Alkaline protease mutant aprBcpM1, recombinant expression vector, recombinant bacteria and application

Abstract

This invention relates to the alkaline protease mutant AprBcpM1, a recombinant expression vector, recombinant bacteria, and their applications. Using the alkaline protease mutant AprBcpM as a starting template, this invention optimizes its three-dimensional conformation through rational design, screens target mutation sites, and obtains the thermostable mutant AprBcpM1 through single-point and combined mutagenesis. Its enzyme activity retention after heat treatment at 60℃, 65℃, and 70℃ for 30 minutes is 1.9 times, 2.6 times, and 3.9 times that of the starting template AprBcpM, respectively. Furthermore, mutant AprBcpM1 exhibits better storage stability; after storage at 25℃ and 37℃ for 30 days, the remaining enzyme activity is 80% and 70%, respectively, which are 1.34 times and 2.33 times that of the starting template AprBcpM. The mutant AprBcpM1 of this invention, when applied to fermented feed for Litopenaeus vannamei, can produce acid-soluble proteins more efficiently.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to the alkaline protease mutant AprBcpM1, recombinant expression vector, recombinant bacteria, and their applications. Background Technology

[0002] Alkaline proteases are a class of enzymes that catalyze the hydrolysis of peptide bonds in proteins under alkaline conditions and are widely found in microorganisms. Due to their high catalytic efficiency, mild reaction conditions, and broad substrate spectrum, alkaline proteases have been widely used in numerous industrial fields such as detergents, leather tanning, feed, food, and environmental protection. In the feed industry, the addition of exogenous proteases is of great significance for improving the digestibility and utilization of feed proteins, improving animal gut health, and reducing nitrogen and phosphorus emissions. Cottonseed protein, as a high-yield and low-cost plant protein source, has the potential to replace fishmeal; however, its large protein molecules require enzymatic hydrolysis to break them down into smaller peptides and free amino acids before efficient utilization. Therefore, developing high-performance alkaline proteases suitable for the efficient enzymatic hydrolysis of cottonseed protein has significant application value.

[0003] However, naturally derived alkaline proteases generally suffer from insufficient thermal stability, severely limiting their potential for industrial applications. As biological macromolecules, proteins are prone to conformational changes and irreversible inactivation under high temperatures, making many alkaline proteases unsuitable for industrial applications requiring high-temperature processing, such as feed processing and high-temperature enzymatic hydrolysis. Therefore, improving the thermal stability of alkaline proteases has always been a research hotspot and challenge in the field of enzyme engineering.

[0004] To improve the thermostability of alkaline proteases, researchers have explored various protein engineering strategies, including directed evolution, rational design, and the introduction of disulfide bonds. Among these, introducing disulfide bonds into the flexible regions of the enzyme's three-dimensional structure to enhance its structural rigidity has proven to be an effective method for improving enzyme thermostability. In previous research, the team used the alkaline protease BCP from Bacillus circulans as a starting template and successfully obtained a mutant AprBcpM with significantly improved thermostability by rationally designing the introduction of disulfide bonds into the flexible region of its three-dimensional conformation (Chinese Patent Publication No.: CN121249629A, Invention Title: An Alkaline Protease Mutant AprBcpM and Its Application). Compared with the starting template BCP, the residual enzyme activity of mutant AprBcpM after heat treatment at 55℃, 60℃, and 65℃ for 30 minutes was 3.58 times, 6.26 times, and 8.09 times that of BCP, respectively; its optimal reaction temperature increased from 60℃ to 65℃, and its relative enzyme activity at 70℃ increased from 15.3% to 42.5%. In terms of application performance, after enzymatic hydrolysis of cottonseed protein at 50℃ for 120 minutes, the acid-soluble protein content of mutant AprBcpM reached 48.9%, which is 2.27 times that of the starting template BCP. The above results show that AprBcpM has significant improvements over the starting template in both thermal stability and application performance.

[0005] However, further research revealed that the thermostability of the mutant AprBcpM above 60°C remains unsatisfactory. After heat treatment at 60°C and 65°C for 30 minutes, its remaining enzyme activity was only 23.8% and 8.9%, respectively. This level of thermostability is insufficient to meet the requirements of practical industrial applications such as feed processing and high-temperature enzymatic hydrolysis, which demand enzyme preparations to maintain activity at higher temperatures. This, to some extent, limits the further promotion and application of the mutant AprBcpM.

