An alkaline protease mutant and its application

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to an alkaline protease mutant with improved enzyme activity and thermostability obtained through molecular modification, and its applications. This invention utilizes site-directed mutagenesis to mutate the wild-type ALK, obtaining the mutant T56C / A90C, whose enzyme activity at 40℃ is increased compared to the wild-type. The protease activity and residual enzyme activity were measured after incubation at 60℃ for 4 hours, demonstrating a certain level of activity under high-temperature conditions. This improves the thermostability of the alkaline protease, facilitating its function in highly alkaline environments. Therefore, it has wide applications in various industries such as detergents, leather, pharmaceuticals, feed, silk, and food.

Description

Technical fields: This invention belongs to the field of bioengineering technology, specifically relating to an alkaline protease mutant with improved enzyme activity and thermal stability obtained through molecular modification, and its applications. Technical background: Proteases are a general term for enzymes that hydrolyze peptide bonds. They are widely found in nature, in animals, plants, and microorganisms, such as animal glands, internal organs, plant fruits, stems, leaves, seeds, as well as molds, Bacillus, and other microorganisms. Currently, proteases account for approximately 60% of the entire industrial enzyme market, with wide applications in industries such as detergents, food, pharmaceuticals, and leather. Proteases can catalyze both hydrolysis and its reverse reactions, exhibiting high activity and specificity, making them ideal for the pharmaceutical industry's need to produce certain specific molecules.

[0001] Based on their optimal pH value, proteases can be classified into alkaline proteases, acidic proteases, and neutral proteases. Alkaline proteases, compared to acidic and neutral proteases, have advantages such as a wider range of action and stronger hydrolytic ability, making them one of the most widely used enzymes in industry. Alkaline proteases were first discovered in pig pancreas, and subsequent studies have shown that they are widely present in animals, plants, and microorganisms. Compared to animal and plant-derived alkaline proteases, a large proportion are extracellular secretory metabolites. Due to their higher production efficiency, simpler product extraction and separation, and ability to be prepared on a large scale through fermentation, they are more likely to meet the needs of industrial production. Therefore, microbial alkaline proteases have received widespread attention from researchers. Currently, the main microbial strains capable of producing alkaline proteases include Bacillus, yeast, actinomycetes, and fungi. Among these strains, Bacillus is the main producing species of alkaline proteases. Numerous Bacillus species have been explored and developed from many different environments for the production of alkaline proteases, such as Bacillus subtilis (…). Bacillus subtilis ), Bacillus clausti ( Bacillus clausii ), Bacillus licheniformis ( Bacillus licheniformis ) and Bacillus moharveni ( Bacillus mohaven )wait.

[0002] Alkaline proteases occupy a crucial position in the protease family. They function effectively in highly alkaline environments, leading to their widespread application in various industries such as detergents, leather, pharmaceuticals, and food. In the laundry industry, the largest market for alkaline proteases is the detergent-related sector, accounting for approximately two-thirds of global demand. The addition of alkaline proteases releases proteins from stains, primarily because they hydrolyze various protein-based stains, including blood, sweat, and milk stains. They also release dirt encapsulated by proteins and stains where proteins increase adhesion to the substrate, exhibiting excellent synergistic cleaning power with surfactants. In the leather industry, the addition of alkaline proteases makes several stages of the leather-making process more environmentally friendly, such as soaking, liming, hair removal, softening, and wastewater treatment. Adding alkaline proteases during soaking breaks the cross-links formed during the drying process of raw hides, dissolving and removing interfiber bonds. In the pharmaceutical industry, alkaline proteases are also of significant value in drug development. For example, alkaline proteases with collagenase activity can be used to treat various diseases such as wound healing, burns, glaucoma, and herniated discs. In the food industry, alkaline proteases are mainly responsible for the hydrolysis of proteins, breaking down large protein molecules into easily absorbed small peptides and amino acids, thereby preparing nutrient-rich hydrolysates. In the baking industry, alkaline proteases 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.

