A mutant of alkaline protease with improved specific activity

Abstract

The present application relates to the technical field of genetic engineering and protein engineering, and particularly relates to a basic protease mutant with improved specific activity. The mutant comprises an amino acid sequence with at least 90% identity with SEQ ID NO: 1, and comprises substitution of amino acids at at least one position selected from the group consisting of 63, 109, 120, 154, 191, 261 and 328, compared with SEQ ID NO: 1. The specific activity of the mutant is generally increased by 13.12%-79.32% compared with wild type, which is beneficial to reduce the production cost of the enzyme and promote its wide application in the field of industrial enzymes.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein engineering, specifically to an alkaline protease mutant with increased specific activity. Background Technology

[0002] Alkaline proteases are a class of enzymes active in alkaline environments, primarily used to catalyze the hydrolysis of proteins. These enzymes typically exhibit optimal activity within a pH range of 8 to 12. The mechanism of action of alkaline proteases involves hydrolyzing protein peptide bonds, breaking down complex macromolecular protein structures into simpler small peptide chains or amino acids, making them easier to absorb or wash away. This characteristic makes them crucial in industries requiring protein breakdown, and they are widely used in detergents, food, medical, brewing, silk, and leather industries.

[0003] In detergents, alkaline proteases can break down protein-based stains into soluble amino acids and small peptides, making them easier to clean. In food processing, they can transform complex macromolecular protein structures into simpler small peptide chains or amino acids, facilitating absorption or removal. Furthermore, alkaline proteases are used in the pharmaceutical industry due to their high activity and specificity, making them suitable for producing specific molecules. In the environmental field, they promote the degradation of environmental pollutants.

[0004] The main source of alkaline proteases is microbial extraction, with Bacillus subtilis being the most widely studied and applied, and other species such as Streptomyces also being used to a small extent. The production capacity, enzyme activity, and stability of natural strains often do not meet the requirements of industrial production, necessitating the screening and improvement of strains. Common methods include mutagenesis, genetic engineering, protein engineering, and spore heat treatment. The main goal is to improve the enzyme's activity, stability (temperature and alkali resistance), antioxidant properties, and anti-chelation properties, thereby promoting the wider application of alkaline proteases. Summary of the Invention

[0005] The purpose of this invention is to provide a mutant alkaline protease with increased specific activity. The mutant exhibits a significantly higher specific activity than the wild type, thereby facilitating the widespread application of alkaline proteases.

[0006] The present invention relates to an alkaline protease mutant comprising an amino acid sequence having at least 90% identity with SEQ ID NO:1, and comprising, compared with SEQ ID NO:1, at at least one position of an amino acid substitution selected from the group consisting of: 63, 109, 120, 154, 191, 261, 328.

[0007] In some embodiments of the invention, the amino acid sequence of the mutant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO:1.

[0008] In some more specific 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 with SEQ ID NO:1.

[0009] In some embodiments of the present invention, the mutant comprises a substitution of at least one amino acid from the following group: Q63K, A109T, K120N, G154W, G191V, A261L, T328G.

[0010] In some embodiments of the present invention, the mutant contains substitutions or combinations of substitutions selected from the following substitutions and combinations of substitutions: Q63K; A109T; K120N; G154W; G191V; A261L; T328G;

[0011] Q63K / A109T;

[0012] Q63K / K120N;

[0013] Q63K / G191V;

[0014] Q63K / A261L;

[0015] Q63K / T328G;

[0016] A109T / G191V;

[0017] A109T / G191V;

[0018] A109T / A261L;

[0019] A109T / T328G;

[0020] K120N / G191V;

[0021] K120N / A261L;

[0022] K120N / T328G;

[0023] G191V / A261L;

[0024] G191V / T328G;

[0025] A261L / T328G;

[0026] Q63K / A109T / K120N;

[0027] Q63K / K120N / G191V;

[0028] Q63K / A261L / T328G;

[0029] Q63K / A109T / T328G;

[0030] A109T / G191V / A261L;

[0031] A109T / K120N / A261L;

[0032] K120N / G191V / A261L;

[0033] K120N / A261L / T328G;

[0034] G191V / A261L / T328G;

[0035] Q63K / A109T / A261L / T328G;

[0036] A109T / G191V / A261L / T328G;

[0037] A109T / K120N / G154W / G191V;

[0038] A109T / G154W / G191V / A261L;

[0039] K120N\G154W\G191V\A261L\T328G;

[0040] Q63K\A109T\K120N\G154W\G191V\A261L;

[0041] A109T\K120N\G154W\G191V\A261L\T328G;

[0042] Q63K\A109T\K120N\G154W\G191V\A261L\T328G.

[0043] The present invention also relates to DNA molecules encoding the above-mentioned alkaline protease mutants.

[0044] The present invention also relates to recombinant expression vectors comprising the above-described DNA molecules.

[0045] In some embodiments of the present invention, a host cell is involved.

[0046] The host cell is Bacillus subtilis.

[0047] This invention provides single-point mutants based on wild-type alkaline protease AKP, containing any one of the mutation sites Q63K, A109T, K120N, G154W, G191V, A261L, or T328G. The specific activity of these single-point mutants is generally increased by 13.12%-38.13%, with the G154W single-point mutant exhibiting the highest specific activity at 13005 U / mg. This achieves unexpected technical benefits, helps reduce the production cost of the enzyme, and promotes its widespread application in the industrial field. Detailed Implementation

[0048] Experimental methods not specified in the embodiments can be performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual* by J. Sambrook et al., or according to the manufacturer's recommendations. Those skilled in the art can better understand and master this invention through these embodiments. However, the methods for implementing this invention should not be limited to the specific steps described in the embodiments.

