pSTOP1622, and recombinant plasmid pSTOP1622-yiappah was constructed.

TABLE 1 primers used for construction of recombinant plasmids

Note: the underlined parts are restriction enzyme cleavage sites, and the boxed parts are mutant bases.

3) Construction of expression vector for Phytase mutant

The three-dimensional modeling of the protein molecular structure of phytase YIAPPA was carried out using the tertiary structure (PDB ID: 4ARV) of phytase YkAPPA derived from Yersinia kristesenii as a template and Swiss-Model (http:// swissmodel. expasy. org), to obtain the protein molecular structure of YIAPPA. Inputting the protein molecular structure information (simulated tertiary structure information) of the YIAPPA into online software Disulfide by Design 2.0(http:// cptpweb. cpt. wayne. edu/DbD2/), predicting amino acid residue pairs which can possibly form new Disulfide bonds in the YIAPPA, and selecting the amino acid residue pairs positioned in the loop structure on the surface of the YIAPPA molecule to perform site-directed mutagenesis so as to obtain the corresponding phytase mutant. The selected pairs of amino acid residues are as follows: S49-W68, V70-Y74, T106-V109, A108-S322, P150-A157, H153-D156, Q311-A314, L350-Q353; the corresponding phytase mutants were: YIAPPA-S49C/W68C, YIAPPA-V70C/Y74C, YIAPPA-T106C/V109C, YIAPPA-A108C/S322C, YIAPPA-P150C/A157C, YIAPPA-H153C/D156C, YIAPPA-Q311C/A314C, and YIAPPA-L350C/Q353C.

According to QuickMution^TMThe instructions of the gene site-directed mutagenesis kit, primers were designed by combining the base sequence of gene yiappa and the amino acid site to be mutated, as shown in table 1. The specific steps for constructing the phytase mutant are as follows: (1) taking the construction of mutant YIAPPA-S49C/W68C as an example, recombinant plasmid pSTOP1622-yiappah is taken as a template, primers S49C-F and S49C-R are adopted, and the QuickMutation is carried out^TMThe instructions of the gene site-directed mutagenesis kit are used for PCR amplification. After the amplification product is treated by Dpn I enzyme, Escherichia coli JM109 is transformed, transformants are screened by ampicillin resistant plates, and recombinant plasmids are extracted. The recombinant plasmid was sent to Shanghai Bioengineering Co., Ltd for sequencing and was compared with the corresponding gene sequence to confirm that the recombinant plasmid pSTOP1622-yiappahS49C was successfully constructed. Then, the recombinant plasmid pSTOP1622-yiappahS49C was used as a template, and primers W68C-F and W68C-R were used, according to Quickmutation^TMThe instructions of the gene site-directed mutagenesis kit are used for PCR amplification. After the amplification product is treated by Dpn I enzyme, Escherichia coli JM109 is transformed, transformants are screened by ampicillin resistant plates, and recombinant plasmids are extracted. Sending the recombinant plasmid to Shanghai biological engineering stockThe sequence was sequenced by the company Limited and aligned with the corresponding gene sequence to confirm that the recombinant plasmid pSTOP1622-yiappahS49C/W68C was successfully constructed. The construction methods of the site-directed mutants YIAPPA-A108C/S322C and YIAPPA-P150C/A157C were performed with reference to the construction method of the mutant YIAPPA-S49C/W68C. (2) Taking the construction of mutant YIAPPA-V70C/Y74C as an example, recombinant plasmid pSTOP1622-yiappah is taken as a template, primers V70C/Y74C-F and V70C/Y74C-R are adopted, and the Quickmutation is carried out^TMThe instructions of the gene site-directed mutagenesis kit are used for PCR amplification. After the amplification product is treated by Dpn I enzyme, Escherichia coli JM109 is transformed, transformants are screened by ampicillin resistant plates, and recombinant plasmids are extracted. The recombinant plasmid is sent to Shanghai biological engineering Co., Ltd for sequencing, and is compared with a corresponding gene sequence, and the success of construction of the recombinant plasmid pSTOP1622-yiappahV70C/Y74C is confirmed. Site-directed mutants YIAPPA-T106C/V109C, YIAPPA-H153C/D156C, YIAPPA-Q311C/A314C and YIAPPA-L350C/Q353C were constructed by reference to the construction method of mutant YIAPPA-V70C/Y74C.