[0003] Rational design is commonly used for targeted modification of enzymes. First, computer simulations are used to determine the spatial structure of the protein and the amino acid regions affecting enzyme function. Then, after obtaining the nucleotide sequence, the amino acid sites to be modified are identified. Precise design is then employed to precisely design the desired mutation sites, altering specific amino acids through substitution, insertion, or deletion to change one or more properties of the enzyme. Compared to irrational design, rational design has the advantages of less workload and easier acquisition of effective mutants. This technique allows for the introduction of disulfide bonds into gene sequences for site-directed mutagenesis, thereby achieving targeted modification of proteins. Disulfide bonds are crucial components of protein secondary and tertiary structures, playing a role in stabilizing the spatial structure of the peptide chain. The more disulfide bonds present, the greater the stability of the protein molecule against external influences.

[0004] 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 offers the following advantages as an expression system: (1) it is easy to culture, grows rapidly, and has no special nutritional requirements; (2) it is non-pathogenic: generally considered to be in a safe (GRAS) state; (3) it can secrete recombinant proteins in culture media; and (4) it does not contain exotoxins or endotoxins. In recent decades, B. subtilis, B. amyloquefaciens, B. licheniformis It has been widely used in the heterogeneous production of industrial and food-grade enzymes; (5) it can directly secrete extracellular enzymes into the culture medium without cell disruption or intracellular protein contamination, which is beneficial for subsequent separation and purification; (6) it has stress resistance and can produce a variety of heat-resistant enzyme preparations.

[0005] Therefore, in this invention, by introducing disulfide bonds to rationally design the alkaline protease gene derived from Bacillus clausti, an alkaline protease mutant gene with improved enzyme activity and thermal stability at high temperatures is obtained. Summary of the Invention: To obtain an alkaline protease with improved enzyme activity and thermostability, its existing properties need further modification. The purpose of this invention is to provide a thermostable alkaline protease mutant. *Bacillus clausti* (… Bacillus clausii The alkaline protease gene derived from TCCC11004 ( apr Site-directed mutagenesis was performed, and the recombinant expression vector pBSA43- was constructed using the shuttle vector pBSA43. apr The expression and preparation of Bacillus subtilis WB600, Bacillus amyloliquefaciens CGMCC No.11218 and Bacillus licheniformis 2709 were achieved.

[0006] The technical approach to achieving the objective of this 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 T56C and A90C mutations on the wild-type alkaline protease mature peptide 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, nucleotide sequence as shown in SEQ ID NO.6.

[0007] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above-mentioned mutant encoding gene; Furthermore, the recombinant plasmid uses pBSA43 as the expression vector; 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 ligating the mutant coding gene with the expression vector pBSA43 and then expressing it in host Escherichia coli.

[0008] The third technical solution provided by this invention is the application of the above-mentioned recombinant plasmid or recombinant strain, especially its application in the alkaline protease mutant described in one of the production technical solutions.

[0009] The fourth technical solution provided by this invention is the application of the alkaline protease mutant described in technical solution one. It can function in a relatively high-temperature alkaline environment, and therefore has wide applications in many industries such as washing, leather, medicine, feed, silk, and food.

[0010] In this invention, the following definitions are used: 1. Nomenclature of amino acids and DNA nucleic acid sequences The IUPAC nomenclature, a widely accepted system for naming amino acid residues, is used, employing single-letter or three-letter codes. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0011] 2. Identification of alkaline protease mutants The mutated amino acid in the ALK mutant is represented by "original amino acid + position + substituted 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 wild-type ALK. The position number corresponds to the amino acid sequence number of the mature peptide of wild-type ALK in SEQ ID NO.1.

[0012] In this invention, lowercase italics apr This indicates the gene encoding the wild-type alkaline protease ALK, in lowercase italics. aprm T56C / A90C represents the gene encoding the mutant T56C / A90C, and the specific information is shown in the table below.

[0013]

[0014] Beneficial effects: 1. This invention utilizes site-directed mutagenesis to mutate the wild-type ALK to obtain the mutant T56C / A90C, whose enzyme activity at 40℃ is increased compared to the wild-type. The protease activity and residual enzyme activity were measured after incubation at 60℃ for 4 hours. It has a certain activity under high temperature environment, thereby improving the thermal stability of alkaline protease. Attached image description: Figure 1 Electrophoresis diagram of PCR amplification of wild-type alkaline protease zymogen gene Where: M is the DNA Marker, and 1 is the alkaline protease progenitor gene. apr ; Figure 2 pBSA43- apr Plasmid digestion verification diagram Where: M represents DNA Marker, and 1 represents pBSA43- apr through Bam HI and Not I. Double enzyme digestion diagram.

