Alkaline protease mutant with improved specific activity

Abstract

The invention relates to the technical field of genetic engineering and protein engineering modification, in particular to an alkaline protease mutant with improved specific activity. 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 is reduced, and wide application of the mutant in the field of industrial enzymes is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering and protein engineering, and in particular to an alkaline protease mutant with improved specific activity. Background Art

[0002] Alkaline proteases are a class of enzymes that are active in alkaline environments and are mainly used to catalyze the hydrolysis of proteins. These enzymes usually exhibit optimal activity at pH values ​​ranging from 8 to 12. The mechanism of action of alkaline proteases is to hydrolyze protein peptide bonds, converting complex macromolecular protein structures into simple small molecular peptide chains or amino acids, making them easy to absorb or wash away. This characteristic makes it important in industries that require protein decomposition, and is widely used in detergents, food, medical, brewing, silk, leather and other industries.

[0003] In detergents, alkaline protease can decompose protein stains into soluble amino acids and small molecule peptides, making them easier to clean. In food processing, it can turn complex macromolecular protein structures into simple small molecule peptide chains or amino acids, making them easier to absorb or remove. In addition, alkaline protease is also used in the pharmaceutical industry. It has high activity and specificity and is suitable for the production of specific molecules. In the field of environmental protection, it promotes the degradation of environmental pollutants.

[0004] The main source of alkaline protease is extracted from microorganisms. The most studied and applied ones are mainly Bacillus, with Bacillus subtilis being the most important, and there are also a small number of other strains, such as Streptomyces. The production capacity of natural strains and the enzyme activity and stability they contain often cannot meet the needs of industrial production, and the strains need to be screened and improved. Commonly used methods include mutagenesis, genetic engineering, protein engineering, spore heat treatment, etc. The main goal is to improve the activity, stability (heat and alkali resistance), antioxidant and anti-chelating properties of the enzyme, thereby promoting the wider application of alkaline protease. Summary of the invention

[0005] The purpose of the present invention is to provide an alkaline protease mutant with improved specific activity. The specific activity of the mutant is significantly improved compared with the wild type, thereby facilitating the wide application of alkaline protease.

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

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

[0008] In some more specific embodiments, the amino acid sequence of the mutant is at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identical to SEQ ID NO:1.

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

[0010] In some embodiments of the present invention, the mutant comprises a substitution or a combination 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 a DNA molecule encoding the above alkaline protease mutant.

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

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

[0046] The host cell is Bacillus subtilis.

[0047] The present invention is based on the wild-type alkaline protease AKP and provides a single-point mutant containing any one mutation site among Q63K, A109T, K120N, G154W, G191V, A261L and T328G. The specific activity of the single-point mutant is generally increased by 13.12%-38.13%, among which the specific activity of the G154W single-point mutant is the highest, reaching 13005U / mg, achieving an unexpected technical effect, being conducive to reducing the production cost of the enzyme and promoting its wide application in the industrial field. DETAILED DESCRIPTION

[0048] The experimental methods for which specific conditions are not specified in the examples can be run under conventional conditions, such as the conditions described in the Molecular Cloning Experiment Guide compiled by J. Sambrook, etc., or under the conditions recommended by the manufacturer. Those skilled in the art can better understand and grasp the present invention with the help of the examples. However, the methods for implementing the present invention should not be limited to the specific method steps recorded in the examples of the present invention.

[0049] The formula of the culture medium involved in the embodiments of the present invention 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*minimum salt solution, 2.5 mL of 20% glucose, 0.4 mL of 5% hydrolyzed casein, and 1 mL of 10% yeast powder juice; the preparation method of 1*minimum salt solution is as follows: K2HPO4 14 g / L, KH2PO4 6 g / L, (NH4)2SO4 2 g / L, trisodium citrate 1 g / L, and MgSO4·7H2O 0.2 g / L, which are dissolved in distilled water in sequence;

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

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

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

[0056] The present invention will be further described below in conjunction 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. First, the nucleotide sequence of the enzyme was optimized according to the codon preference of Bacillus, and the optimized sequence was synthesized by Beijing Liuhe BGI Technology Co., Ltd.