4) Expression and purification of phytase YIAPPA and its mutant in bacillus subtilis

And respectively transforming the successfully constructed expression vectors of the YIAPPA and the mutants thereof into Bacillus subtilis WB600 competent cells, and simultaneously transforming pSTOP1622 as a negative control Contr.

The seed culture conditions of the recombinant bacillus subtilis are as follows: LB liquid culture medium is adopted, and a 250mL triangular flask is used for culture, wherein the liquid loading of the culture medium is 25mL, the culture temperature is 37 ℃, the rotation speed is 180rpm, and the culture time is 24 h. The fermentation culture conditions of the recombinant bacillus subtilis are as follows: LB liquid medium was used, and culture was carried out in a 250mL Erlenmeyer flask, in which the medium was 25mL, the inoculum size was 1%, the culture temperature was 37 ℃ and the rotation speed was 180 rpm. When cultured to the OD of the cells_600nmWhen the concentration reaches 1, xylose with the final concentration of 0.5% is added, and the induction time is 30 h.

By using Ni²⁺Purifying the target protein in the fermentation supernatant by an affinity chromatography column, and eluting with 250mmol/L imidazole elution buffer solution to obtain the purified recombinant phytic acidAn enzyme. The purity of the recombinant phytase was checked by SDS-PAGE and the concentration of the recombinant phytase was determined by Bradford method.

5) Enzyme activity assay of recombinant phytase

And (3) enzyme activity determination of the recombinant phytase: 250. mu.L of the enzyme solution was put into a centrifuge tube, and 750. mu.L of 0.25mol/L sodium acetate buffer (pH 4.5) was added thereto and mixed well. 2mL of 1.5mmol/L sodium phytate solution (0.25mol/L sodium acetate buffer, pH 4.5) was added to the tube of the experimental group, 2mL of color/end point mixture (ammonium molybdate/ammonium vanadate/nitric acid) was added to the control group, and the mixture was shaken well. After 30min reaction at 37 ℃, 2mL of the color/end point mixture was immediately added to the experimental group, 2mL of 1.5mmol/L sodium phytate solution was added to the control group, and the mixture was mixed well and the light absorption value was measured at 415 nm. The conversion formula of the inorganic phosphorus content in the reaction liquid and the light absorption value of the reaction liquid at 415nm is as follows: inorganic phosphorus (mmol/L) ═ 26.5510 XOD_415nm+0.3113. Phytase activity units (U) are defined as: the phytase amount required for releasing 1 mu mol/L inorganic phosphorus from 1.5mmol/L sodium phytate solution per minute at 37 ℃ and pH 4.5 is one enzyme activity unit (U). The results of the enzyme activity assay of the recombinant phytase are shown in Table 2.

TABLE 2 determination of enzyme Activity of recombinant Phytase

6) Thermal stability of recombinant phytase at 80 ℃

Thermostability of recombinant phytase at 80 ℃: 250 μ L of enzyme solution was put into a centrifuge tube, and 750 μ L of 0.25mol/L sodium acetate buffer (pH 4.5) was added thereto and mixed well. And (3) after the enzyme solution is subjected to heat preservation at 80 ℃ for 15min, determining the enzyme activity of the sample according to a method of determining the enzyme activity of the recombinant phytase, and calculating the residual enzyme activity of the sample after heat preservation treatment by taking the enzyme activity of the sample without heat preservation treatment as 100%. The results of the determination of the residual enzyme activity of the recombinant phytase after incubation for 15min at 80 ℃ are shown in Table 3. After the recombinant phytase is insulated for 15min at 80 ℃, the residual enzyme activity of the phytase mutants, namely YIAPPA-S49C/W68C, YIAPPA-T106C/V109C and YIAPPA-H153C/D156C is slightly lower than that of YIAPPA; the residual enzyme activity of the phytase mutants YIAPPA-V70C/Y74C, YIAPPA-P150C/A157C, YIAPPA-Q311C/A314C and YIAPPA-L350C/Q353C is slightly higher than that of YIAPPA; the residual enzyme activity of the phytase mutant YIAPPA-A108C/S322C is obviously higher than that of the YIAPPA.