[0015] Figure 3 The residual enzyme activity of wild-type ALK and mutant T56C / A90C after incubation at 60℃ is recorded. Detailed implementation method: The technical content of the present invention will be further described below with reference to the embodiments. However, the present invention is not limited to these embodiments, and the scope of protection of the present invention cannot be limited by the following embodiments.

[0016] The culture media used in some embodiments of this invention are as follows: LB medium (g / L): yeast extract 5.0, tryptone 10.0, NaCl 10.0, the remainder being water, sterilized at 121℃ for 20 min.

[0017] Fermentation medium (g / L): corn flour 64, soybean meal 40, with 2.7 amylase, 4 Na2HPO4, 0.3 KH2PO4 added, and the remainder being water; incubate at 90℃ for 30 min and then sterilize at 121℃ for 20 min.

[0018] Add 1.5% agar to the solid culture medium.

[0019] In this invention, the mature peptide sequence of the wild-type alkaline protease ALK is shown in SEQ ID NO.1: AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

[0020] In this invention, the zymogen region sequence of the wild-type alkaline protease ALK is shown in SEQ ID NO.3: AEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTMAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKV LGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

[0021] In this invention, the zymogen region sequence of the alkaline protease T56C / A90C mutant is shown in SEQ ID NO. 5: AEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTMAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSCQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYCVKV LGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

[0022] The present invention will be further explained and illustrated below through specific embodiments.

[0023] Example 1: Obtaining the wild-type alkaline protease gene 1. Use the kit (OMEGA: Bacterial DNA Kit) to extract Bacillus croceae ( Bacillus clausii The genomic DNA extraction steps for TCCC11004 are as follows: (1) Inoculate the strain onto LB solid plates with an inoculation loop and incubate overnight at 37 °C.

[0024] (2) Pick a single colony from the culture plate and inoculate it into a liquid test tube culture medium. Incubate overnight at 37 °C with shaking at 220 r / min.

[0025] (3) Take 3 mL-5 mL of bacterial solution and place it in a sterilized EP tube. Centrifuge at 12000 r / min for 2 min and discard the supernatant.

[0026] (4) Add 200 μL of sterile water to the EP tube to resuspend the bacterial cells, then add 50 μL of lysozyme, mix by blowing and aspiration, and keep warm at 37 ℃ for 20 min.

[0027] (5) Add 100 μL of BTL buffer and 20 μL of proteinase K to the EP tube, vortex to mix, incubate at 55 °C for 40 min, and vortex to mix every 20 min.

[0028] (6) Add 5 μL of RNase, invert and mix several times, and let stand at room temperature for 10 min. (7) Centrifuge at 12000 r / min for 2 min, remove the undigested part, transfer the supernatant to a new EP tube, add 220 μL BDL buffer, and incubate in a 65 °C water bath for 15 min.

[0029] (8) Add 220 μL of anhydrous ethanol and mix by blowing and sucking.

[0030] (9) Transfer the liquid in the EP tube to the recovery column and let it stand for 1 min. Centrifuge at 12000 r / min for 1 min. Pour the filtrate back into the recovery column and repeat twice. Discard the waste liquid.

[0031] (10) Add 500 μL HBC buffer, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0032] (11) Add 700 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0033] (12) Add 500 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0034] (13) 12000 r / min for 2 min, discard the waste liquid tube, and put the recovery column into a new EP tube.

[0035] (14) Dry in a metal bath at 55 °C for 10 min.

[0036] (15) Add 50 μL of sterile water at 55 °C, let stand at room temperature for 5 min, centrifuge at 12000 r / min for 2 min, discard the recovery column, and the liquid in the EP tube is the genome.

[0037] 2. Using the extracted genome of Bacillus clausti as a template, a pair of primers were designed upstream and downstream of the ORF frame to introduce restriction enzyme sites, respectively. BamH I, Not I. The 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' Using P1 and P2 as upstream and downstream primers, amplification was performed using the Bacillus clausti genome as a template.

