Alkaline protease mutant and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an alkaline protease mutant which is obtained through molecular modification and has improved enzyme activity and thermal stability and application of the alkaline protease mutant. An ALK wild type is mutated by utilizing a site-directed mutagenesis technology to obtain a mutant T56C / A90C of which the enzyme activity is improved relative to that of the wild type at 40 DEG C, and the mutant T56C / A90C is subjected to heat preservation at 60 DEG C for 4 hours to determine the protease activity and residual enzyme activity and has certain activity in a high-temperature environment, so that the heat stability of alkaline protease is improved, and the alkaline protease can play a role in a relatively high alkaline environment; therefore, the method can be widely applied to a plurality of industries such as washing, leather, medicine, feed, silk, food and the like.

Description

Technical field: The invention belongs to the technical field of bioengineering, and specifically relates to an alkaline protease mutant with improved enzyme activity and thermal stability obtained through molecular modification and application thereof. Technical background: Protease is a general term for enzymes that have the ability to hydrolyze peptide bonds. Proteases are widely found in animals, plants and microorganisms in nature, such as animal glands, internal organs, plant fruits, stems and leaves, seeds, as well as molds, Bacillus and other microorganisms. Currently, proteases account for about 60% of the entire industrial enzyme market and are widely used in many industries such as washing, food, medicine, leather, etc. Proteases can catalyze both hydrolysis reactions and their reverse reactions, and have high activity and specificity, which is very suitable for the production needs of certain specific molecules in the pharmaceutical industry.

[0003] According to the different optimal pH values, proteases can be divided into alkaline proteases, acidic proteases and neutral proteases. Compared with acidic and neutral proteases, alkaline proteases have the advantages of a wide range of action and strong hydrolysis ability. They are currently one of the most widely used enzymes in industry. Alkaline proteases were first discovered in pig pancreas. Subsequent studies have shown that alkaline proteases are widely present in animals, plants and microorganisms. Compared with those from animals and plants, a large part of alkaline proteases from microbial sources are extracellular secretory metabolites. Due to their higher production efficiency, simple product extraction and separation, and large-scale fermentation preparation, they are easier to meet the needs of industrial production. Therefore, alkaline proteases from microbial sources have received widespread attention from researchers. At present, microbial strains that can produce alkaline proteases mainly include Bacillus, yeast, actinomycetes and fungi. Among these strains, Bacillus is the main producer of alkaline proteases. People have explored and developed a large number of Bacillus species from many different environments for the production of alkaline proteases, such as Bacillus subtilis ( Bacillus subtilis ), Bacillus clausii ( Bacillus clausii )、Bacillus licheniformis( Bacillus licheniformis ) and Bacillus mojavensis ( Bacillus mohaven )wait.

[0004] Alkaline proteases occupy a very important position in the protease family. They can function in a high alkaline environment, so they are widely used in many industries such as washing, leather, medicine, and food. In the laundry industry, the largest application market for alkaline proteases is the detergent-related industry, which accounts for about two-thirds of the global demand for alkaline proteases. The addition of alkaline proteases can release proteins from stains. The main reason is that it can hydrolyze various types of protein stains, including blood, sweat stains, milk stains, etc. It can also release dirt wrapped by proteins and stains whose adhesion to the substrate is increased by proteins. It can also play a good synergistic decontamination ability with surfactants. In the leather industry, the addition of alkaline proteases makes many links of the leather making process more environmentally friendly, such as soaking, liming, dehairing, softening and wastewater treatment. Adding alkaline proteases during the soaking process can cut the cross-linking bonds formed during the drying process of raw hides and dissolve and remove the fibers. In the pharmaceutical industry, alkaline protease also has important application value in the development of pharmaceutical products. For example, alkaline protease with collagenase activity can be used to treat and promote wound healing, burns, glaucoma, herniated disc and other diseases. In the food industry, alkaline protease is mainly responsible for the hydrolysis of proteins, breaking down large molecular proteins into small molecular peptides and amino acids that are easily absorbed, thereby preparing nutritious hydrolyzates. In the baking industry, alkaline protease can improve the properties of gluten, reduce the viscosity of dough, improve its mechanical properties, and at the same time reduce production costs and energy consumption.