TABLE 3 residual enzyme activity of recombinant phytase after incubation at 80 ℃ for 15min

Example 2: verification of enzymological properties of phytase mutant YIAPPA-A108C/S322C

Determination of the optimum reaction pH of the recombinant phytase: and (3) determining the enzyme activity of the sample by using the enzyme solution under the conditions of different pH values (1.0-8.0) according to a method of determining the enzyme activity of the recombinant phytase, and mapping the pH value by using the relative enzyme activity to determine the optimum reaction pH value. The buffers used were as follows: 0.25mol/L glycine-hydrochloric acid buffer solution, pH 1.0-3.5; 0.25mol/L sodium acetate-acetic acid buffer solution, pH 3.5-6.0; 0.25 mol/LTris-hydrochloric acid buffer, pH 6.0-8.5. The results of pH determination of the optimal reaction of the recombinant phytases YIAPPA and YIAPPA-A108C/S322C are shown in FIG. 1.

pH stability of recombinant phytase: diluting the enzyme solution with buffer solutions with different pH values (1.0-12.0), treating the enzyme solution for 2h at 37 ℃ under different pH conditions, then diluting the enzyme solution with the buffer solution with the optimal pH value, and measuring the enzyme activity of the sample according to a method of measuring the enzyme activity of the recombinant phytase. And calculating the residual enzyme activity of the treated sample by taking the enzyme activity of the untreated sample as 100%, and plotting the residual enzyme activity against the pH to determine the pH stability of the treated sample. The buffers used were as follows: 0.25mol/L glycine-hydrochloric acid buffer solution, pH 1.0-3.5; 0.25mol/L sodium acetate-acetic acid buffer solution, pH 3.5-6.0; 0.25mol/L Tris-hydrochloric acid buffer solution, pH 6.0-8.5; 0.25mol/L glycine-sodium hydroxide buffer solution, pH 8.5-12.0. The results of the pH stability measurements of the recombinant phytases YIAPPA and YIAPPA-A108C/S322C are shown in FIG. 2.

Determination of the optimal reaction temperature of the recombinant phytase: and (3) measuring the enzyme activity of the sample according to a method of measuring the enzyme activity of the recombinant phytase, reacting for 30min at 30-90 ℃, measuring the enzyme activity of the sample under different temperature conditions, and mapping the relative enzyme activity to the temperature to determine the optimal reaction temperature. The results of the determination of the optimal reaction temperature for the recombinant phytases YIAPPA and YIAPPA-A108C/S322C are shown in FIG. 3.

Determination of the thermostability of the recombinant phytase: and (3) preserving the temperature of the enzyme solution at 85 ℃, taking out a part of samples in a time-sharing gradient manner, and determining the enzyme activity of the samples according to a method of determining the enzyme activity of the recombinant phytase. The enzyme activity of the untreated enzyme solution was defined as 100%, and the thermal stability of the enzyme was evaluated by plotting the percentage of the remaining enzyme activity against time. The results of the thermal stability assay of the recombinant phytases YIAPPA and YIAPPA-A108C/S322C are shown in FIG. 4.

