CN121022803B - A thermostable acidic protease mutant, PepA4, and its applications. - Google Patents
A thermostable acidic protease mutant, PepA4, and its applications.Info
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Abstract
The invention discloses an acidic protease mutant PepA4 with improved heat resistance and application thereof, and relates to the technical fields of genetic engineering and enzyme engineering. The amino acid sequence of the acid protease mutant PepA4 is obtained by performing D67A, E103D, E304K, S368L point mutation on the amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence of the acid protease mutant PepA4 is shown in SEQ ID NO. 2. The high-temperature resistant acid protease can still maintain more than 80% of enzyme activity at 75 ℃ for 5 min. The acid protease has better acid resistance, and can be widely applied to the industries of food processing, leather processing and feed processing.
Description
Technical Field
The invention relates to the technical fields of genetic engineering and enzyme engineering, in particular to an acidic protease mutant PepA4 with improved heat resistance and application thereof.
Background
Acid proteases (aspartic proteases) are a class of enzymes that catalyze the hydrolysis of proteins under acidic conditions, with the active center containing two aspartic acid residues that are capable of capturing and activating water molecules in the environment, and then the activated water molecules attack the substrate peptide bonds, resulting in cleavage of the substrate peptide chain. Typically, the optimum pH of such proteases is between 2 and 5, i.e.it is stable in an acidic environment, but is prone to loss of activity when the pH in solution is too high.
In recent years, the use of acid proteases has become more and more widespread. The acid protease is added in the word stock, so that the digestion and absorption rate of the nutrient components of the word stock can be improved, the physique of young livestock and poultry is enhanced, the word stock with relatively low energy and protein can be utilized for the adult livestock and poultry, the cost is reduced, and the addition of the acid protease in leather production is beneficial to dehairing and the gloss and softness of leather are maintained, so that the quality of a finished product is improved.
The current commercial acid protease generally has the problems of poor heat stability and narrow pH application range, and severely restricts the application of the acid protease in industry. In order to solve the problem, the method can be optimized from multiple layers, namely, the molecular structure of the enzyme is modified through protein engineering means (such as rational design and directed evolution), the heat resistance and the pH stability of the enzyme are improved, a fermentation process is optimized, high-yield strains are screened, stabilizers such as metal ions and the like are added, the industrial applicability of the enzyme is enhanced by adopting an immobilization technology or chemical modification, and a compound enzyme preparation is developed to widen the application scene. These improvements are expected to promote the wider application of acid proteinase in the fields of food processing (such as cheese production, juice clarification), feed additives and the like.
The invention discloses an acid protease mutant with improved stability, a product and application thereof in China patent CN119552846A, which is a high-temperature resistant acid protease mutant IAIC6M, wherein the mutant can still maintain more than 60% of enzyme activity at 75 ℃ for 3min, and the IAIC can only maintain 20% of enzyme activity at 75 ℃ for 3min, and the acid protease mutant IAIC6M has the following properties of optimal pH value of 3.0, higher enzyme activity under high-temperature conditions, and wide application value in the aspects of industry, food and the like. But its thermal stability is still poor.
The traditional acidic protease fermentation production has the defects of low yield, poor heat resistance, low enzyme activity, high production cost and the like, and the conventional breeding method is used for improving the yield and the heat resistance of the acidic protease. Therefore, it is necessary to provide an acidic protease mutant with high thermostability by genetic engineering techniques.
Disclosure of Invention
The invention aims to provide an acid protease mutant PepA4 with improved heat resistance and a coding gene thereof.
In order to achieve the above object, the present invention has the following technical scheme:
In one aspect, the invention provides an acid protease mutant PepA4, wherein the amino acid sequence of the acid protease mutant PepA4 is obtained by carrying out D67A, E103D, E304K, S368L point mutation on the amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence of the acid protease mutant PepA4 is shown in SEQ ID NO. 2.
SEQ ID NO:1:
APAQIVGRSTFQIDQVASGKVYKNGPMAMMQTYNKYAHVGAVAPAAVVAAAAAAQTGEVSANPEQYDESYLCPVTIGDQTLNLDFDTGSADLWVFSTLTPSSESTGHTLYNPADSGTEKQGYTWNITYGDGSGAAGVVYADKVVVGGVTATSQAVEAATSVSSEFTQDTKNDGLLGLAFSSINTVQPVQQTTFFDTVKDTLAKKLFTADLKKGAAGSYGFGYIDSSKYTGTITYVPVNNENGFWQFTAGGYSIGGGNGTSGSNATTGSIGTSIADTGTTLLLLPSNVVTAYYKQVSGASYNSAQGGYTYPCGATLPDFNVAIGGKTFVVPGTDLNYAPINSAGTTCFGGIQANTGIGFNIFGDIFLKSVYAVFDQTQSSPRLGFAEQS.