[0059] The synthesized alkaline protease AKP gene sequence was used as a template to design primers, and PCR amplification was performed using the GeneMorph II random mutation PCR kit (Bomais). The PCR product was recovered by gel extraction, double-digested with KpnI and MLuI, and ligated with the pSZX101 vector that had been digested with the same enzymes. The product was transformed into Escherichia coli DH5α, coated on LB+Amp plates, and inverted for culture at 37°C. After transformants appeared, the plasmid was extracted and transformed into Bacillus subtilis.

[0060] After the transformants grow out, they are picked up one by one with a toothpick into a 48-well plate, 20 μg / mL kanamycin is added to each well, and cultured at 37°C and 500 rpm for about 48 h. The supernatant is centrifuged for high-throughput analysis to measure the protease activity and protein content, and calculate the specific activity of different mutants.

[0061] The experimental results show that some mutations can significantly increase the specific activity of alkaline protease AKP, while some mutations cause its specific activity to become lower; in addition, some mutations, although they can increase the specific activity of alkaline protease AKP, their enzymatic properties have changed significantly after mutation, which do not meet the requirements. Finally, the applicant screened and obtained mutation sites that can significantly increase the specific activity of alkaline protease AKP without significantly affecting its original enzymatic properties, namely: Q63K, A109T, K120N, G154W, G191V, A261L, T328G.

[0062] Based on the wild-type alkaline protease AKP, the present invention provides mutants respectively comprising single mutation sites of Q63K, A109T, K120N, G154W, G191V, A261L and T328G.

[0063] The present invention further provides a mutant comprising a combination of at least two mutation sites selected from Q63K, A109T, K120N, G154W, G191V, A261L, and T328G. For example: Q63K / A109T, Q63K / K120N, Q63K / G191V, Q63K / A261L, Q63K / T328G, A109T / G191V, A109T / G191V, A109T / A261L, A109T / T328G, K120N / G191V, K120N / A261L, K120N / T328G. 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 triple mutants; Q63K / A109T / A261L / T328G, A109T / G191V / A261L / T328G, A109T / K120N / G154W / G191V, A109T / G154W / G191V / A261L quadruple mutants; K120N\ G154W\G191V\A261L\T328G five-point mutant; Q63K\A109T\K120N\G154W\G191V\A261L, A109T\K120N\G154W\G191V\A261L\T328G six-point mutants and Q63K\A109T\K120N\G154W\G191V\A261L\T328G seven-point mutant.

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

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

[0066] According to 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 synthetic 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 recovery and ligated with T4 ligase overnight. The ligation product was transformed into Escherichia coli DH5α competent cells and spread on LB+Amp plates. Single colonies grew after culturing at 37°C overnight. Colony PCR was used to verify the correct connection of the transformant, and the plasmid was extracted and sent to Beijing BGI Research Center for sequencing analysis.

[0068] Plasmids were purified from E. coli clones with correct sequencing results using a plasmid midi-prep kit (Axygen).

[0069] Transformation operation method: inoculate fresh activated Bacillus subtilis 1A75 from LB plate into 5mL GMⅠ solution, shake and culture at 30℃, 125rpm overnight; take 1mL and transfer to 9mL GMI on the next day, culture at 37℃, 220rpm for 3.5h; take 1mL of the culture solution in the previous step and transfer to 9mL GMⅡ solution, culture at 37℃, 125rpm for 90min, centrifuge at 5000g, 10min to collect bacteria; gently suspend the bacteria with 1mL GMⅡ solution, the suspended bacteria are competent cells, which can be used for transformation. Storage of competent cells: add 30% sterilized glycerol to a final concentration of 10%, mix well and dispense into centrifuge tubes, and then store at -70℃.

[0070] 1 μg of recombinant plasmid was mixed evenly with 200 μL of the competent cells, shaken (200 rpm) at 37°C for 30 min, and then spread on a skim milk plate containing 30 μg / mL kanamycin and cultured overnight at 37°C. The single colony grown overnight is the engineered strain of Bacillus subtilis containing alkaline protease AKP and its single point mutant.

[0071] Example 3 Analysis of specific activity of alkaline protease mutants

[0072] The Bacillus subtilis engineered bacteria expressing wild-type alkaline protease AKP or its mutants constructed in Example 2 were inoculated into liquid fermentation medium, respectively, and after shaking flask fermentation for 48 hours, the supernatant was collected by centrifugation at 5000 rpm for 10 minutes, and the protease activity and protein content in the supernatant were determined, 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] It can be seen from the data in Table 1 that compared with the wild-type alkaline protease AKP, the specific activity of the alkaline protease single point mutants 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 13005U / mg, achieving unexpected technical effects.

