Alkaline protease mutant and application thereof

Abstract

The invention relates to the technical field of genetic engineering and protein engineering modification, in particular to an alkaline protease mutant and application thereof. The present invention relates to a mutant comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 1 and comprising a substitution of an amino acid at at least one position selected from the group consisting of 63, 109, 120, 154, 191, 261, 328 compared to SEQ ID NO: 1. The specific activity of the mutant is generally improved by 13.12%-79.32% compared with that of a wild type, the production cost of the enzyme can be reduced, and wide application of the enzyme in the industrial field is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering and protein engineering, and particularly relates to a basic protease mutant and application thereof. BACKGROUND

[0002] Basic protease is a kind of enzyme active in alkaline environment, mainly used for catalyzing the hydrolysis of proteins. This kind of enzyme usually shows the best activity under the condition of pH value ranging from 8 to 12. The mechanism of basic protease is to hydrolyze the peptide bond of proteins, so as to convert the complex macromolecular protein structure into simple small molecular peptide chains or amino acids, thereby making them easy to be absorbed or washed away. This characteristic makes it play an important role in the industry requiring protein decomposition, and is widely used in the industries of detergent, food, medicine, brewing, silk, leather making, etc.

[0003] In the detergent, basic protease can decompose protein stains into soluble amino acids and small molecular peptides, thereby being easy to be cleaned. In food processing, it can make the complex macromolecular protein structure into simple small molecular peptide chains or amino acids, thereby being easy to be absorbed or removed. In addition, basic protease is also used in the pharmaceutical industry, has high activity and specificity, and is suitable for producing specific molecules. In the environmental protection field, it promotes the degradation of environmental pollutants. The main source of basic protease is microbial extraction, and the main research and application is Bacillus, with Bacillus subtilis being the most, and a small amount of other strains, such as Streptomyces. The production capacity and enzyme activity and stability of natural strains often cannot meet the needs of industrial production, and the strains need to be screened and improved. Common methods include mutagenesis, genetic engineering, protein engineering, spore heat treatment, etc. The main goal is to improve the activity, stability (temperature resistance and alkali resistance), oxidation resistance and chelation resistance of the enzyme, so as to promote the more extensive application of basic protease. SUMMARY

[0004] The purpose of the present application is to provide a basic protease mutant with high specific activity. The specific activity of the mutant is significantly improved compared with the wild type, thereby facilitating the extensive application of basic protease.

[0005] The present application relates to a basic protease mutant comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1, and comprising a substitution of an amino acid at at least one position selected from the group consisting of 63, 109, 120, 154, 191, 261, 328, compared to SEQ ID NO: 1.

[0006] In some embodiments of the present application, the amino acid sequence of the mutant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 1.

[0007] In some more particular embodiments, the amino acid sequence of the mutant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity to SEQ ID NO: 1.

[0008] In some embodiments of the application, the mutant comprises a substitution of at least one amino acid in the group: Q63K, A109T, K120N, G154W, G191V, A261L, T328G.

[0009] In some embodiments of the application, the mutant comprises a substitution or combination of substitutions selected from the group consisting of: Q63K; A109T; K120N; G154W; G191V; A261L; T328G; Q63K / A109T; Q63K / K120N; Q63K / G191V; Q63K / A261L; Q63K / T328G; A109T / G191V; A109T / G191V; A109T / A261L; A109T / T328G; K120N / G191V; K120N / A261L; K120N / T328G; G191V / A261L; G191V / T328G; A261L / T328G; Q63K / A109T / K120N; Q63K / K120N / G191V; Q63K / A261L / T328G; Q63K / A109T / T328G; A109T / G191V / A261L; A109T / K120N / A261L; K120N / G191V / A261L; K120N / A261L / T328G; G191V / A261L / T328G; Q63K / A109T / A261L / T328G; A109T / G191V / A261L / T328G; A109T / K120N / G154W / G191V; A109T / G154W / G191V / A261L; K120N\G154W\G191V\A261L\T328G; Q63K\A109T\K120N\G154W\G191V\A261L; A109T\K120N\G154W\G191V\A261L\T328G; Q63K\A109T\K120N\G154W\G191V\A261L\T328G.

[0010] The present application also relates to a DNA molecule encoding the above-mentioned alkaline protease mutant.

[0011] The present application also relates to a recombinant expression vector comprising the above-mentioned DNA molecule.

[0012] In some embodiments of the present application, a host cell is involved.

[0013] The host cell is Bacillus subtilis (B. subtilis) Bacillus subtilis ).