SEQ ID NO:2:
APAQIVGRSTFQIDQVASGKVYKNGPMAMMQTYNKYAHVGAVAPAAVVAAAAAAQTGEVSANPEQYAESYLCPVTIGDQTLNLDFDTGSADLWVFSTLTPSSDSTGHTLYNPADSGTEKQGYTWNITYGDGSGAAGVVYADKVVVGGVTATSQAVEAATSVSSEFTQDTKNDGLLGLAFSSINTVQPVQQTTFFDTVKDTLAKKLFTADLKKGAAGSYGFGYIDSSKYTGTITYVPVNNENGFWQFTAGGYSIGGGNGTSGSNATTGSIGTSIADTGTTLLLLPSNVVTAYYKQVSGASYNSAKGGYTYPCGATLPDFNVAIGGKTFVVPGTDLNYAPINSAGTTCFGGIQANTGIGFNIFGDIFLKLVYAVFDQTQSSPRLGFAEQS.
The invention carries out site-directed mutagenesis on the acid protease pepA gene of thermophilic fungi (Bispora sp.) and the amino acid sequence of the acid protease pepA obtained from the thermophilic fungi is SEQ ID NO.1.
The high-temperature resistant acidic protease mutant PepA4 has the characteristics of high enzyme activity, high temperature resistance and the like under acidic and neutral conditions. The high-temperature resistant acidic protease mutant has the optimal pH value of 2.0, is relatively stable within the pH range of 2.0-7.0 (residual enzyme activity is more than 80 percent), and has activity of more than 80 percent after being treated for 5 minutes at 75 ℃.
In yet another aspect, the present invention provides a method for preparing an acid protease mutant PepA4, comprising the steps of:
1) Transforming host cells with the recombinant vector to obtain recombinant strains;
2) Culturing the recombinant strain and inducing the expression of recombinant acid protease;
3) Recovering and purifying the expressed high temperature resistant acid protease PepA4.
Specifically, the preparation method of the recombinant vector in the step 1) comprises the steps of inserting the high-temperature resistant acidic protease gene into a proper restriction enzyme cutting site of an expression vector, so that the nucleotide sequence of the high-temperature resistant acidic protease gene is operably connected with an expression regulatory sequence.
According to some embodiments of the invention, the recombinant vector pPIC9K-pepA4 is prepared by inserting the high temperature resistant acid protease gene of the invention between EcoRI and NotI restriction sites on plasmid pPIC9K, and positioning the nucleotide sequence downstream of and under the control of an AOX1 promoter to obtain pPIC9K-pepA4.
Specifically, the host cell in the step 1) is selected from any one of pichia pastoris cells, beer yeast cells or polytype yeast cells.
Further, the host cell in step 1) is a pichia pastoris cell.
According to some embodiments of the invention, step 1) transforming a recombinant yeast expression plasmid into Pichia pastoris cell (Pichia pastoris) GS115 to obtain recombinant strain GS115/pepA4.
Specifically, the recombinant strain is cultured in step 2) under the condition of being inoculated into BMGY culture solution and shake-cultured at 30 ℃ 200 rpm for 48 hours.
In yet another aspect, the invention provides a gene encoding the acid protease mutant PepA 4.
Further, the gene is pepA4 gene, and the sequence of the pepA4 gene is shown in SEQ ID NO. 3.