[0005] Rational design is often used for the directional modification of enzymes. First, it is necessary to simulate the spatial structure of the protein and the amino acid region that affects the relevant functions of the enzyme through a computer. Then, after knowing the nucleotide sequence, determine the amino acid site to be modified, accurately design the required mutation site, and change the amino acid at a specific site by substitution, insertion, deletion, etc., thereby changing one or more aspects of the enzyme's properties. Compared with irrational design, rational design has the advantages of less workload and easier to obtain effective mutants. This technology can be used to introduce disulfide bonds into the gene sequence for site-directed mutagenesis, thereby achieving directional modification of proteins. Disulfide bonds are an important component of the secondary and tertiary structures of proteins, and play a role in stabilizing the spatial structure of the peptide chain in protein molecules. The more disulfide bonds there are, the greater the stability of the protein molecule against the influence of external factors.

[0006] The genus Bacillus represents a diverse group of Gram-positive endospore-forming bacterial species and is one of the most promising industrial microorganisms discovered to date. Bacillus has the following advantages as an expression system: (1) it is easy to culture, grows rapidly, and has no special requirements for nutrients; (2) it is non-pathogenic: generally regarded as safe (GRAS) status; (3) it can secrete recombinant proteins in the culture medium; and (4) it does not contain exotoxins and endotoxins. In recent decades, B. subtilis, B. amyloquefaciens, B. licheniformis etc. have been widely used in the heterologous production of industrial-grade and food-grade enzymes; (5) Extracellular enzymes can be directly secreted into the culture medium without the need for cell disruption and intracellular protein contamination, which is conducive to subsequent separation and purification; (6) They are stress-resistant and can produce a variety of heat-resistant enzyme preparations.

[0007] Therefore, in the present invention, the alkaline protease gene derived from Bacillus Clausii is rationally designed by introducing a disulfide bond, thereby obtaining an alkaline protease mutant gene with improved enzyme activity and thermal stability at high temperature. Summary of the invention: In order to obtain alkaline protease with improved enzyme activity and thermal stability, its existing properties need to be further modified. The purpose of the present invention is to provide a high temperature resistant alkaline protease mutant. Bacillus clausii ) Alkaline protease gene from TCCC11004 ( apr ) were used for site-directed mutagenesis and the recombinant expression vector pBSA43- apr , and achieved expression and preparation in Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218 and Bacillus licheniformis 2709.

[0009] The technical route for achieving the purpose of the present invention is summarized as follows: One of the technical solutions provided by the present invention is an alkaline protease mutant, wherein the mature peptide of the mutant is obtained by causing T56C and A90C mutations on the basis of the mature peptide of the wild-type alkaline protease shown in SEQ ID NO.1; Furthermore, the alkaline protease mutant is a T56C / A90C mutant, and the amino acid sequence of the zymogen region is shown in SEQ ID NO.5; Furthermore, the coding gene of the T56C / A90C mutant is aprm T56C / A90C, the nucleotide sequence is shown in SEQ ID NO.6.

[0010] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above mutant encoding gene; Furthermore, the expression vector used by the recombinant plasmid is pBSA43; Furthermore, the host used by the recombinant strain includes but is not limited to: Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis; further, the host is Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218 or Bacillus licheniformis 2709; Preferably, the recombinant strain is obtained by connecting the mutant encoding gene to the expression vector pBSA43 and expressing it in the host Escherichia coli.

[0011] The third technical solution provided by the present invention is the application of the above-mentioned recombinant plasmid or recombinant strain, especially the application in producing the alkaline protease mutant described in one of the technical solutions.

[0012] The fourth technical solution provided by the present invention is the application of the alkaline protease mutant described in the first technical solution, which can function in an alkaline environment at a relatively high temperature, and therefore has a wide range of applications in multiple industries such as washing, leather, medicine, feed, silk, and food.

[0013] The following definitions are used in the present invention: 1. Nomenclature of amino acid and DNA sequences The generally accepted IUPAC nomenclature for amino acid residues is used, either as a single letter or three letter code. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0014] 2. Identification of alkaline protease mutants The mutated amino acid in the ALK mutant is represented by "original amino acid + position + replaced amino acid". For example, Thr56Cys (T56C) means that the amino acid at position 56 of the mature peptide is replaced by Cys from Thr in the wild-type ALK, and the position number corresponds to the amino acid sequence number of the wild-type ALK mature peptide in SEQ ID NO.1.

[0015] In the present invention, lowercase italics apr Indicates the gene encoding the wild-type alkaline protease ALK, in lowercase italics aprm T56C / A90C indicates the coding gene of the mutant T56C / A90C. The specific information is as shown in the following table.