[0006] Therefore, based on the previously obtained mutant AprBcpM, how to further improve its thermal stability so that it can maintain high enzyme activity at higher temperatures (especially above 60°C) is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to provide an alkaline protease mutant, AprBcpM1, a recombinant expression vector, recombinant bacteria, and its applications. Using the alkaline protease mutant AprBcpM as a starting template, this invention optimizes its three-dimensional conformation through rational design, screens target mutation sites, and obtains a thermostable mutant, AprBcpM1, through single-point and combined mutagenesis. The enzyme activity retention rates after heat treatment at 60℃, 65℃, and 70℃ for 30 minutes are 1.9 times, 2.6 times, and 3.9 times that of the starting template AprBcpM, respectively.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The first object of the present invention is to provide a protease mutant AprBcpM1, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] AQSVPWGISRVQAPAAHNRGLCGSGEKVAVLDTGISPHPCLNIRGGASFVPGAPSFQDGNGHGTHVAGTIAALNNSICVLGVAPCAELYAVNVLGADGRGSISSIAHGLEWTGNNNMHVANLSLGSPSPSATLEQA VNSATSRGVLVVAASGNSGASSISYPARYPNAMAVGATDQNNNRASFSQYGAGLDIVAPGVGVQSSYPGSTYASLNGTSMATPHVAGVAALVKHKNPSWSNTQIRNHLENTATSLGSTNLYVSGLVKAEAATR (SEQ ID NO.1) Preferably, the sequence encoding the amino acid is a polynucleotide sequence, as shown in SEQ ID NO.2.

[0010] (SEQ ID NO.2) A second objective of this invention is to provide a recombinant expression vector pHY17-aprbcpm27 containing the encoding gene of the protease mutant AprBcpM1 described above.

[0011] Another object of the present invention is to provide a recombinant engineered bacterium comprising the above-described recombinant expression vector pHY17-aprbcpm27.

[0012] Preferably, the strain is Bacillus subtilis.

[0013] Preferably, the strain is Bacillus subtilis 1285.

[0014] Another object of the present invention is to provide the application of the protease mutant AprBcpM1 as described above in the preparation of fermented feed for Litopenaeus vannamei.

[0015] Preferably, the application includes adding the alkaline protease mutant AprBcpM1 to the feed ingredients of Litopenaeus vannamei and performing fermentation treatment.

[0016] Preferably, the fermentation temperature is 25~35℃.

[0017] Compared with the prior art, the present invention has the following beneficial effects: Based on the previously obtained alkaline protease mutant AprBcpM (Chinese Patent Publication No.: CN121249629A, Invention Title: An Alkaline Protease Mutant AprBcpM and Its Application), this invention, through rational design and site-directed mutagenesis at the target site, yielded a mutant AprBcpM1 with further enhanced thermostability. Experimental results show that the enzyme activity retention rate of the mutant AprBcpM1 obtained in this invention after heat treatment at 55℃, 60℃, 65℃, and 70℃ for 30 minutes is 1.62 times, 1.9 times, 2.6 times, and 3.9 times that of the starting template AprBcpM, respectively.

[0018] Furthermore, the protease mutant AprBcpM1 exhibits better storage stability than the starting template AprBcpM. After 30 days of storage at 25°C and 37°C, the remaining enzyme activities were 80% and 70%, respectively, which are 1.34 times and 2.33 times that of the starting template AprBcpM.

[0019] Finally, the mutant AprBcpM1 was applied to fermented feed for Litopenaeus vannamei, with the starting template AprBcpM as the control. Under the same enzyme addition amount (1 U / g feed), after 35 days of fermentation, the acid-soluble protein content of the sample corresponding to the mutant AprBcpM1 was 28.1%, while the acid-soluble protein content of the sample corresponding to the starting template AprBcpM was 20.89%. Attached Figure Description

[0020] Figure 1 Three-dimensional conformational diagrams of AprBcpM and its mutant AprBcpM1 are shown; among them, Figure 1 In this context, A represents the three-dimensional conformation of AprBcpM. Figure 1 B in the diagram represents the three-dimensional conformation of AprBcpM1. Figure 2Thermal stability diagrams of AprBcpM and its mutant AprBcpM1; Figure 3 The graphs show the storage stability of AprBcpM and its mutant AprBcpM1 at 25°C and 37°C; among them, Figure 3 In the figure, A represents the storage stability of AprBcpM and its mutant AprBcpM1 at 25°C. Figure 3 B in the figure represents the storage stability of AprBcpM and its mutant AprBcpM1 at 37°C. Figure 4 The graph shows the changes in acid-soluble protein in fermented feed corresponding to AprBcpM and its mutant AprBcpM1. Detailed Implementation

[0021] The present invention will be further described in detail below through specific embodiments. However, it should not be construed that the scope of the present invention is limited to the following embodiments.