SEQ ID NO:3:
GCGCCTGCTCAGATTGTTGGGCGATCCACATTTCAGATCGACCAAGTCGCGTCCGGGAAGGTATACAAGAATGGCCCAATGGCAATGATGCAGACGTACAACAAATATGCACATGTGGGCGCCGTCGCGCCGGCTGCGGTAGTCGCAGCCGCAGCGGCTGCCCAAACGGGGGAAGTCTCGGCGAATCCTGAACAGTACGCTGAGTCATATTTGTGCCCCGTGACCATCGGGGATCAAACACTGAATCTCGATTTCGACACTGGAAGTGCAGATTTGTGGGTTTTCTCTACCCTAACTCCTTCCTCGGACTCTACAGGCCACACGCTATACAACCCTGCAGACTCAGGTACTGAGAAGCAAGGCTATACGTGGAACATTACCTACGGGGATGGCTCGGGCGCCGCTGGCGTGGTATATGCGGACAAGGTCGTGGTAGGGGGCGTCACGGCAACCTCGCAAGCAGTTGAGGCTGCAACATCCGTCTCTAGTGAATTTACTCAAGATACTAAGAATGACGGACTTCTAGGACTCGCCTTTAGCAGCATAAACACAGTTCAGCCCGTACAACAAACGACTTTTTTTGACACCGTGAAAGACACGCTCGCTAAAAAACTTTTTACCGCTGACTTAAAGAAAGGAGCCGCCGGTAGCTATGGATTTGGATACATAGATAGTTCCAAGTACACCGGTACAATCACATACGTACCCGTAAATAACGAGAACGGGTTCTGGCAATTCACTGCGGGGGGTTATAGCATTGGAGGTGGCAATGGGACCTCTGGAAGCAACGCTACGACGGGGTCAATTGGTACATCAATAGCAGACACGGGAACTACCCTTTTACTGTTGCCAAGTAATGTGGTTACCGCTTACTATAAACAGGTGAGCGGCGCTTCTTATAATTCCGCGAAGGGAGGTTATACGTACCCGTGTGGTGCCACCCTACCGGATTTCAACGTAGCTATAGGGGGAAAGACTTTTGTTGTTCCAGGGACAGATTTAAACTACGCACCAATAAACTCAGCCGGAACTACTTGCTTCGGTGGTATCCAGGCGAATACAGGCATCGGCTTTAATATCTTCGGTGACATTTTTCTGAAATTAGTTTATGCCGTCTTCGATCAGACACAAAGTTCGCCCCGCTTAGGTTTCGCGGAACAGAGT.
The invention synthesizes the high temperature resistant acid protease mutant gene pepA4 by a gene synthesis method, and the DNA complete sequence analysis result shows that the full length 1164bp of the acid protease pepA4 structural gene pepA 4.
In yet another aspect, the present invention provides a recombinant vector comprising the acid protease mutant PepA4 described above or the gene described above.
Specifically, the vector is selected from pET-28a, pPIC9K or pPICZα.
Further, the vector is pPIC9K.
According to some embodiments of the invention, the recombinant vector is pPIC9K-pepA4.
According to some embodiments of the invention, the recombinant vector is prepared by inserting the high temperature resistant acid protease gene of the invention between appropriate restriction sites of an expression vector, so that the nucleotide sequence of the gene is operably linked with an expression regulatory sequence.
According to some embodiments of the invention, the recombinant vector pPIC9K-pepA4 is prepared by inserting the high temperature resistant acid protease gene of the invention between EcoRI and NotI restriction sites on plasmid pPIC9K, and positioning the nucleotide sequence downstream of and under the control of an AOX1 promoter to obtain pPIC9K-pepA4.
In yet another aspect, the present invention provides a recombinant strain comprising the above gene.
Specifically, the strain is selected from escherichia coli, saccharomycetes, bacillus or lactobacillus.
Further, the recombinant strain is GS115/pepA4.
In a further aspect, the invention provides the use of the acid protease PepA4, gene, recombinant vector, recombinant strain described above in food processing, leather processing or feed processing.
The beneficial effects of the invention are as follows:
The invention provides high-temperature resistant acid protease with excellent properties. The optimal pH of the high-temperature resistant acid protease is 2.0, the high-temperature resistant acid protease is stable within the pH range of 2.0-8.0, and the high-temperature resistant acid protease can still maintain 80% of enzyme activity at 75 ℃ for 5 min. Acid protease producing bacteria are generally fungi such as mold and yeast, and there are few strains producing acid protease among bacteria. The acid protease has better acid resistance, so that the acid protease is widely applied to the industries of food processing, leather processing and feed processing.
Drawings
FIG. 1 is the optimum pH for recombinant acid protease mutants.
FIG. 2 is a graph showing the pH stability of recombinant acid protease mutants.
FIG. 3 is the thermostability of recombinant acid protease mutants.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purpose and the effect of the present invention easy to understand, the present invention will be further elucidated with reference to the specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all of them. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. In the following examples, unless otherwise specified, the methods of operation used were conventional, the equipment used was conventional, and the materials used in the examples were the same.