[0038] The amplification reaction system is as follows:

[0039] The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 7 s, for 30 cycles; 72℃ extension for 10 min. The PCR amplification products were subjected to 0.8% agarose gel electrophoresis, yielding a 1062 bp band. Figure 1 The PCR product was recovered using a small-volume DNA recovery kit to obtain the wild-type alkaline protease progenitor gene of this invention. apr (SEQ ID NO.4) apr The pBSA43 plasmid was treated with restriction endonucleases. BamH I and Not I performed double enzyme digestion and recovered the gel. apr Ligated with the pBSA43 vector, the recombinant plasmid pBSA43- was obtained. apr Enzyme digestion verification, such as Figure 2 As shown, it was transformed into Escherichia coli JM109.

[0040] Example 2: Preparation of alkaline protease mutants 1. A novel alkaline protease was constructed by site-directed mutagenesis using overlap PCR technology. The following mutation primers were designed: Mutate upstream primer 56-F: 5'- AACCATCCTGTCAAGATGGG -3' Mutate downstream primer 56-R: 5'- CCCATCTTGA CAGGATGGTT -3' In the first step of the overlap PCR reaction system, P1 and 56-R were used as upstream and downstream primers, respectively, and P2 and 56-F were used as upstream and downstream primers, respectively. The plasmid pBSA43- apr Using a template, a PCR1 reaction was performed to obtain the upstream and downstream fragments, respectively.

[0041] The reaction system for upstream fragment amplification is as follows:

[0042] The reaction system for downstream fragment amplification is as follows:

[0043] The amplification program was as follows: 98℃ pre-denaturation for 30 min; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 7 s for 30 cycles; 72℃ extension for 10 min.

[0044] 2. After gel extraction and recovery of upstream and downstream fragments, PCR 2 was performed. The reaction system was as follows:

[0045] The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 7 s, for 5 cycles; 72℃ extension for 10 min.

[0046] 3. After PCR 2, add 2 μL each of primers P1 and P2 to the system. The PCR 3 amplification program is as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 10 s, for 5 cycles; 72℃ extension for 10 min. The PCR amplification products are subjected to 0.8% agarose gel electrophoresis, and the PCR products are recovered using a small-volume DNA recovery kit to obtain the alkaline protease encoding gene with the T56C site-directed mutation. aprm T56C.

[0047] 4. Based on the T56C site-directed mutagenesis, a novel alkaline protease was constructed by performing an A90C site-directed mutagenesis using overlap PCR technology. The mutagenesis primers are designed as follows: Mutation of upstream primer 90-F: 5'- CGGAACTATACTGTGTTAAAGTATTAGG -3' Mutated downstream primer 90-R: 5'- CCTAATACTTTAACACAGTATAGTTCCG -3' In the first step of the overlap PCR reaction system, P1 and 90-R were used as upstream and downstream primers, respectively, and P2 and 90-F were used as upstream and downstream primers, respectively, to perform PCR 4 reaction and obtain upstream and downstream fragments.

[0048] The reaction system for upstream fragment amplification is as follows:

[0049] The reaction system for downstream fragment amplification is as follows:

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

[0051] 5. After gel extraction and recovery of upstream and downstream fragments, PCR 5 was performed. The reaction system was as follows:

[0052] The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 7 s, for 5 cycles; 72℃ extension for 10 min.

[0053] After PCR 5, 2 μL each of primers P1 and P2 were added to the system. The PCR 6 amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 10 s, for 5 cycles; 72℃ extension for 10 min. The PCR products were subjected to 0.8% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit to obtain the gene encoding the alkaline protease T56C / A90C mutant. aprm T56C / A90C.

[0054] 6. Obtain the site-directed mutant gene of alkaline protease. aprm The T56C / A90C and pBSA43 plasmids were used with restriction endonucleases. BamH I and Not I performed double enzyme digestion and recovered the gel. aprm T56C / A90C was ligated into the pBSA43 vector and transformed into E. coli JM109. The resulting single colonies of transformants were inoculated into 5 mL LB accumulator tubes to obtain the recombinant plasmid pBSA43- aprm The T56C / A90C variant was verified by double enzyme digestion and then sent to Genewiz for sequencing. The sequencing results were compared with the alkaline protease WT gene, confirming that it was a mutation occurring at position 56 (Thr→Cys) and position 90 (Ala→Cys) of the mature peptide in the WT variant. This alkaline protease was named the T56C / A90C mutant, and its encoding gene was named... aprm T56 / A90C.