[0022] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions; the reagents and biological materials mentioned are commercially available unless otherwise specified. Experimental materials and reagents involved in this invention: 1. Strains and vectors The top 10 strains of Escherichia coli were purchased from Tiangen Biotech (Beijing) Co., Ltd.; Bacillus subtilis 1285 was purchased from Baori Biotechnology (Beijing) Co., Ltd.

[0023] The expression vector pHY17-aprbcpm was previously constructed by our team (Chinese Patent Publication No.: CN121249629A, Invention Title: An alkaline protease mutant AprBcpM and its application).

[0024] The sequence of the expression vector pHY17-aprbcpm is shown in SEQ ID NO.3 below, where: the bolded area below the sequence is the promoter sequence, the underlined area is the alkaline protease aprbcpm gene sequence, and the area between the two is the signal peptide sequence.

[0025] CGCAATCAGTGCCATGGGGAATTAGCCG TGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGTGTGGTTCTGGTGAAAAAGTTGCTGTCCTCGATACAGGTATT TCCACTCATCCATGTTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGCACCATCCACTCAAGATGGGAATG GGCATGGCACGCATGTGGCCGGGACGATTGCTGCTTTAAACAATTCGATTTGTGTTCTTGGCGTAGCGCCGTGTGC GGAACTATACGCTGTTAATGTTTTAGGAGCCGACGGGAGAGGTGCAATCAGCTCGATTGCCCATGGGTTGGAATGG ACAGGGAACAATAACATGCACGTTGCTAATTTAAGTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTG TTAATAGCGCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGGAATTCAGGTGCAAGCTCAATCAGCTATCC GGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGC GCAGGGCTTGACATTGTCGCACCAGGGGTAGGCGTGCAGAGCTCATACCCAGGTTCAACGTATGCCAGCTTAAACG GTACATCGATGGCTACTCCTCATGTTGCAGGTGTCGCAGCCCTTGTTAAACACAAGAACCCATCTTGGTCCAATAC ACAAATCCGCAACCATCTAGAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGTAAGCGGACTTGTCAAA GCAGAAGCGGCAACACGCTAA 2. Enzymes and Kits Q5 high-fidelity Taq enzyme MIX was purchased from NEB; plasmid extraction kit (#DP103-03) and gel purification kit (#DP209-02) were purchased from Tiangen Biotech (Beijing) Co., Ltd.; Taq enzyme MIX (EmeraldAmp® MAX PCR MasterMix) was purchased from Baori Biotech (Beijing) Co., Ltd.; kanamycin and ampicillin were purchased from Maclean's Reagent Company.

[0026] 3. Culture medium The culture medium for *E. coli* was LB (1% (w / v) peptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, pH 7.0). LBK was LB medium supplemented with kanamycin at a concentration of 30 μg / mL.

[0027] The shake-flask fermentation medium for Bacillus subtilis spores is maltose medium, and its composition is as follows: maltose 4%, peptone 1.5%, yeast extract 2.5%, dipotassium hydrogen phosphate 1%, sodium citrate 1%, and calcium chloride 0.3%.

[0028] Example 1: Rational Design of Target Sites The lower the free energy of a protein's three-dimensional conformation, the more stable its structure and the better its thermal stability. Therefore, improving enzyme thermal stability by reducing protein free energy is one of the effective strategies in protein engineering. Using the alkaline protease AprBcpM obtained in the team's previous research (patent application number: CN121249629A) as a starting template, we rationally designed site-directed mutagenesis based on its three-dimensional conformation.

[0029] This invention uses the free energy analysis software FoldX (see website: https: / / foldxsuite.crg.eu / , version number FoldX3) to analyze the three-dimensional conformation of AprBcpM (e.g., ... Figure 1 Stability calculations and analyses were performed on the model shown. First, FoldX3 software was downloaded and installed. Second, according to the FoldX3 software operation requirements, water molecules were removed from the AprBcpM three-dimensional conformation. At the same time, random mutations were performed on each amino acid site, and the change in free energy after mutation was calculated. Finally, the prediction results were analyzed.