[0014] The present application provides a single-point mutant comprising any one of the mutation sites of Q63K, A109T, K120N, G154W, G191V, A261L and T328G based on wild-type alkaline protease AKP. The specific activity of the single-point mutant is generally increased by 13.12%-38.13%, and the specific activity of the G154W single-point mutant is the highest, reaching 13005 U / mg, which achieves an unexpected technical effect, is conducive to reducing the production cost of the enzyme, and promotes its wide application in the industrial field. DETAILED DESCRIPTION

[0015] The experimental methods not specified in the examples can be operated according to the conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Manual written by J. Sambrook et al., or the conditions suggested by the manufacturer. The relevant personnel in the field can better understand and master the present application with the help of the examples. However, the method for realizing the present application should not be limited to the specific method steps recorded in the examples of the present application.

[0016] The formula of the culture medium involved in the examples of the present application is as follows: LB medium: tryptone 1%, yeast extract 0.5%, NaCl 0.5%; LB plate: tryptone 1%, yeast extract 0.5%, NaCl 0.5%, agar 2%; The preparation method of GM I is as follows: 1* minimal salt solution 95.6 mL, 20% glucose 2.5 mL, 5% hydrolyzed casein 0.4 mL, 10% yeast extract 1 mL; wherein the preparation method of 1* minimal salt solution is as follows: K2HPO4 14 g / L, KH2PO4 6 g / L, (NH4)2SO4 2 g / L, trisodium citrate 1 g / L, MgSO4•7H2O 0.2 g / L, sequentially dissolved in distilled water; The preparation method of GM II is as follows: 1* minimal salt solution 96.98 mL, 20% glucose 2.5 mL, 5% hydrolyzed casein 0.08 mL, 10% yeast extract 0.04 mL, 1 M MgCl2 0.25 mL, 1 M CaCl2 0.05 mL; Skim milk plate: tryptone 1%, yeast extract 0.5%, NaCl 0.5%, skim milk 1%, agar 1.5%; Liquid fermentation medium: yeast extract 0.5%, tryptone 0.5%, glucose 1%, K2HPO4 1.8%.

[0017] The application will be further described below in combination with specific examples.

[0018] Example 1 Screening of high specific activity alkaline protease mutants The amino acid sequence of the alkaline protease gene AKP is SEQ ID NO: 1, and the encoding nucleotide sequence is SEQ ID NO: 2. First, the nucleotide sequence of the enzyme is optimized according to the codon bias of Bacillus, and the optimized sequence is synthesized by Beijing Lihe Huada Gene Technology Co., Ltd.

[0019] The synthesized alkaline protease AKP gene sequence is used as a template, primers are designed, PCR amplification is performed by using a GeneMorph II random mutation PCR kit (Biomass), the PCR product is recovered by gel, KpnI and MLuI are used for double enzyme digestion, and the enzyme treatment is connected with the pSZX101 vector which has been subjected to the same enzyme digestion, transformed into Escherichia coli DH5α, coated on LB+Amp plates, and cultured at 37°C, and after the appearance of the transformants, the plasmid is extracted and transformed into Bacillus subtilis. Bacillus

[0020] ​After the transformants grew, they were picked up one by one with a toothpick and transferred to a 48-well plate. 20 μg / mL kanamycin was added to each well. The plate was incubated at 37℃ and 500 rpm for about 48 hours. The supernatant was centrifuged and high-throughput analysis was performed to determine the enzyme activity and protein content of the transformants. The specific activity of different mutants was calculated.

[0021] Experimental results showed that some mutations significantly increased the specific activity of alkaline protease AKP, while others resulted in a decrease in its specific activity. Additionally, some mutations, although increasing the specific activity of AKP, significantly altered its enzymatic properties, which did not meet the requirements. The applicant ultimately selected mutation sites that could significantly increase the specific activity of AKP without significantly affecting its original enzymatic properties: Q63K, A109T, K120N, G154W, G191V, A261L, and T328G.

[0022] Based on wild-type alkaline protease AKP, this invention provides mutants containing single mutation sites of Q63K, A109T, K120N, G154W, G191V, A261L, and T328G respectively.