[0016]

[0017] Beneficial effects: 1. The present invention utilizes site-directed mutagenesis technology to mutate the ALK wild type, and obtains a mutant T56C / A90C whose enzyme activity at 40°C is improved relative to the wild type. The protease activity and residual enzyme activity are measured at 60°C for 4 hours, and the mutant has a certain activity in a high temperature environment, thereby improving the thermal stability of the alkaline protease. Description of the drawings: Figure 1 The electrophoresis diagram of PCR amplification of wild-type alkaline protease zymogen gene Among them: M is DNA Marker, 1 is alkaline protease zymogen gene apr ; Figure 2 For pBSA43- apr Plasmid restriction enzyme cutting verification diagram Where: M is DNA Marker, 1 is pBSA43- apr through Bam HI and Not I double enzyme digestion map.

[0019] Figure 3 It is the residual enzyme activity of wild-type ALK and mutant T56C / A90C after incubation at 60°C. Specific implementation method: The technical content of the present invention is further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0021] Part of the culture medium used in the embodiments of the present invention is as follows: LB medium (g / L): 5.0% yeast extract, 10.0% tryptone, 10.0% NaCl, and the rest water, sterilized at 121°C for 20 min.

[0022] Fermentation medium (g / L): 64 corn flour, 40 soybean meal, add 2.7 amylase, 4 Na2HPO4, 0.3 KH2PO4, and the rest is water; keep warm at 90℃ for 30min and then sterilize at 121℃ for 20min.

[0023] Solid medium was supplemented with 1.5% agar.

[0024] In the present invention, the mature peptide sequence of the wild-type alkaline protease ALK is shown in SEQ ID NO.1: AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

[0025] In the present invention, the zymogen region sequence of the wild-type alkaline protease ALK is shown in SEQ ID NO.3: AEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTMAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKV LGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

[0026] In the present invention, the zymogen region sequence of the alkaline protease T56C / A90C mutant is shown in SEQ ID NO.5: AEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTMAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSCQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYCVKV LGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

[0027] The present invention will be further explained below through specific examples.

[0028] Example 1 Obtaining wild-type alkaline protease gene 1. Use the kit (OMEGA: Bacterial DNA Kit) to extract Bacillus Clausii ( Bacillus clausii )TCCC11004 genomic DNA, extraction steps are as follows: (1) Use an inoculation loop to inoculate the strain onto a LB solid plate and culture at 37 °C overnight.

[0029] (2) Pick a single colony from the culture plate and inoculate it into liquid test tube culture medium. Incubate overnight at 37 °C and 220 rpm with shaking.

[0030] (3) Place 3 mL-5 mL of bacterial solution in a sterilized EP tube, centrifuge at 12,000 rpm for 2 min, and discard the supernatant.

[0031] (4) Add 200 μL of sterile water to the EP tube to resuspend the bacteria, then add 50 μL of lysozyme, pipette to mix, and keep at 37 °C for 20 min.

[0032] (5) Add 100 μL BTL buffer and 20 μL proteinase K to the EP tube, vortex to mix, and incubate at 55 °C for 40 min, vortexing to mix every 20 min.

[0033] (6) Add 5 μL of RNase, invert several times to mix, and leave at room temperature for 10 min. (7) Centrifuge at 12,000 r / min for 2 min to remove the undigested portion. Transfer the supernatant to a new EP tube, add 220 μL of BDL buffer, and incubate at 65 °C in a water bath for 15 min.

[0034] (8) Add 220 μL of anhydrous ethanol and mix by pipetting.

[0035] (9) Transfer the liquid in the EP tube into the recovery column and let it stand for 1 min. Centrifuge at 12,000 r / min for 1 min. Pour the filtrate back into the recovery column. Repeat twice and discard the waste liquid.

[0036] (10) Add 500 μL HBC buffer, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0037] (11) Add 700 μL DNA wash buffer, let stand for 1 min, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0038] (12) Add 500 μL DNA wash buffer, let stand for 1 min, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0039] (13) Incubate at 12,000 r / min for 2 min, discard the waste liquid tube, and place the recovery column in a new EP tube.

[0040] (14) Dry in a metal bath at 55 °C for 10 min.

[0041] (15) Add 50 μL of 55°C sterile water, let stand at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, discard the recovery column, and the liquid in the EP tube is the genome.