Protease resistance of phytase: 0.1mg/mL of pepsin and trypsin were prepared with 0.25mol/L Gly-HCl buffer (pH 2.0) and 0.25mol/L Tris-HCl buffer (pH 7.0), respectively. Respectively adding pepsin and trypsin into phytase according to the mass ratio of 1:10 of the protease to the phytase, respectively preserving the heat at 37 ℃ for 2 hours, adding a protease inhibitor to stop the reaction, then diluting a sample treated by the protease by 100 times by using an optimal pH buffer solution (0.25mol/L sodium acetate buffer solution, pH 4.5), determining the enzyme activity of the sample according to a method of 'determination of enzyme activity of recombinant phytase', and calculating the residual enzyme activity of the treated sample by taking the enzyme activity of an untreated enzyme solution as 100%. The results of the protease resistance assay for the recombinant phytases YIAPPA and YIAPPA-A108C/S322C are shown in FIG. 5.

The results of the above enzymatic property measurements show that: the optimal reaction temperature of the phytase mutant YIAPPA-A108C/S322C is 55 ℃, the optimal reaction pH is 4.5, and the relative enzyme activity is more than 80% in the pH range of 1.0-10.0. The optimal reaction temperature, the optimal reaction pH and the pH stability of the phytase mutant YIAPPA-A108C/S322C are basically consistent with those of YIAPPA. In addition, the phytase mutant YIAPPA-A108C/S322C has obviously improved thermal stability and protease resistance. Wherein the half-life of YIAPPA-A108C/S322C at 85 ℃ is improved to 15min from 6min of a control (before mutation) and is improved by 1.5 times, and the pepsin resistance and the trypsin resistance of YIAPPA-A108C/S322C are respectively improved by 16 percent and 12 percent.

Example 3: granulation application experiment

The recombinant phytases YIAPPA and YIAPPA-A108C/S322C are respectively prepared into 5000U/g enzyme powder. Each enzyme sample is added into a compound feed for medium and large pigs (50 kg-marketing) according to the addition amount of 360g/t, and then the compound feed is produced into granulated feed through a granulator. The formula of the compound feed for the middle and large pigs (50 kg-slaughter) is as follows: 48% of corn, 20% of wheat, 17% of wheat middling, 12% of soybean meal, 1.1% of stone powder, 0.6% of calcium hydrophosphate, 0.3% of salt and 1% of premix. The granulation processing conditions are that the granulation temperature is 85 ℃, the humidity is 16-18%, the steam pressure is 0.28MPa, the hardening and tempering time is 30 seconds, and the annular membrane compression ratio is 7: 1. According to the conditions of the granulator, the granulated feed at the unstable stage of the granulation and the granulated feed possibly mixed with other test materials due to the continuous production are discarded, the feed with the specification of 40 kg/bag is packaged into 3 bags, a sample with the weight more than or equal to 600g is taken, and 8 samples are continuously taken. After the 8 samples are mixed evenly, the mixture is intermittently crushed by a low-temperature crusher until the mixture completely passes through a standard sieve of 0.45mm, and then the mixture is mixed evenly again. And measuring the phytase activity of the sample after the granulation processing, and calculating the residual enzyme activity of the sample after the granulation processing by taking the phytase activity of the sample before the granulation processing as 100%. The results of the residual phytase activity measurements of the samples after the granulation process are shown in table 4.

TABLE 4 residual enzyme activity after pelleting experiment

According to the pelletization application experimental result, the residual enzyme activity of the phytase mutant YIAPPA-A108C/S322C after pelletization processing at 85 ℃ is about 98.21 percent and is obviously higher than that of the phytase YIAPPA. Compared with phytase YIAPPA, the mutant YIAPPA-A108C/S322C is more suitable for the feed additive industry.

The above description is only for the preferred embodiment of the present invention, and not intended to limit the present invention, and any changes or substitutions that can be easily conceived by one skilled in the art within the technical scope of the present invention are also within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.