[0030] Computational analysis revealed that mutations at 30 sites in different regions of the AprBcpM three-dimensional conformation could theoretically optimize its protein free energy. These 30 sites and their corresponding optimized free energies are as follows: E26V (-5.01kJ / mol), T37P (-1.1kJ / mol), A47I (-1.36kJ / mol), T56F (-1.69kJ / mol) ), T56Y (-1.57kJ / mol), C72A (-0.38kJ / mol), C81G (-2.12kJ / mol), N92K (-2.09kJ / mol), R99S (0.01kJ / mol), A101S (-2.06kJ / mol), T112A (-0.34kJ / mol), T112V (-1. 44kJ / mol), G113A (-1.21kJ / mol), H118D (0.19kJ / mol), V119I (-0.82kJ / mol), L122 M (0.45kJ / mol), A131P (-1.51kJ / mol), T132L (-1.28kJ / mol), S139Y (-1.7kJ / mol) , S151A (-0.92kJ / mol), A166P (-1.54kJ / mol), A168V (0.09kJ / mol), T174V (-2.14k J / mol), V199I (-0.73kJ / mol), Q200L (-1.25kJ / mol), S206N (-0.42kJ / mol), S210Q (-1.1kJ / mol), T254P (-1.02kJ / mol), V258G (-2.24kJ / mol) and S259N (-2.11kJ / mol).

[0031] Based on the theory that lower free energy corresponds to better protein stability, this invention selected sites with free energies less than -1 kJ / mol for further screening. Through screening, 19 sites were ultimately identified for experimental verification. These 19 sites and their corresponding free energies are as follows: E26V (-5.01kJ / mol), T37P (-1.1kJ / mol), A47I (-1.36kJ / mol), T56F (-1.69kJ / mol), V82G (-2.12kJ / mol) , N92K (-2.09kJ / mol), A101S (-2.06kJ / mol), T112V (-1.44kJ / mol), G113A (-1.21kJ / mol), A131P (-1.51k J / mol), T132L (-1.28kJ / mol), S139Y (-1.7kJ / mol), A166P (-1.54kJ / mol), T174V (-2.14kJ / mol), Q200L ( -1.25kJ / mol), S210Q (-1.1kJ / mol), T254P (-1.02kJ / mol), V258G (-2.24kJ / mol) and S259N (-2.11kJ / mol).

[0032] Example 2 Construction of Single-Point Mutant Expression Vector Primers were designed to construct corresponding mutants based on the 19 sites (E26V, T37P, A47I, T56F, V82G, N92K, A101S, T112V, G113A, A131P, T132L, S139Y, A166P, T174V, Q200L, S210Q, T254P, V258G, and S259N) obtained from the analysis in Example 1. The primers corresponding to the 19 mutants are shown in Table 1, and the primer sequences are shown in SEQ ID NO. 4~41.

[0033] Table 1 Primers corresponding to point mutants

[0034] The construction process of expression vectors for the 19 point mutants is as follows (taking mutant E26V as an example, and so on for the others): Using the vector pHY17-aprbcpm as a template, PCR amplification was first performed using upstream and downstream primers E26V-fw and E26V-rev. The upstream primer sequence information is shown in SEQ ID NO. 4, and the downstream primer sequence information is shown in SEQ ID NO. 5. The PCR reaction system is shown in Table 2 below. The PCR amplification conditions were: 98℃ pre-denaturation for 20 seconds, 98℃ denaturation for 10 seconds, 56℃ annealing for 20 seconds, 72℃ extension for 50 seconds, 33 amplification cycles, and a final extension at 72℃ for 5 minutes.

[0035] Table 2 PCR reaction system

[0036] The PCR amplification results were detected by agarose gel electrophoresis, and the target PCR product was purified and recovered. The PCR product recovery process is roughly as follows: (1) the target product was cut into a gel and placed into a 2mL centrifuge tube; (2) the sol solution was added and reacted at 60℃ for 10 minutes; (3) the sol solution from (2) was added to a collection tube and centrifuged at 10000rpm for 1 minute; (4) the product was washed twice with 75% ethanol and then dried; (5) 50μL of water was added and centrifuged for 3 minutes.

[0037] The presence of the template plasmid pHY17-aprbcpm can lead to false positives during transformation and culture PCR; therefore, the template plasmid pHY17-aprbcpm needs to be removed. The purified PCR product is then digested with the restriction endonuclease DpnI.