[0042] 2. Using the extracted genome of Bacillus clausii as a template, design a pair of primers upstream and downstream of the ORF frame to introduce restriction enzyme sites respectively. Bm I. Not I. Alkaline protease gene of the present invention apr The amplification primers are as follows: Upstream primer P1: 5'- CGCGGATCCGCTGAAGAAGCAAAAGA -3' Downstream primer P2: 5'-AAGGAAAAAAGCGGCCCGCTTAGCGTGTTGCCGCTTCT -3' P1 and P2 were used as upstream and downstream primers, and the genome of Bacillus clausii was used as template for amplification.

[0043] The amplification reaction system is:

[0044] The amplification program was as follows: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 54°C annealing for 20 s, 72°C extension for 7 s, 30 cycles; 72°C extension for 10 min. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis, and a 1062 bp band was obtained ( Figure 1 ), using a small amount of DNA recovery kit to recover the PCR product, and obtain the wild-type alkaline protease zymogen region gene of the present invention. apr (SEQ ID NO.4), apr , pBSA43 plasmid Bm I and Not I was double-digested, and the gel-recovered apr Connect with pBSA43 vector to obtain recombinant plasmid pBSA43- apr , enzyme digestion verification Figure 2 and transformed into E. coli JM109.

[0045] Example 2 Preparation of alkaline protease mutants 1. Based on overlapping PCR technology, site-directed mutagenesis was performed to construct a new alkaline protease. The mutation primers were designed as follows: Mutation upstream primer 56-F: 5'-AACCATCCTGTCAAGATGGG-3' Mutation downstream primer 56-R: 5'-CCCATCTTGA CAGGATGGTT -3' In the first step of overlapping PCR reaction system, P1 and 56-R were used as upstream and downstream primers, and P2 and 56-F were used as upstream and downstream primers. apr As a template, PCR1 reaction was carried out to obtain the upstream fragment and the downstream fragment respectively.

[0046] The reaction system for upstream fragment amplification is:

[0047] The reaction system for downstream fragment amplification is:

[0048] The amplification program was as follows: pre-denaturation at 98°C for 30 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 7 s; and extension at 72°C for 10 min.

[0049] 2. After gel excision and recovery of upstream and downstream fragments, PCR 2 was performed. The reaction system was:

[0050] The amplification program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 7 s, for 5 cycles; and extension at 72°C for 10 min.

[0051] 3. After PCR 2, 2 μL of primers P1 and P2 were added to the system, and PCR 3 was performed with the following procedures: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 54°C annealing for 20 s, 72°C extension for 10 s, 5 cycles; 72°C extension for 10 min. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis, and the PCR product was recovered using a small amount of DNA recovery kit to obtain the alkaline protease encoding gene with T56C site-directed mutation. aprm T56C.

[0052] 4. Based on the T56C site-directed mutagenesis, the A90C site-directed mutagenesis was performed using overlapping PCR technology to construct a new alkaline protease. The mutation primers were designed as follows: Mutation upstream primer 90-F: 5'- CGGAACTATACTGTGTTAAAGTATTAGG -3' Mutation downstream primer 90-R: 5'- CCTAATACTTTAACACAGTATAGTTCCG -3' In the first step of overlapping PCR reaction system, P1 and 90-R were used as upstream and downstream primers, and P2 and 90-F were used as upstream and downstream primers, respectively, and PCR 4 reactions were performed to obtain upstream fragments and downstream fragments, respectively.

[0053] The reaction system for upstream fragment amplification is:

[0054] The reaction system for downstream fragment amplification is:

[0055] The amplification program was as follows: pre-denaturation at 98°C for 30 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 7 s; and extension at 72°C for 10 min.

[0056] 5. After gel cutting and recovery of upstream and downstream fragments, PCR 5 was performed. The reaction system was:

[0057] The amplification program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 7 s, for 5 cycles; and extension at 72°C for 10 min.

[0058] After PCR 5, 2 μL of primers P1 and P2 were added to the system, and the PCR 6 amplification program was as follows: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 54°C annealing for 20 s, 72°C extension for 10 s, and 72°C extension for 10 min. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis, and the PCR product was recovered using a small amount of DNA recovery kit to obtain the gene encoding the alkaline protease T56C / A90C mutant. aprm T56C / A90C.