[0049] The culture medium formulation involved in the embodiments of the present invention is as follows:

[0050] LB medium: 1% tryptone, 0.5% yeast extract, 0.5% NaCl;

[0051] LB agar: 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 2% agar;

[0052] The preparation method for GM I is as follows: 95.6 mL of 1* minimum salt solution, 2.5 mL of 20% glucose, 0.4 mL of 5% hydrolyzed casein, and 1 mL of 10% yeast extract; wherein the preparation method for the 1* minimum salt solution is as follows: 14 g / L K2HPO4, 6 g / L KH2PO4, 2 g / L (NH4)2SO4, 1 g / L trisodium citrate, and 0.2 g / L MgSO4·7H2O are dissolved in distilled water in sequence;

[0053] The GMII preparation method is as follows: 96.98 mL of 1* minimum salt solution, 2.5 mL of 20% glucose, 0.08 mL of 5% hydrolyzed casein, 0.04 mL of 10% yeast extract, 0.25 mL of 1M MgCl2, and 0.05 mL of 1M CaCl2.

[0054] Skim milk agar plates: 1% tryptone, 0.5% yeast, 0.5% NaCl, 1% skim milk, 1.5% agar;

[0055] Liquid fermentation medium: yeast extract 0.5%, tryptone 0.5%, glucose 1%, K2HPO4 1.8%.

[0056] The invention will be further illustrated below with specific examples.

[0057] Example 1: Screening of high specific activity alkaline protease mutants

[0058] 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. The nucleotide sequence of this enzyme was first optimized based on the codon bias of Bacillus. The optimized sequence was synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0059] Using the synthesized alkaline protease AKP gene sequence as a template, primers were designed, and PCR amplification was performed using the GeneMorph II random mutagenesis PCR kit (Bomais). The PCR product was recovered from the gel, and KpnI and MLuI were used for double digestion. The product was then ligated into the pSZX101 vector, which had been digested with the same enzymes. The ligation was performed and transformed into Escherichia coli DH5α. The transformed products were plated on LB+Amp plates and incubated upside down at 37°C. After the transformants appeared, the plasmid was extracted and transformed into Bacillus subtilis.

[0060] 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°C 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] Referring to the amino acid sequence of the mutant, the encoding nucleotide sequence of the alkaline protease mutant was obtained.

[0065] Example 2: Expression of alkaline protease mutant in Bacillus subtilis

[0066] 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.

[0067] 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.

[0068] Plasmids were purified from correctly sequenced E. coli clones using a plasmid medium-quantity preparation kit (Axygen).

[0069] Transformation procedure: Freshly activated Bacillus subtilis 1A75 was inoculated onto LB agar plates into 5 mL of GMⅠ solution and cultured overnight at 30°C and 125 rpm with shaking. The next day, 1 mL of the culture was transferred to 9 mL of GMMI and cultured at 37°C and 220 rpm for 3.5 h. Then, 1 mL of the culture from the previous step was transferred to 9 mL of GMⅡ solution and cultured at 37°C 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°C.

[0070] 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 plate 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.

[0071] Example 3: Specific activity analysis of alkaline protease mutants

[0072] 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 hours of shake-flask fermentation, the supernatant was collected by centrifugation at 5000 rpm for 10 minutes. 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.

[0073] Table 1 Comparison of specific activities of alkaline protease mutants

[0074]

[0075]

[0076] 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.

[0077] 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.

[0078] (I) Methods for determining protease activity:

[0079] 1. Principle

[0080] 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, and thus the enzyme activity of the product can be calculated.

[0081] 2. Definition of enzyme activity

[0082] 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. This is 1 unit of enzyme activity, expressed as u / g (u / mL).

[0083] 3. Reagents and Solutions

[0084] (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.

[0085] 4. Measurement Method

[0086] (1) Preparation of the 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 in a 10 mm cuvette, with a blank tube (0 tube) containing no tyrosine, to measure the absorbance of each solution. Plot the standard curve with absorbance A as the ordinate and the concentration C of tyrosine as the abscissa (this line should pass through the zero point).

[0087] (2) Enzyme activity assay

[0088] 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.

[0089] (3) Calculation

[0090] 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:

[0091] X = (A × K × 4 × n) / 10.

[0092] Where: X — enzyme activity of the sample (U / g or U / ML);

[0093] A—The average absorbance of the sample in parallel tests;

[0094] K—absorption constant;

[0095] 4 — Total volume of reaction reagents (mL);

[0096] 10 — Reaction time 10 min, calculated as 1 min;

[0097] n – dilution factor.

[0098] (II) Protein content determination method:

[0099] The Bradford Brilliant Blue binding method for protein content determination is a combined colorimetric and pigment method. Coomassie Brilliant Blue G-250 is brownish-red in acidic solution, turning blue upon binding with 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 specific activity is calculated.

[0100] "Specific Activity" refers to the number of enzyme activity units per unit weight of protein, usually expressed as U / mg protein.

[0101] 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 obtained by mutating the 328th amino acid of the alkaline protease with the amino acid sequence SEQ ID NO:1 from Thr to Gly.

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, and the host cell is Bacillus subtilis (…). Bacillus subtilis ).

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