[0023] The present invention further provides mutants comprising at least two mutation sites selected from Q63K, A109T, K120N, G154W, G191V, A261L, and T328G. Examples include: Q63K / A109T, Q63K / K120N, Q63K / G191V, Q63K / A261L, Q63K / T328G, A109T / G191V, A109T / G191V, A109T / A261L, A109T / T328G, K120N / G191V, K120N / A261L, K120N / T328G, and G191V / A2. 61L, G191V / T328G, A261L / T328G two-point mutants; Q63K / A109T / K120N, Q63K / K120N / G191V, Q63K / A261L / T328G, Q63K / A109T / T328G, A109T / G191V / A261L, A109T / K120N / A261L, K120N / G191V / A2 61L, K120N / A261L / T328G, G191V / A261L / T328G three-point mutants; Q63K / A109T / A261L / T328G, A109T / G191V / A261L / T328G, A109T / K120N / G154W / G191V, A109T / G154W / G191V / A261L four-point mutants; K120N\ The five-point mutant G154W\G191V\A261L\T328G; the six-point mutant Q63K\A109T\K120N\G154W\G191V\A261L, A109T\K120N\G154W\G191V\A261L\T328G; and the seven-point mutant Q63K\A109T\K120N\G154W\G191V\A261L\T328G.

[0024] Referring to the amino acid sequence of the mutant, the encoding nucleotide sequence of the alkaline protease mutant was obtained.

[0025] Example 2: Expression of alkaline protease mutant in Bacillus subtilis Based on the codon preference of Bacillus, the gene sequences of alkaline protease AKP and its single-point mutant were optimized and synthesized, and two restriction sites, KpnI and MLuI, were added to the 5' and 3' ends of the synthesized sequence, respectively.

[0026] The alkaline protease gene fragment and the expression vector pSZX101 were double-digested with KpnI and MLUI, respectively, and the target fragment was recovered by gel electrophoresis. The fragments were ligated overnight with T4 ligase. The ligation products were transformed into E. coli DH5α competent cells, plated on LB+Amp plates, and incubated overnight at 37°C. Single colonies grew after incubation. Colony PCR was used to verify the correct ligation of the transformants. Plasmids were extracted from the transformed cells and sent to the Beijing BGI Genomics Research Center for sequencing analysis.

[0027] Plasmids were purified from correctly sequenced E. coli clones using a plasmid medium-quantity preparation kit (Axygen).

[0028] Transformation procedure: Freshly activated Bacillus subtilis 1A75 was inoculated onto LB agar plates into 5 mL of GMⅠ solution and cultured overnight at 30℃ and 125 rpm with shaking. The next day, 1 mL of the culture was transferred to 9 mL of GMMI and cultured at 37℃ and 220 rpm for 3.5 h. Then, 1 mL of the culture medium from the previous step was transferred to 9 mL of GMⅡ solution and cultured at 37℃ and 125 rpm for 90 min. The cells were then collected by centrifugation at 5000g for 10 min. The cells were gently resuspended in 1 mL of GMⅡ solution. The resuspended cells are competent cells and can be used for transformation. Preservation of competent cells: 30% sterile glycerol was added to a final concentration of 10%, mixed well, and aliquoted into centrifuge tubes. The tubes were then stored at -70℃.

[0029] Mix 1 μg of recombinant plasmid with 200 μL of the above competent cells thoroughly, incubate at 37°C with shaking (200 rpm) for 30 min, then spread the mixture onto skim milk plates containing 30 μg / mL kanamycin and incubate overnight at 37°C. The single colony that grows overnight is the engineered Bacillus subtilis strain containing alkaline protease AKP and its single-point mutant.

[0030] Example 3: Specific activity analysis of alkaline protease mutants The recombinant Bacillus subtilis strains expressing wild-type alkaline protease AKP or its mutants obtained in Example 2 were inoculated into liquid fermentation medium. After 48 h of shake-flask fermentation, the supernatant was collected by centrifugation at 5000 rpm for 10 min. The protease activity and protein content in the supernatant were measured, and the specific activity was calculated. The specific results are shown in Table 1.

[0031] Table 1 Comparison of specific activities of alkaline protease mutants Alkaline protease Specific activity (U / mg) Wild type AKP 9415 Q63K single point mutant 11981 A109T single point mutant 11082 K120N single point mutant 10650 G154W single point mutant 13005 G191V single point mutant 12554 A261L single point mutant 12206 T328G single point mutant 12758 As can be seen from the data in Table 1, compared with wild-type alkaline protease AKP, the specific activity of the single-point mutant alkaline protease provided by the present invention is increased by 13.12%-38.13%, among which the G154W single-point mutant has the highest specific activity, reaching 13005 U / mg, achieving unexpected technical effects.

[0032] The above results indicate that the mutation sites Q63K, A109T, K120N, G154W, G191V, A261L, and T328G provided by this invention can significantly improve the specific activity of wild-type alkaline protease AKP, which is beneficial to reducing the production cost of the enzyme and promoting its widespread application in the field of industrial enzymes.