Test materials and reagents
1. The acid protease mutant gene pepA4 is synthesized by the biological technology limited company of the Boxing family Beijing Rui, and the pichia pastoris expression vector pPIC9K and the strain GS115 are purchased from Invitrogen company.
2. Enzymes and other biochemical reagents endonucleases were purchased from TaKaRa, and ligases were purchased from Invitrogen. Proteases were purchased from Sigma, the others being domestic reagents (all available from general Biochemical reagents).
3. Culture medium:
(1) Yeast medium YPD 1% peptone, 0.5% yeast extract, 1% glucose, 2% agar, pH 7.0.
(2) Coli culture medium LB, 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.
(3) BMGY medium 1% yeast extract, 2% peptone, 1.34% YNB,0.00004% Biotin,1% glycerol (V/V).
(4) BMMY culture medium divided by 0.5% methanol to replace glycerol, and the rest components are the same as BMGY.
The molecular biological experiments described in the examples below, which are not specifically described, were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) J.Sam Brookfield, or according to the kit and product specifications.
EXAMPLE 1 Synthesis of the acid protease mutant encoding Gene pepA4 from thermophilic fungi (Bispora sp.)
The invention takes acid proteinase pepA gene from thermophilic fungi as reference, the sequence is subjected to the following mutation (D67A, E103D, E304K, S368L), ecoRI and NotI restriction enzyme sites are respectively added at the 5 'end and the 3' end of the mutated sequence, and the sequence is sent to Beijing Rui Boxing family biotechnology company for artificial synthesis. The amino acid sequence of the artificially synthesized acid protease mutant is shown as SEQ ID NO.2, and the nucleotide sequence of the artificially synthesized acid protease mutant is shown as SEQ ID NO. 3.
SEQ ID NO:2:
APAQIVGRSTFQIDQVASGKVYKNGPMAMMQTYNKYAHVGAVAPAAVVAAAAAAQTGEVSANPEQYAESYLCPVTIGDQTLNLDFDTGSADLWVFSTLTPSSDSTGHTLYNPADSGTEKQGYTWNITYGDGSGAAGVVYADKVVVGGVTATSQAVEAATSVSSEFTQDTKNDGLLGLAFSSINTVQPVQQTTFFDTVKDTLAKKLFTADLKKGAAGSYGFGYIDSSKYTGTITYVPVNNENGFWQFTAGGYSIGGGNGTSGSNATTGSIGTSIADTGTTLLLLPSNVVTAYYKQVSGASYNSAKGGYTYPCGATLPDFNVAIGGKTFVVPGTDLNYAPINSAGTTCFGGIQANTGIGFNIFGDIFLKLVYAVFDQTQSSPRLGFAEQS.
SEQ ID NO:3:
GCGCCTGCTCAGATTGTTGGGCGATCCACATTTCAGATCGACCAAGTCGCGTCCGGGAAGGTATACAAGAATGGCCCAATGGCAATGATGCAGACGTACAACAAATATGCACATGTGGGCGCCGTCGCGCCGGCTGCGGTAGTCGCAGCCGCAGCGGCTGCCCAAACGGGGGAAGTCTCGGCGAATCCTGAACAGTACGCTGAGTCATATTTGTGCCCCGTGACCATCGGGGATCAAACACTGAATCTCGATTTCGACACTGGAAGTGCAGATTTGTGGGTTTTCTCTACCCTAACTCCTTCCTCGGACTCTACAGGCCACACGCTATACAACCCTGCAGACTCAGGTACTGAGAAGCAAGGCTATACGTGGAACATTACCTACGGGGATGGCTCGGGCGCCGCTGGCGTGGTATATGCGGACAAGGTCGTGGTAGGGGGCGTCACGGCAACCTCGCAAGCAGTTGAGGCTGCAACATCCGTCTCTAGTGAATTTACTCAAGATACTAAGAATGACGGACTTCTAGGACTCGCCTTTAGCAGCATAAACACAGTTCAGCCCGTACAACAAACGACTTTTTTTGACACCGTGAAAGACACGCTCGCTAAAAAACTTTTTACCGCTGACTTAAAGAAAGGAGCCGCCGGTAGCTATGGATTTGGATACATAGATAGTTCCAAGTACACCGGTACAATCACATACGTACCCGTAAATAACGAGAACGGGTTCTGGCAATTCACTGCGGGGGGTTATAGCATTGGAGGTGGCAATGGGACCTCTGGAAGCAACGCTACGACGGGGTCAATTGGTACATCAATAGCAGACACGGGAACTACCCTTTTACTGTTGCCAAGTAATGTGGTTACCGCTTACTATAAACAGGTGAGCGGCGCTTCTTATAATTCCGCGAAGGGAGGTTATACGTACCCGTGTGGTGCCACCCTACCGGATTTCAACGTAGCTATAGGGGGAAAGACTTTTGTTGTTCCAGGGACAGATTTAAACTACGCACCAATAAACTCAGCCGGAACTACTTGCTTCGGTGGTATCCAGGCGAATACAGGCATCGGCTTTAATATCTTCGGTGACATTTTTCTGAAATTAGTTTATGCCGTCTTCGATCAGACACAAAGTTCGCCCCGCTTAGGTTTCGCGGAACAGAGT.