[0055] Finally, pBSA43- aprm T56 / A90C and pBSA43- apr The bacteria were transformed into Bacillus subtilis WB600, and after screening for kanamycin (Kan) resistance and enzyme digestion verification, recombinant strain WB600 / pBSA43- was obtained. aprm T56C / A90C, and WB600 / pBSA43- apr .

[0056] Example 3: Determination of alkaline protease activity using the national standard method (1) Experimental group: Take 1 mL of enzyme solution, preheat at 40 ℃ and pH=10.5 for 2 min; then add 1 mL of 1% casein substrate, mix thoroughly, 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 working solution, and measure the absorbance value at 680 nm. Each sample is performed in triplicate. Control group: The procedure for the control group was similar to that for the experimental group, but 2 mL of TCA solution was added before adding the substrate. The reaction was carried out at 40 °C for 10 min, then 1 mL of casein substrate was added. After thorough mixing, the reaction was terminated, and the mixture was allowed to stand for 10 min. After centrifugation, the supernatant was collected, and Na2CO3 solution and Folin reagent solution were added. The absorbance at 680 nm was measured using a UV spectrophotometer.

[0057] (2) Definition of enzyme activity: The amount of ALK required to catalyze the production of 1 μg of L-tyrosine within 1 min under the conditions of 40 ℃ and pH=10.5 is defined as one unit of enzyme activity U.

[0058] The activity of alkaline protease is calculated using the following formula: ,in: X: Enzyme activity value, U / mL A680: Absorbance value at 680 nm K: Absorbance constant, i.e., 95.85 N: Dilution factor 10: Reaction time 10 min 4: Total reaction volume 4 mL.

[0059] Example 4: Evaluation of the enzyme activity of high-activity alkaline protease mutants The recombinant strain WB600 / pBSA43- obtained in Example 2 above. aprm T56 / A90C and WB600 / pBSA43- apr Inoculate each culture into 5 mL of LB liquid medium (containing kanamycin, 50 µg / mL) and incubate overnight at 37°C and 220 r / min. Then, transfer the culture to 50 mL of fresh LB medium (containing kanamycin, 50 µg / mL) at a 2% inoculation rate and continue incubation at 37°C and 220 r / min for 48 h.

[0060] The fermentation broth was centrifuged, and the supernatant was collected. First, impurities were removed by precipitation with 25% saturated ammonium sulfate. Then, the saturation was increased to 65% to precipitate the target protein. After dissolution, the supernatant was dialyzed to remove salt. The active component obtained after salting-out was dissolved in 0.02 mol / L Tris-HCl (pH 7.0) buffer and loaded onto a cellulose ion exchange chromatography column. Unadsorbed proteins were eluted with the same buffer, followed by gradient elution with 0.02 mol / L Tris-HCl (pH 7.0) buffers containing different concentrations of NaCl (0–1 mol / L). The target protein was collected. The active component obtained from ion exchange was equilibrated with 0.02 mol / L Tris-HCl (pH 7.0) buffer containing 0.15 mol / L NaCl, loaded onto a Sephadex G25 gel chromatography column, and eluted with the same buffer at a rate of 0.5 mL / min to obtain purified enzyme solution for enzyme activity assay.

[0061] The enzyme activity assay method is as described in Example 3.

[0062] Protein concentration was determined using the BCA protein concentration assay kit, following the instructions. Alkaline protease activity = the ratio of enzyme activity (U / ml) to protein concentration (mg / ml).

[0063] The final calculated enzyme activities of wild-type ALK and mutant at 40℃ are shown in the table below.

[0064]

[0065] The purified enzyme solution was diluted 100-fold, and 5 ml was placed in a 60℃ water bath for 4 hours. The initial enzyme activity before placing the solution in the water bath and the enzyme activity after incubation at 60℃ for 4 hours were measured. Using the initial enzyme activity as 100%, the residual enzyme activity after incubation 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℃ for 4 hours are shown in the table below.