[0033] (a) Methods for determining protease activity: 1. Principle Under specific temperature and pH conditions, proteases hydrolyze casein substrates to produce amino acids containing phenolic groups (such as tyrosine and tryptophan). Under alkaline conditions, Folin reagent is reduced to produce molybdenum blue and tungsten blue. The absorbance of the solution is measured at a wavelength of 680 nm using a spectrophotometer. Enzyme activity is directly proportional to absorbance, thus allowing the calculation of the product's enzyme activity.

[0034] 2. Definition of enzyme activity The definition of protease activity, expressed in units, is as follows: 1 g of solid enzyme powder (or 1 mL of liquid enzyme) hydrolyzes casein to produce 1 μg of tyrosine in 1 minute under certain temperature and pH conditions, which is 1 unit of enzyme activity, expressed as u / g (u / mL).

[0035] 3. Reagents and solutions (1) Folin reagent (Folin:water = 1:2); (2) 42.4 g / L sodium carbonate solution; (3) 0.5 mol / L sodium hydroxide solution; (4) borate buffer (pH 10.5); (5) 10.0 g / L casein solution; (6) 100 g / mL and 1 mg / mL L-tyrosine standard solutions; (7) 6.54% trichloroacetic acid.

[0036] 4. Measurement Method (1) Preparation of standard curve: Prepare L-tyrosine standard solutions with concentrations of 0 g / mL, 10 g / mL, 20 g / mL, 30 g / mL, 40 g / mL and 50 g / mL. Take 1.00 mL of each standard solution, add 5.00 mL of 0.4 mol / L sodium carbonate solution and 1.00 mL of Folin reagent working solution, shake well, and place in a 40℃ water bath for color development for 20 min. Remove and use a spectrophotometer at a wavelength of 680 nm with a 10 mm cuvette, using a tyrosine-free tube (C) as a blank, to measure the absorbance of each. Plot the standard curve with absorbance A as the ordinate and the concentration of tyrosine C as the abscissa (this line should pass through the zero point).

[0037] (2) Enzyme activity assay Take a pre-diluted amount of enzyme solution, then add an equal volume of 10% casein preheated at 40℃, and react at 40℃ for 10 min. Then add an equal volume of trichloroacetic acid (6.54% concentration) to the reaction system, mix well, and let stand at room temperature for 10 min to terminate the reaction. Take 1 mL of the terminated reaction solution, then add 5 mL of 42.4 g / L sodium carbonate solution, followed by 1 mL of Folin reagent, and perform a colorimetric reaction at 40℃ for 20 min. Finally, measure the OD608 value.

[0038] (3) Calculation Read the enzyme activity of the final diluted sample from the standard curve, in units of u / mL. The enzyme activity of the sample is calculated using the following formula: X = (A × K × 4 × n) / 10.

[0039] Where: X — enzyme activity of the sample (U / g or U / ML); A—The average absorbance of the sample in parallel tests; K—absorption constant; 4 — Total volume of reaction reagents (mL); 10 — Reaction time 10 min, calculated as 1 min; n – dilution factor.

[0040] (II) Methods for determining protein content: The Bradford binding method for protein determination is a combined colorimetric and dye-based method. Coomassie Brilliant Blue G-250 is brownish-red in acidic solution, turning blue upon binding to protein. Within a certain protein concentration range, it follows Beer's Law and can be measured colorimetrically at 595 nm. It exhibits significant absorption within 3–5 minutes and remains stable for at least 1 hour. In the range of 10–1000 μg / mL, the absorbance is directly proportional to the protein concentration. The enzyme solution and Coomassie Brilliant Blue solution are mixed at a volume ratio of 1:5, allowed to stand for 10 minutes, and then the protein content is determined using the Bradford binding method. (III) Specific vitality calculation: "Specific Activity" refers to the number of enzyme activity units per unit weight of protein, usually expressed as U / mg protein.

[0041] Specific activity calculation formula: Specific activity (U / mg) = enzyme activity (U / mL) / protein content (mg / mL).

Claims

1. An alkaline protease mutant, characterized in that, The mutant is an alkaline protease with the amino acid sequence SEQ ID NO:1, in which the 109th amino acid is mutated from Ala to Thr.

2. A DNA molecule encoding the alkaline protease mutant of claim 1.

3. A recombinant expression plasmid, characterized in that, The recombinant expression plasmid comprises the DNA molecule of claim 2.

4. A host cell, characterized in that, The host cell comprises the recombinant expression plasmid as described in claim 3; the host cell is a non-animal or non-plant variety.

5. The host cell as described in claim 4, characterized in that, The host cell is Bacillus subtilis (B. subtilis) Bacillus subtilis ).