[0038] The enzymatically digested product was transformed into *E. coli* Top10 using the heat shock method, and the recombinant transformants were verified by bacterial culture PCR. The specific steps for the bacterial culture PCR verification experiment were as follows: a single colony was picked up with an autoclaved toothpick and placed in 500 μL of LBK medium, incubated at 37°C and 200 rpm for 4 hours; 2 μL of bacterial culture was used as the PCR template, and the PCR reaction system is shown in Table 3; the primers used for bacterial culture PCR were bcp-fw and bcp-rev, and their sequence information is shown in SEQ ID NO. 42-43; the PCR amplification conditions were: 95°C pre-denaturation for 3 minutes, 94°C denaturation for 30 seconds, 50°C annealing for 30 seconds, 72°C extension for 60 seconds, 33 cycles, and a final extension at 72°C for 5 minutes.

[0039] The results of bacterial PCR amplification were observed by agarose gel electrophoresis. Plasmids of correctly transformed individuals were extracted and sequenced to verify their PCR performance. The vector pHY17-aprbcpm1 corresponding to the mutant E26V was ultimately obtained.

[0040] Table 3 Bacterial PCR Reaction System

[0041] The sequence information for primers bcp-fw and bcp-rev is as follows: bcp-fw:GACAGGTTCTGGGTGAAAAAG (SEQ ID NO.42); bcp-rev: CGCTTACATACAAGTTCGTG (SEQ ID NO. 43).

[0042] The expression vectors corresponding to the remaining 18 point mutants were obtained using the same method, namely: pHY17-aprbcpm2 (T37P), pHY17-aprbcpm3 (A47I), pHY17-aprbcpm4 (T56F), pHY17-aprbcpm5 (V82G), pHY17-aprbcpm6 (N92K), pHY17-aprbcpm7 (A101S), pHY17-aprbcpm8 (T112V), pHY17-aprbcpm9 (G113A), and pHY17-aprbcpm2 (T37P). 10 (A131P), pHY17-aprbcpm11 (T132L), pHY17-aprbcpm12 (S139Y), pHY17-aprbcpm13 (A166P), pHY17-aprbcpm14 (T174V), pHY17-aprbcpm15 (Q200L), pHY17-aprbcpm16 (S210Q), pHY17-aprbcpm17 (T254P), pHY17-aprbcpm18 (V258G), and pHY17-aprbcpm19 (S259N).

[0043] Example 3 Construction and screening of point mutant recombinant engineered bacteria The expression vectors corresponding to the 19 point mutants obtained in Example 2 were pHY17-aprbcpm1 (E26V), pHY17-aprbcpm2 (T37P), pHY17-aprbcpm3 (A47I), pHY17-aprbcpm4 (T56F), pHY17-aprbcpm5 (V82G), pHY17-aprbcpm6 (N92K), pHY17-aprbcpm7 (A101S), pHY17-aprbcpm8 (T112V), pHY17-aprbcpm9 (G113A), and pHY17-aprbcpm10 (E26V), pHY17-aprbcpm6 (V82G), pHY17-aprbcpm7 (A101S), pHY17-aprbcpm8 (T112V), pHY17-aprbcpm9 (G113A), and pHY17-aprbcpm10 (E26V), ... The following strains were transformed into Bacillus subtilis 1285: pHY17-aprbcpm11 (T132L), pHY17-aprbcpm12 (S139Y), pHY17-aprbcpm13 (A166P), pHY17-aprbcpm14 (T174V), pHY17-aprbcpm15 (Q200L), pHY17-aprbcpm16 (S210Q), pHY17-aprbcpm17 (T254P), pHY17-aprbcpm18 (V258G), and pHY17-aprbcpm19 (S259N).

[0044] The construction process of recombinant Bacillus subtilis mainly includes two experimental steps: preparation of Bacillus subtilis 1285 competent cells and electroconversion.

[0045] The preparation process of Bacillus subtilis 1285 competent cells is roughly as follows: (1) Pick a single colony (2~3 mm in diameter) from a plate cultured at 37℃ for 20 hours, transfer it to a 50 mL centrifuge tube containing 5 mL LB medium, and shake it vigorously overnight at 37℃; (2) Inoculate 1% of the culture medium into 50 mL GM (LB + 0.5 M sorbitol), measure the OD in the shaking tube, and control the inoculation amount so that the OD of the culture medium after inoculation is between 0.19 and 0.2. (3) Incubate at 37℃ and 200rpm until OD600=1.0; (4) Take all the bacterial culture and incubate in an ice water bath for 10 minutes, then centrifuge at 5000rpm for 8 minutes at 4℃ to collect the bacterial cells; (5) Wash the bacterial cells with 30mL of pre-cooled electroporation buffer ETM (0.5M sorbitol, 0.5M mannitol, 10% glycerol, and 0.5M trehalose to improve efficiency), centrifuge at 5000rpm for 8 minutes at 4℃ to remove the supernatant, and repeat this washing process 3 times; (6) Resuspend the washed bacterial cells in 500μL of ETM and dispense 100μL into each tube.