[0066]

[0067] The results above show that the initial enzyme activity of T56C / A90C was higher than that of WT at 40℃, and the stability of T56C / A90C was significantly improved compared to WT after incubation in a 60℃ water bath for 4 hours. Specifically, the residual enzyme activity of T56C / A90C after incubation in a 60℃ water bath for 4 hours reached 74.3%.

[0068] Example 5: Expression and preparation of alkaline protease mutant in recombinant Bacillus amyloliquefaciens strains The recombinant plasmid pBSA43- aprm T56 / A90C and pBSA43- apr The bacteria were transformed into Bacillus amyloliquefaciens CGMCC No. 11218, and after screening for kanamycin (Kan) resistance and enzyme digestion verification, wild-type recombinant strain CGMCC No. 11218 / pBSA43- was obtained. apr , and the mutant recombinant bacteria CGMCC No.11218 / pBSA43- aprm T56 / A90C.

[0069] The recombinant mutant strain of Bacillus amyloliquefaciens, CGMCC No. 11218 / pBSA43-, was separately tested. 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), incubate overnight at 37°C and 220 r / min, then transfer to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at a 2% inoculation rate, and continue incubation at 37°C and 220 r / min for 48 h. (Fermentation medium (g / L): corn flour 64, soybean meal 40, with added 2.7 g / L amylase, 4 g / L Na₂HPO₄, 0.3 g / L KH₂PO₄, and the remainder water; incubate at 90°C for 30 min, then sterilize at 121°C for 20 min.) The activity of alkaline protease obtained from Bacillus amyloliquefaciens fermentation was determined using the national standard method described in Example 3 (the supernatant was collected after centrifugation of the fermentation broth for enzyme activity determination). The enzyme activity of alkaline protease in the fermentation broth of Bacillus amyloliquefaciens was as follows: ALK wild-type enzyme activity was 10760.5 U / ml, and the enzyme activity of the ALK mutant T56C / A90C was 12685.6 U / ml.

[0070] Example 6: Expression and preparation of alkaline protease mutant in recombinant Bacillus licheniformis strains The recombinant plasmid pBSA43- aprm T56 / A90C and pBSA43- apr The strains were transformed into Bacillus licheniformis 2709, and after screening for kanamycin (Kan) resistance and enzyme digestion verification, wild-type recombinant strain 2709 / pBSA43- was obtained. apr , and the mutant recombinant strain 2709 / pBSA43- aprm T56 / A90C.

[0071] The Bacillus licheniformis mutant recombinant strain 2709 / pBSA43- was separately tested. aprm T56 / A90C and wild-type recombinant bacteria 2709 / pBSA43- aprInoculate into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL), incubate overnight at 37°C and 220 r / min, then transfer to 50 mL of fresh fermentation medium (containing kanamycin, 50 µg / mL) at a 2% inoculation rate, and continue incubation at 37°C and 220 r / min for 48 h. (Fermentation medium (g / L): corn flour 64, soybean meal 40, with added 2.7 g / L amylase, 4 g / L Na₂HPO₄, 0.3 g / L KH₂PO₄, and the remainder water; incubate at 90°C for 30 min, then sterilize at 121°C for 20 min.) The activity of alkaline protease obtained from Bacillus licheniformis fermentation was determined using the national standard method described in Example 3 (the supernatant of the fermentation broth was collected after centrifugation to determine enzyme activity). The enzyme activity of alkaline protease in the fermentation broth of Bacillus licheniformis was as follows: ALK wild-type enzyme activity was 14383.8 U / ml, and the enzyme activity of the ALK mutant T56C / A90C was 16956 U / ml.

[0072] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. An alkaline protease mutant, characterized in that, Based on the wild-type amino acid shown in SEQ ID NO.3, a mutation was performed, and the mutated amino acid sequence is shown in SEQ ID NO.

5.

2. The encoding gene of the alkaline protease mutant of claim 1.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

6.

4. A recombinant vector or recombinant strain containing the encoding gene of claim 2.

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

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

7. The use of the recombinant vector or recombinant strain of claim 4 in the production of the alkaline protease mutant of claim 1.

8. The application of the alkaline protease mutant according to claim 1, characterized in that, It is used in the washing, leather, feed, silk, and food industries.

Images