Alkaline protease mutants and uses thereof

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 to 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 improved by 13.12% to 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] The present application relates to the 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 action of basic protease is to convert complex macromolecular protein structure into simple small molecular peptide chains or amino acids by hydrolyzing the peptide bond of proteins, so that it is easy to be absorbed or washed away. This property makes it play an important role in industries requiring protein decomposition, and is widely used in detergent, food, medical, brewing, silk, leather and other industries.

[0003] In detergents, basic protease can decompose protein stains into soluble amino acids and small molecular peptides, so that it is easy to be cleaned. In food processing, it can convert complex macromolecular protein structure into simple small molecular peptide chains or amino acids, so that it is easy to be absorbed or removed. In addition, basic protease is also used in the pharmaceutical industry, which has high activity and specificity, and is suitable for producing specific molecules. In the field of environmental protection, it promotes the degradation of environmental pollutants.

[0004] The main source of basic protease is microbial extraction. 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 improved by screening. 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

[0005] 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.

[0006] 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.

[0007] In some embodiments of the 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.

[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 to SEQ ID NO: 1.

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

[0010] 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;

[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 application also relates to a DNA molecule encoding the above-mentioned alkaline protease mutants.

[0044] The present application also relates to a recombinant expression vector comprising the above-mentioned DNA molecule.

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

[0046] The host cell is Bacillus subtilis.

[0047] The application provides a single-point mutant containing any one of the following mutation sites: 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% to 38.13%, wherein the specific activity of the G154W single-point mutant is the highest, reaching 13005 U / mg, and an unexpected technical effect is achieved, which is beneficial to reducing the production cost of the enzyme and promoting the wide application of the enzyme in the industrial field. DETAILED DESCRIPTION

[0048] 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 edited by J. Sambrook et al., or the conditions suggested by the manufacturers. The related personnel in the field can better understand and master the application with the help of the examples. However, the method for realizing the application should not be limited to the specific method steps recorded in the examples of the application.

[0049] The formula of the culture medium involved in the examples of the application is as follows:

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

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

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

[0053] The preparation method of GM II is as follows: 96.98 mL of 1* minimal salt solution, 2.5 mL of 20% glucose, 0.08 mL of 5% hydrolyzed casein, 0.04 mL of 10% yeast powder juice, 0.25 mL of 1M MgCl2, and 0.05 mL of 1M CaCl2;

[0054] Skim milk plate: 1% tryptone, 0.5% yeast powder, 0.5% NaCl, 1% skim milk, and 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 20 min for color development. Remove and use a spectrophotometer at a wavelength of 680 nm in a 10 mm cuvette, with a tyrosine-free tube (C) as a blank, to measure the absorbance of each solution. 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).

[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) Methods for determining protein content:

[0099] The Bradford Brilliant Blue binding method for protein content determination is a combined colorimetric and dye 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 and allowed to stand for 10 minutes. (III) Specific Activity Calculation for the Bradford Brilliant Blue Binding Method for Protein Content Determination:

[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. A mutant of alkaline protease, characterized in that, The mutant is a basic protease with amino acid sequence of SEQ ID NO: 1, in which the amino acid at position 154 is mutated from Gly to Trp.

2. A DNA molecule encoding the basic 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 of claim 3; and the host cell is a non-animal or non-plant species.

5. The host cell of claim 4, wherein The host cell is Bacillus subtilis (B. subtilis) Bacillus subtilis ).

Images