[0046] The transformation process is roughly as follows: (1) Add 6 μL of expression vectors of different site variants to 100 μL competent cells, incubate on ice for 10 minutes, add to a pre-cooled electroporation cuvette (1 mm), and electroporate once. Electroporator settings: 2.0 kV, 25 μF, 200 Ω, 1 mm, electroporation once (duration between 4.5 ms and 5 ms); (2) After electroporation, immediately add 0.5 mL of recovery medium RM (LB + 0.5 M sorbitol + 0.38 M mannitol), 37 °C, 120 rpm, after recovery for 3 hours, spread the transformant on LBK solid plates and incubate overnight at 37 °C.

[0047] Using toothpicks, each recombinant Bacillus subtilis transformant corresponding to a single-point mutant was individually transferred to a 24-well plate containing 1.6 mL of maltose medium per well. After incubation at 37°C and 200 rpm for 24 hours, the supernatant was collected by centrifugation for enzyme activity determination. For each single-point mutant, the recombinant engineered strain with the highest enzyme activity was selected for shake-flask culture. Shake-flask culture was conducted in 250 mL Erlenmeyer flasks. First, the corresponding recombinant engineered strain was inoculated into a 50 mL centrifuge tube containing 5 mL of maltose medium and incubated at 30°C and 220 rpm for approximately 24 hours. Then, the cultured recombinant Bacillus subtilis engineered strain was inoculated at a rate of 1% (v / v) into a 250 mL Erlenmeyer flask containing 50 mL of maltose medium. Shake-flask culture was carried out at 37°C and 200 rpm for 48 hours, after which samples were collected for activity determination and thermostability testing.

[0048] The alkaline protease activity was determined in accordance with the national standard GB / T 23527.1-2023 "Quality Requirements for Enzyme Preparations Part 1: Protease Preparations". One unit of enzyme activity, denoted as U, is defined as 1 μg of tyrosine produced per minute from the hydrolysis of casein.

[0049] The thermal stability test method is as follows: the fermentation supernatant enzyme solution is diluted 5 times, and the diluted enzyme solution is incubated in a 55℃ water bath for 30 minutes before the residual enzyme activity is determined. The sample without heat treatment is used as a control. The enzyme activity and preliminary thermal stability of the starting template AprBcpM and the point mutant recombinant bacteria after 48 hours of shake-flask culture are shown in Table 4.

[0050] As shown in Table 4, compared with the starting template AprBcpM, 6 out of the 20 single-point mutants have better thermal stability. These 6 single-point mutants are T37P, T56F, V82G, A101S, A166P and V258G.

[0051] After incubation in a 55℃ water bath for 30 minutes, the residual enzyme activities of mutants T37P, T56F, V82G, A101S, A166P, and V258G were 1.09, 1.11, 1.07, 1.14, 1.1, and 1.08 times that of AprBcpM, respectively.

[0052] Table 4. Enzyme activity and thermostability of different point mutants in shake flasks

[0053] Example 4 Construction and screening of combined mutants Example 3 identified six single-point mutants with better thermal stability than the starting template AprBcpM. To further improve thermal stability, these six effective mutants were subjected to combined mutations. Since mutant A101S showed the most significant improvement, it was used as the starting template for combined mutations.

[0054] First, double mutant combinations were performed, namely A101S-T37P, A101S-T56F, A101S-V82G, A101S-A166P, and A101S-V258G.

[0055] The construction process of different dual-combination expression vectors was carried out in accordance with Example 2, and the process is as follows: (1) Using pHY17-aprbcpm7 as a template, PCR amplification was performed with the corresponding primers, and the PCR product was purified and recovered; (2) The PCR product was purified by treatment with restriction endonuclease DpnI and then transformed into Escherichia coli Top10; (3) Positive transformants were obtained by bacterial PCR verification, and different dual-combination mutant expression vectors pHY17-aprbcpm20 (A101S-T37P), pHY17-aprbcpm21 (A101S-T56F), pHY17-aprbcpm22 (A101S-C81G), pHY17-aprbcpm23 (A101S-A166P) and pHY17-aprbcpm24 (A101S-V258G) were obtained by sequencing verification.