EXAMPLE 2 cloning of the high temperature acid protease resistant Gene pepA4
The synthesized gene vector is preserved in the form of a puncture fungus, the puncture fungus is picked up by a sterile toothpick in an ultra clean bench, and is placed in an LB shake tube containing Amp (working concentration: 100 mug/ml) antibiotics, and is cultured at 37 ℃ and 220rpm overnight, and the experiment is carried out according to the specification step of a century plasmid extraction kit PurePlasmid Mini Kit (CW 0500) for the next day to extract the vector containing the gene.
Based on the acid protease gene sequence, the following primers were designed and synthesized:
P1: 5'-CCGGAATTCCGGGCGCCTGCTCAGATTGTTG-3'(SEQ ID NO:4);
P2: 5'-TTGCGGCCGCAAACTCTGTTCCGCGAAACCTAA-3'(SEQ ID NO:5)。
And carrying out PCR amplification by taking the extracted carrier as a template. The PCR parameters were 94℃denaturation 5min, then 94℃denaturation 30 sec,55℃annealing 30 sec,72℃extension 2 min,30 cycles later 72℃incubation 10 min. An approximately 1164bp fragment was obtained, which was recovered and ligated with the pMD19 vector and sequenced by the biological technology Co., ltd. Of the Boxing family, beijing, predicting a protein molecular weight of 40.1kDa.
According to the nucleotide sequence obtained by sequencing, the obtained nucleotide sequence is compared with the pepA4 sequence by DNA Man software, and the error is confirmed.
EXAMPLE 3 preparation of recombinant high temperature resistant acid protease PepA4
Double digestion (EcoRI+NotI) is carried out on the expression vector pPIC9K, meanwhile, double digestion (EcoRI+NotI) is carried out on the gene pepA4 encoding the high temperature resistant acid protease, a gene fragment encoding the mature high temperature resistant acid protease is obtained through enzyme digestion and connected with the expression vector pPIC9K, the recombinant plasmid pPIC 9K-pepA 4 containing the high temperature resistant acid protease gene pepA4 is obtained, and the pichia pastoris GS115 is transformed, so that the recombinant pichia pastoris strain GS115/pepA4 is obtained.
The GS115 strain containing the recombinant plasmid and the control strain (namely, the unmutated strain GS 115/pepA) are inoculated into a 300 mL BMGY culture solution, and after shaking culture is carried out for 48 hours at the temperature of 30 ℃ and 200 rpm, thalli are collected by centrifugation. Then resuspended in 150mL BMMY medium and cultured with shaking at 30℃200 rpm. After induction for 72 hours, the supernatant was collected by centrifugation and the activity of the high temperature resistant acid protease was measured.
EXAMPLE 4 Activity assay of high temperature resistant acid protease PepA4
The method for measuring the acid protease activity comprises the following steps of adding 1ml of a proper diluted enzyme solution at a pH of 3.0,40 ℃, adding 1ml of a substrate, reacting 10 min, adding 2 ml of trichloroacetic acid solution to terminate the reaction, adding 2.5ml of sodium carbonate solution and 1ml of Fulin reagent into filtrate, and measuring the OD value by 680 nm after water bath 20 min. 1 enzyme activity unit (U) is defined as 1g solid enzyme powder (or 1ml liquid enzyme) under certain temperature and pH value conditions, 1 min hydrolyzed casein to generate 1 ug tyrosine, and 1 enzyme activity unit is expressed as U/g (U/ml).