Sequence listing

<110> institute of microbiology of academy of sciences of Jiangxi province

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ctgcaacaga tcgagactgc cctcgtcctt cagcgtgatg ctcaggggca aacattgcca 960

ttatgccctc aaaccaaaat tctgttcctc gggggacatg atacaaacat cgccaatatt 1020

gctggaatgt tgggggctaa ctggcaatta ccacagcagc ccgataatac cccacctggg 1080

gggggattgg tcttcgagct atggcaaaac ccagataatc atcaacgtta tgtcgcggtg 1140

aaaatgttct atcaaacaat gggccaattg cgaaatgctg agaaactaga cctgaaaaac 1200

aatccggctg gtagggtccc tgttgcaata gacggttgtg aaaatagtgg tgatgacaaa 1260

ctttgtcagc ttgatacctt ccaaaagaaa gtagctcagg cgattgaacc tgcttgccat 1320

atttaa 1326

<210> 3

<211> 441

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 3

Met Thr Ile Thr Val Asp Ser Leu Arg Leu Ser Val Leu Thr Leu Ile

1 5 10 15

Leu Asn Ser Tyr Ala Ile Ser Ala Ala Pro Val Ala Ile Gln Pro Thr

20 25 30

Gly Tyr Thr Leu Glu Arg Val Val Ile Leu Ser Arg His Gly Val Arg

35 40 45

Ser Pro Thr Lys Gln Thr Gln Leu Met Asn Asp Val Thr Pro Asp Thr

50 55 60

Trp Pro Gln Trp Pro Val Ala Ala Gly Tyr Leu Thr Pro Arg Gly Ala

65 70 75 80

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

85 90 95

Gln Gly Leu Leu Ala Ala Gly Cys Pro Thr Asp Ala Val Ile Tyr Ala

100 105 110

Gln Ala Asp Val Asp Gln Arg Thr Arg Leu Thr Gly Gln Ala Phe Leu

115 120 125

Asp Gly Ile Ala Pro Gly Cys Gly Leu Lys Val His Tyr Gln Ala Asp

130 135 140

Leu Lys Lys Val Asp Pro Leu Phe His Pro Val Asp Ala Gly Val Cys

145 150 155 160

Lys Leu Asp Ser Thr Gln Thr His Lys Ala Val Glu Glu Arg Leu Gly

165 170 175

Gly Pro Leu Ser Glu Leu Ser Lys Arg Tyr Ala Lys Pro Phe Ala Gln

180 185 190

Met Gly Glu Ile Leu Asn Phe Ala Ala Ser Pro Tyr Cys Lys Ser Leu

195 200 205

Gln Gln Gln Gly Lys Thr Cys Asp Phe Ala Asn Phe Ala Ala Asn Lys

210 215 220

Ile Thr Val Asn Lys Pro Gly Thr Lys Val Ser Leu Ser Gly Pro Leu

225 230 235 240

Ala Leu Ser Ser Thr Leu Gly Glu Ile Phe Leu Leu Gln Asn Ser Gln

245 250 255

Ala Met Pro Asp Val Ala Trp His Arg Leu Thr Gly Glu Asp Asn Trp

260 265 270

Ile Ser Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Met Ala Lys

275 280 285

Thr Pro Tyr Ile Ala Arg His Lys Gly Thr Pro Leu Leu Gln Gln Ile

290 295 300

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

305 310 315 320

Leu Ser Pro Gln Thr Lys Ile Leu Phe Leu Gly Gly His Asp Thr Asn

325 330 335

Ile Ala Asn Ile Ala Gly Met Leu Gly Ala Asn Trp Gln Leu Pro Gln

340 345 350

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

355 360 365

Gln Asn Pro Asp Asn His Gln Arg Tyr Val Ala Val Lys Met Phe Tyr

370 375 380

Gln Thr Met Gly Gln Leu Arg Asn Ala Glu Lys Leu Asp Leu Lys Asn

385 390 395 400

Asn Pro Ala Gly Arg Val Pro Val Ala Ile Asp Gly Cys Glu Asn Ser

405 410 415

Gly Asp Asp Lys Leu Cys Gln Leu Asp Thr Phe Gln Lys Lys Val Ala

420 425 430

Gln Ala Ile Glu Pro Ala Cys His Ile

435 440

<210> 4

<211> 1326

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 4

atgacaataa cagtagatag tctgcgatta tccgtactga ccttgatact caatagttat 60

gcgattagtg ccgcgccggt tgccatacaa cccacgggct atacattgga gcgagtggtt 120

attttgagcc gccatggtgt tcgctcgcca accaaacaaa cacagttaat gaatgatgtt 180

acccctgaca cgtggccgca atggccggtc gccgcaggat acttaacccc ccgaggtgca 240