[0056] The construction process of different dual-combination mutant recombinant Bacillus subtilis was the same as in Example 3. The dual-combination expression vectors were transformed into Bacillus subtilis 1285 by electroporation. The screening and thermal stability determination of the recombinant transformants were carried out in accordance with Example 3, and the experimental results are shown in Table 5.

[0057] The thermostability improvement effect of the dual mutants A101S-C81G and A101S-V258G was not significant. However, the dual mutants A101S-T37P, A101S-T56F, and A101S-A166P could further improve thermostability. After heat treatment at 55℃ for 30 minutes, the residual enzyme activities were 1.25 times, 1.21 times, and 1.19 times that of AprBcpM, respectively.

[0058] Table 5. Enzyme activity and thermal stability of the dual-mutant strains in shake flasks.

[0059] Based on the two-combination mutation, multiple-combination mutations were further performed. Using A101S-T37P as the starting template, multiple-combination mutant expression vectors were constructed, and the construction process was consistent with that in Example 2. Through experiments, expression vectors corresponding to the three-combination mutants were obtained: pHY17-aprbcpm25 (A101S-T37P-T56F), pHY17-aprbcpm26 (A101S-T37P-A166P), and pHY17-aprbcpm27 (A101S-T37P-T56F-A166P).

[0060] Multiple mutant expression vectors were transformed into Bacillus subtilis 1285. The screening and thermal stability determination of recombinant transformants were carried out in accordance with Example 3. The experimental results are shown in Table 6.

[0061] Experimental results showed that multiple mutant combinations could further improve thermal stability. After heat treatment at 55℃ for 30 minutes, the residual enzyme activities of the multiple mutant combinations A101S-T37P-T56F, A101S-T37P-A166P and A101S-T37P-T56F-A166P were 1.43 times, 1.51 times and 1.62 times that of AprBcpM, respectively.

[0062] For ease of writing, A101S-T37P-T56F-A166P is abbreviated as AprBcpM1. The three-dimensional conformation of the mutant AprBcpM1 is obtained through 3D modeling, as follows... Figure 1 As shown in B, all four single-point mutants were found to be located in the irregularly curled region of the three-dimensional conformation.

[0063] Table 6. Enzyme activity and thermal stability of multiple mutant combinations in shake flasks

[0064] Example 5: Thermal stability determination of mutant AprBcpM1 The thermal stability of the combined mutant AprBcpM1 was determined, with the starting template AprBcpM serving as a control throughout the determination process.

[0065] The thermostability of the combined mutant AprBcpM1 was determined as follows: Residual enzyme activity was measured after incubation in a water bath at 55℃, 60℃, 65℃, and 70℃ for 30 minutes, 60 minutes, and 90 minutes, respectively. The enzyme activity of the untreated sample was taken as 100%, and the relative residual enzyme activity at other temperatures was calculated. The experimental results are as follows: Figure 2 As shown.

[0066] Depend on Figure 2 It was found that after heat treatment at 55℃ for 30 minutes, the remaining enzyme activities of AprBcpM and the mutant AprBcpM1 were 41.2% and 66.8%, respectively. When the temperature was increased to 60℃ and heat treated for 30 minutes, the remaining enzyme activities of AprBcpM and the mutant AprBcpM1 were 23.8% and 45.2%, respectively, with the remaining enzyme activity of the mutant AprBcpM1 being 1.9 times that of AprBcpM. As the heat treatment temperature increased, the thermostability advantage of the mutant AprBcpM1 became more obvious. After heat treatment at 65℃ for 30 minutes, the remaining enzyme activities of the two were 8.9% and 23.1%, respectively, with the remaining enzyme activity of the mutant AprBcpM1 being 2.6 times that of AprBcpM. After heat treatment at 70℃ for 30 minutes, the remaining enzyme activity of AprBcpM was only 2.1%, while the mutant AprBcpM1 still retained 8.2% residual enzyme activity, which was 3.9 times that of AprBcpM.

[0067] Example 6 Storage stability determination of mutant AprBcpM1 The storage stability of the mutant AprBcpM1 was assessed, with the starting template AprBcpM serving as a control.