Example 5 determination of Properties of high temperature resistant acid protease PepA4
And (3) measuring and comparing the enzymatic properties of the recombinant high-temperature resistant acid protease PepA4 and the unmutated acid protease PepA. Meanwhile, mutant recombinant acid protease PepAM2 is additionally added for enzyme property comparison, the sequence PepAM is D67A and G343E mutation based on SEQ ID NO.1, and the specific PepA4 mutant and the preparation method of the PepA4M2 recombinase are as in the above examples.
1. The method for determining the optimum pH and the pH stability of the recombinant acid protease PepA4 is as follows:
the purified acid proteases PepA, pepA4M2 were subjected to enzymatic reactions at different pH to determine their optimum pH. And (3) carrying out enzymolysis reaction on the properly diluted enzyme solution under different pH (1.0-10.0) conditions to determine the optimal reaction pH. The buffer solution is composed of glycine-hydrochloric acid buffer solution with pH of 1.0-3.0:0.1mol/L, citric acid-disodium hydrogen phosphate buffer solution with pH of 4.0-7.0:0.1mol/L, and glycine-sodium hydroxide buffer solution with pH of 8.0-10.0:0.1 mol/L.
The pH adaptability results (figure 1) of the acid protease PepA measured at 40 ℃ under the buffer systems with different pH values show that the optimal pH values of the acid protease PepA and the buffer systems are 2.0, and the optimal pH ranges are the same.
Purified acid proteases PepA, pepA4M2 were treated in the above-mentioned buffers of various pH at 40 ℃ for 180min, and then the enzyme activity was measured at 40 ℃ in a buffer system of pH3.0 to investigate the pH tolerance of the enzyme. The result (figure 2) shows that the recombinant acid protease, pepA4, is very stable between pH 2.0 and 7.0, and the activity of the residual enzyme is over 80% after being treated for 60min in the pH range, and the relative enzyme activity is improved to a certain extent compared with that of PepA and PepA4M2, which shows that the recombinant enzyme has better pH stability in a wider pH range.
2. The method for measuring the thermal stability of the recombinant acid protease PepA4 comprises the following steps:
The temperature resistance of the acid protease was measured by treating the acid protease at 75℃for various periods of time and then measuring the enzyme activity at 40 ℃. The thermal stability test of the enzyme shows that (FIG. 3), pepA4 has good thermal stability, and can keep 80% of enzyme activity after being incubated for 5min at 75 ℃. And under the temperature of 75 ℃, the residual enzyme activity of the PepA is only 60 percent, and the residual enzyme activity of the PepA4M2 is only 68 percent, so that compared with the PepA4, the thermal stability of the PepA4 is obviously improved, and the heat resistance is good.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The acid protease mutant PepA4 is characterized in that the amino acid sequence of the acid protease mutant PepA4 is obtained by carrying out D67A, E103D, E304K, S L point mutation on the amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence of the acid protease mutant PepA4 is shown in SEQ ID NO. 2.
2. The method for producing an acid protease mutant PepA4 according to claim 1, comprising the steps of:
1) Transforming host cells with the recombinant vector to obtain recombinant strains;
2) Culturing the recombinant strain and inducing the expression of recombinant acid protease;
3) Recovering and purifying the expressed high-temperature resistant acidic protease mutant PepA4.
3. The method according to claim 2, wherein the host cell in step 1) is selected from any one of Pichia pastoris cells, saccharomyces cerevisiae cells and polytype of the cells.
4. A gene encoding the acid protease mutant PepA4 of claim 1.
5. The gene according to claim 4, wherein the gene is pepA4 gene, and the sequence of pepA4 gene is shown in SEQ ID NO. 3.
6. A recombinant vector comprising the gene of any one of claims 4-5.
7. The recombinant vector according to claim 6, wherein the vector is selected from pET-28a, pPIC9K or ppiczα.
8. A recombinant strain comprising the gene of any one of claims 4-5.
9. The recombinant strain of claim 8, wherein the strain is escherichia coli, yeast, bacillus, or lactobacillus.
10. Use of the acid protease mutant PepA4 of claim 1, the gene of any one of claims 4-5, the recombinant vector of any one of claims 6-7, the recombinant strain of any one of claims 8-9 in food processing, leather processing or feed processing.
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