caattagtga cattgatggg cggattctat ggtgattact tccgtagcca agggttactc 300

gcagcagggt gcccaactga cgcggttatt tatgctcagg ccgatgttga tcaacgaacg 360

cgtttaacgg ggcaggcatt ccttgatgga atagcaccgg ggtgtggact gaaagtacat 420

tatcaggctg atttgaaaaa agtggatccg ctgtttcatc ccgtcgacgc gggggtgtgt 480

aagttagatt cgacacaaac ccataaggct gttgaggagc gactaggtgg gccattaagt 540

gaactgagca aacgctatgc taagcccttt gcccagatgg gtgagattct gaattttgcg 600

gcatctcctt actgtaaatc actgcaacag caagggaaaa cctgtgattt tgccaacttt 660

gcagcgaata agatcacggt gaacaagccg gggacaaaag tctcgctcag cggaccactg 720

gcactgtcat caaccttagg tgagatcttt ttgctacaaa attcacaagc gatgcctgat 780

gttgcctggc atcggttaac gggagaagat aattggatct cgttattatc gttgcacaat 840

gcgcaatttg atttaatggc aaaaacacct tatatcgctc gtcataaggg cacaccgttg 900

ctgcaacaga tcgagactgc cctcgtcctt cagcgtgatg ctcaggggca aacattgcca 960

ttatcacctc aaaccaaaat tctgttcctc gggggacatg atacaaacat cgccaatatt 1020

gctggaatgt tgggggctaa ctggcaatta ccacagcagc ccgataatac cccacctggg 1080

gggggattgg tcttcgagct atggcaaaac ccagataatc atcaacgtta tgtcgcggtg 1140

aaaatgttct atcaaacaat gggccaattg cgaaatgctg agaaactaga cctgaaaaac 1200

aatccggctg gtagggtccc tgttgcaata gacggttgtg aaaatagtgg tgatgacaaa 1260

ctttgtcagc ttgatacctt ccaaaagaaa gtagctcagg cgattgaacc tgcttgccat 1320

atttaa 1326

Claims (7)

1. A phytase mutant with improved heat stability and protease resistance is characterized in that the amino acid sequence is shown as SEQ ID NO: 1 is shown.

2. A gene encoding the phytase mutant with increased thermostability and protease resistance according to claim 1.

3. The gene of claim 2, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO: 2, respectively.

4. A vector expressing a phytase mutant producing the increased thermostability and protease resistance of claim 1.

5. A genetically engineered bacterium expressing a phytase mutant producing the phytase of claim 1 with improved thermostability and protease resistance.

6. The method for preparing phytase mutant with improved thermostability and protease resistance according to claim 1, characterized in that the specific steps are as follows:

1) according to the gene sequence of the phytase YiAPPA of Yersinia intermedia, the gene sequence is shown as SEQ ID NO: 4, synthesizing an optimized gene by adopting a chemical total synthesis method, cloning the optimized gene into a recombinant plasmid pSTOP1622, and constructing the recombinant plasmid pSTOP 1622-yiappah;

2) replacing a 322 th base G with a base T, a 323 rd base C with a base G, a 324 th base G with a base C, a 965 th base C with a base G and a 966 th base A with a base C in a phytase YIAPPA gene sequence by adopting a site-directed mutagenesis method to construct an expression vector pSTOP1622-yiappahA108C/S322C of a mutant YIAPPA-A108C/S322C;

3) transforming the expression vector pSTOP1622-yiappahA108C/S322C into Bacillus subtilis WB600 to obtain Bacillus subtilis genetic engineering bacteria, and performing induced expression to obtain the phytase mutant YIAPPA-A108C/S322C.

7. The use of the phytase mutant YiAPPA-a108C/S322C with improved thermostability and protease resistance according to claim 1 in a feed additive.

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