[0059] 6. Obtaining alkaline protease site-directed mutant gene aprm T56C / A90C and pBSA43 plasmids were stained with restriction endonucleases Bm I and Not I was double-digested, and the gel-recovered aprm T56C / A90C was connected with the vector pBSA43 and transformed into Escherichia coli JM109. The transformed product of the single colony was inoculated into a 5 mL liquid LB resistance test tube to obtain the recombinant plasmid pBSA43- aprm The mutant was double-digested and then sent to GeneWeiZ for sequencing. The sequencing results were compared with the alkaline protease WT gene, and it was determined that the mutations of Thr at position 56→Cys and Ala at position 90→Cys occurred on the basis of WT. The alkaline protease was named T56C / A90C mutant, and its encoding gene was named aprm T56 / A90C.

[0060] Finally, pBSA43- aprm T56 / A90C and pBSA43- apr The recombinant strain WB600 / pBSA43- aprm T56C / A90C, and WB600 / pBSA43- apr .

[0061] Example 3: Determination of alkaline protease activity using the national standard method (1) Experimental group: Take 1 mL of enzyme solution, preheat at 40 ℃, pH = 10.5 for 2 min; then add 1 mL of 1% casein substrate, mix thoroughly and react at 40 ℃ for 10 min, add 2 mL of trichloroacetic acid (TCA) solution to terminate the reaction, take out and let stand for 10 min, centrifuge at 12000 rpm for 1 min, take 1 mL of supernatant, add 5 mL of Na2CO3 solution and 1 mL of Folin reagent solution, and measure the absorbance at 680 nm. Make 3 parallels for each sample. Control group: The operation of the control group was similar to that of the experimental group, but 2 mL of TCA solution was added before adding the substrate, and the reaction was carried out at 40 °C for 10 min. Then 1 mL of casein substrate was added, and the reaction was terminated after sufficient mixing. The mixture was allowed to stand for 10 min, centrifuged, and the supernatant was taken. Na2CO3 solution and Folin reagent solution were added, and the absorbance at 680 nm was measured using a UV spectrophotometer.

[0062] (2) Definition of enzyme activity: The amount of ALK required to catalyze the production of 1 μg of L-tyrosine within 1 min at 40°C and pH 10.5 is defined as one enzyme activity unit (U).

[0063] The activity of alkaline protease was calculated according to the following formula: ,in: X: Enzyme activity value, U / mL A680: absorbance at 680 nm K: Absorbance constant, i.e. 95.85 N: dilution multiple 10: Reaction time 10 min 4: Total reaction volume 4 mL.

[0064] Example 4 Evaluation of the specific enzyme activity of high-activity mutants of alkaline protease The recombinant strain WB600 / pBSA43- aprm T56 / A90C and WB600 / pBSA43- apr Inoculate each cell into 5 mL of LB liquid medium (containing kanamycin, 50 µg / mL), culture at 37°C, 220 r / min overnight, transfer to 50 mL of fresh LB medium (containing kanamycin, 50 µg / mL) at a 2% inoculum, and continue to culture at 37°C, 220 r / min for 48 h.

[0065] The fermentation broth was centrifuged to obtain the supernatant, and the impurities were first removed by salting out with ammonium sulfate at 25% saturation, and then the saturation was increased to 65% to precipitate the target protein. After dissolution, the salt was removed by dialysis, and the active components obtained after salting out and desalting were dissolved in 0.02mol / LTris-HCl (pH 7.0) buffer, loaded onto a cellulose ion exchange chromatography column, and then the unadsorbed proteins were first eluted with the same buffer, and then gradient eluted with 0.02mol / L Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0~1mol / L) to collect the target protein. The active components obtained by ion exchange were first equilibrated with 0.02mol / LTris-HCl (pH 7.0) buffer containing 0.15mol / L NaCl, loaded onto a sephadex g25 gel chromatography column, and then eluted with the same buffer at a rate of 0.5mL / min to obtain a purified enzyme solution for enzyme activity determination.

[0066] The enzyme activity determination method is the same as in Example 3.

[0067] The protein concentration was determined by BCA protein concentration assay kit according to its instructions; Alkaline protease specific enzyme activity = the ratio of enzyme activity (U / ml) to protein concentration (mg / ml).

[0068] The final calculated enzyme activity of wild-type ALK and mutant at 40°C is shown in the following table.