[0068] The stability assessment method of mutant AprBcpM1 is as follows: (1) Sample preparation: mutant AprBcpM1 and AprBcpM liquid enzyme were prepared according to Example 2; (2) Sample processing: mutant AprBcpM1 and AprBcpM liquid enzyme were sterilized by filtration through a 0.22-micron filter membrane and then dispensed into 1.5 mL centrifuge tubes; (3) Stability test at different temperatures: the dispensed enzyme solutions were placed at 25℃ and 37℃ respectively, and samples were taken every 5 days for enzyme activity determination. The activity determination was carried out according to Example 3; (4) Data statistics: the enzyme activity on day 0 was used as a control, and the percentage of enzyme activity measured by samples at different storage times divided by the enzyme activity on day 0 was used to calculate the enzyme activity remaining rate.

[0069] Storage stability of mutant AprBcpM1 at different temperatures, such as Figure 3 As shown.

[0070] Depend on Figure 3 As shown in A, at 25℃, the mutant AprBcpM1 has better stability than the starting template AprBcpM. After 30 days of storage, its remaining enzyme activity is 80.3%, while that of the starting template AprBcpM is 60.1%.

[0071] Depend on Figure 3 As shown in B, at 37°C, the mutant AprBcpM1 also has better stability than the starting template AprBcpM. After 30 days of storage, its remaining enzyme activity is 70.5%, while that of the starting template AprBcpM is 30.2%.

[0072] Example 7: Application of mutant AprBcpM1 in fermented feed for Litopenaeus vannamei Proteases play a crucial role in shrimp feed fermentation, breaking down feed proteins into small peptides. These peptides provide a nitrogen source for fermenting microorganisms and possess various biological activities that help enhance shrimp immunity. 1.0mm particle size Litopenaeus vannamei feed was purchased from Jiangmen Aohua Biotechnology Co., Ltd. for fermentation. 800g of feed was weighed and mixed with 180g of sterile water and 3g of a compound microbial agent (purchased from Wuhan Xinhua Yang Biotechnology Co., Ltd., product model Anteno Series 1) containing Bacillus coagulans, Enterococcus faecalis, and Clostridium butyricum, with a viable count of 10-1. 10The mixture was prepared using CFU / g of feed and either the mutant AprBcpM1 or the starting template AprBcpM. Both the mutant AprBcpM1 and the starting template AprBcpM were added at a concentration of 1 U / g of feed. The thoroughly mixed material was then placed in fermentation bags equipped with a single vent valve, heated and sealed, and stored at 30°C. Samples were taken every 7 days to determine the acid-soluble protein content. The method for determining the acid-soluble protein content followed GB / T 22492-2008 "Soybean Peptide Powder".

[0073] The changes in acid-soluble protein content in fermented feed samples of Litopenaeus vannamei corresponding to mutant AprBcpM1 and starting template AprBcpM are as follows: Figure 4 As shown. By Figure 4 It can be seen that the acid-soluble protein content of the sample corresponding to the mutant AprBcpM1 was higher than that of the sample corresponding to the starting template AprBcpM throughout the fermentation process. After 35 days of fermentation, the acid-soluble protein content of the sample corresponding to the mutant AprBcpM1 was 28.1%, while that of the sample corresponding to the starting template AprBcpM was 20.8%.

[0074] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A protease mutant AprBcpMl, characterized in that, The amino acid sequence of the protease mutant AprBcpM1 is shown in SEQ ID NO.

1.

2. The protease mutant AprBcpMl according to claim 1, characterized in that The sequence encoding the amino acid is a polynucleotide sequence, as shown in SEQ ID NO.

2.

3. A recombinant expression vector pHY17-aprbcpm27, characterized by, It includes the encoding gene of the protease mutant AprBcpM1 as described in claim 1.

4. A recombinant engineered bacterium, characterized in that, It includes the recombinant expression vector pHY17-aprbcpm27 as described in claim 3.

5. The recombinant engineered bacteria according to claim 4, characterized in that, The strain is Bacillus subtilis.

6. The recombinant engineered bacteria according to claim 5, characterized in that, The strain is Bacillus subtilis 1285.

7. The application of the protease mutant AprBcpM1 as described in claim 1 in the preparation of fermented feed for Litopenaeus vannamei.

8. The application according to claim 7, characterized in that, The application includes adding the alkaline protease mutant AprBcpM1 to the feed ingredients of Litopenaeus vannamei and performing fermentation treatment.

9. The application according to claim 8, characterized in that, The fermentation process is carried out at a temperature of 25-35°C.

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