[0069]

[0070] The purified enzyme solution was diluted 100 times and 5 ml was taken and placed in a 60℃ water bath for 4 hours. The initial enzyme activity of the enzyme solution before being placed in the water bath and the enzyme activity after being kept at 60℃ for 4 hours were measured. The initial enzyme activity was taken as 100%, and the residual enzyme activity of the enzyme solution after being kept at 60℃ for 4 hours was calculated ( Figure 3 ). The final calculated residual enzyme activities of wild-type ALK and mutant T56C / A90C after incubation at 60°C for 4 hours are shown in the following table.

[0071]

[0072] From the above results, it can be seen that the initial enzyme activity of T56C / A90C is higher than that of WT at 40°C, and the stability of T56C / A90C is significantly improved compared with WT when kept in a water bath at 60°C for 4 hours. Among them, the residual enzyme activity of T56C / A90C after 4 hours of keeping in a water bath at 60°C for 4 hours reached 74.3%.

[0073] Example 5 Expression and preparation of alkaline protease mutants in recombinant strains of Bacillus amyloliquefaciens The recombinant plasmid pBSA43- aprm T56 / A90C and pBSA43- apr The wild-type recombinant strain CGMCC No.11218 / pBSA43- apr , and mutant recombinant bacteria CGMCC No.11218 / pBSA43- aprm T56 / A90C.

[0074] The mutant recombinant strain of Bacillus amyloliquefaciens CGMCC No.11218 / pBSA43- aprm T56 / A90C and wild-type recombinant bacteria CGMCC No.11218 / pBSA43- apr Inoculate into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL), culture at 37°C, 220 r / min overnight, transfer to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at 2% inoculum, and continue to culture at 37°C, 220 r / min for 48 h. (Fermentation medium (g / L): corn flour 64, soybean meal 40, add 2.7 amylase, Na2HPO44, KH2PO40.3, the rest is water; keep warm at 90°C for 30 min and sterilize at 121°C for 20 min.) The activity of alkaline protease obtained by fermentation of Bacillus amyloliquefaciens was determined using the national standard method in Example 3 (the fermentation broth was centrifuged and the supernatant was taken to determine the enzyme activity). The enzyme activity of alkaline protease in the fermentation broth of Bacillus amyloliquefaciens: the enzyme activity of ALK wild type was 10760.5 U / ml, and the enzyme activity of ALK mutant T56C / A90C was 12685.6 U / ml.

[0075] Example 6 Expression and preparation of alkaline protease mutants in recombinant strains of Bacillus licheniformis The recombinant plasmid pBSA43- aprm T56 / A90C and pBSA43- apr The wild-type recombinant strain 2709 / pBSA43- apr , and mutant recombinant strain 2709 / pBSA43- aprm T56 / A90C.

[0076] The mutant recombinant strains of Bacillus licheniformis 2709 / pBSA43- aprm T56 / A90C and wild-type recombinant bacteria 2709 / pBSA43- aprInoculate into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL), culture at 37°C, 220 r / min overnight, transfer to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at 2% inoculation, and continue to culture at 37°C, 220 r / min for 48 h. (Fermentation medium (g / L): corn flour 64, soybean meal 40, add 2.7 amylase, Na2HPO44, KH2PO40.3, the rest is water; keep warm at 90°C for 30 min and sterilize at 121°C for 20 min.) The activity of alkaline protease obtained by fermentation of Bacillus licheniformis was determined using the national standard method in Example 3 (the fermentation broth was centrifuged and the supernatant was measured for enzyme activity). The enzyme activity of alkaline protease in fermentation broth of Bacillus licheniformis: the enzyme activity of wild-type ALK was 14383.8 U / ml, and the enzyme activity of ALK mutant T56C / A90C was 16956 U / ml.

[0077] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. An alkaline protease mutant, characterized in that: The mutant is a T56C / A90C mutant, and the amino acid sequence of the zymogen region is shown in SEQ ID NO.

5.

2. A gene encoding the alkaline protease mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID NO.

6.

4. A recombinant vector or recombinant strain comprising the coding gene according to claim 2.

5. The recombinant vector according to claim 4, characterized in that The expression plasmid used was pBSA43.

6. The recombinant strain according to claim 4, characterized in that The host used is Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218 or Bacillus licheniformis 2709.

7. Use of the recombinant vector or recombinant strain according to claim 4 in producing the alkaline protease mutant according to claim 1.

8. Use of the alkaline protease mutant according to claim 1.

9. The use according to claim 8, characterized in that It is used in washing, leather, medicine, feed, silk and food industries.

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