[0077] The above results show that the mutation sites Q63K, A109T, K120N, G154W, G191V, A261L, and T328G provided by the present invention can significantly improve the specific activity of wild-type alkaline protease AKP, which is beneficial to reduce the production cost of the enzyme and promote its wide application in the field of industrial enzymes.

[0078] (I) Protease activity assay method:

[0079] 1. Principle

[0080] Under certain temperature and pH conditions, proteases hydrolyze casein substrates to produce amino acids containing phenol groups (such as tyrosine, tryptophan, etc.). Under alkaline conditions, Folin reagent is reduced to generate molybdenum blue and tungsten blue. The absorbance of the solution is measured at a wavelength of 680nm using a spectrophotometer. The enzyme activity is proportional to the absorbance, and the enzyme activity of the product can be calculated from this.

[0081] 2. Definition of enzyme activity

[0082] The definition of protease activity is that protease activity is expressed in protease activity units, which is defined as 1g solid enzyme powder (or 1mL liquid enzyme) hydrolyzing casein for 1min to produce 1ug tyrosine under certain temperature and pH conditions, which is 1 enzyme activity unit, expressed in 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) boric acid 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. Determination Method

[0086] (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 solution, shake evenly, place in a 40°C water bath for color development for 20 min, take out and use a spectrophotometer at a wavelength of 680 nm, a 10 mm cuvette, and use tube 0 without tyrosine as a blank to measure its absorbance. Use absorbance A as the ordinate and tyrosine concentration C as the abscissa to draw a standard curve (this line should pass through the zero point).

[0087] (2) Enzyme activity assay

[0088] Take an appropriate amount of pre-diluted enzyme solution, then add an equal volume of 40℃ preheated 10% casein, and react at 40℃ for 10 minutes; then add an equal volume of trichloroacetic acid (concentration 6.54%) to the reaction system, mix well, and let stand at room temperature for 10 minutes to terminate the reaction. Take out 1mL of the terminated reaction solution, then add 5mL42.4g / L sodium carbonate solution, then add 1mL Folin reagent, and react at 40℃ for 20 minutes; finally measure the OD608 value.

[0089] (3) Calculation

[0090] Read the enzyme activity of the final dilution of the sample from the standard curve in u / mL. The enzyme activity of the sample is calculated according to 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——average absorbance of parallel samples;

[0094] K——absorption constant;

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

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

[0097] n——dilution multiple.

[0098] (II) Protein content determination method:

[0099] The Coomassie Brilliant Blue (Bradford) binding method for determining protein content is a composite method that combines colorimetry with pigmentation. Coomassie Brilliant Blue G-250 is brown-red in acidic solution, and turns blue when combined with protein. It conforms to Beer's law within a certain protein concentration range and can be colorimetrically determined at 595nm. A large amount of absorption is achieved in 3 to 5 minutes and is stable for at least 1 hour. In the range of 10 to 1000 μg / mL, the absorbance is proportional to the protein concentration. Mix the enzyme solution and Coomassie Brilliant Blue solution in a volume ratio of 1:5, let stand for 10 minutes, and determine the protein content by the Coomassie Brilliant Blue (Bradford) binding method (III) Calculation of specific activity:

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

[0101] The specific activity calculation formula is: specific activity (U / mg) = enzyme activity (U / mL) / protein content (mg / mL).

Claims

1. An alkaline protease mutant, characterized in that The mutant comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 1, and comprises a substitution of amino acid at position 328: T328G compared with SEQ ID NO:

1.

2. The alkaline protease mutant according to claim 1, characterized in that The mutant comprises a substitution or a combination of substitutions selected from the following substitutions and combinations of substitutions: K120N / T328G; A261L / T328G; Q63K / A109T / T328G; Q63K / A261L / T328G; Q63K / A109T / A261L / T328G; K120N / G154W / G191V / A261L / T328G; A109T / K120N / G154W / G191V / A261L / T328G; Q63K / A109T / K120N / G154W / G191V / A261L / T328G.

3. A DNA molecule encoding the alkaline protease mutant according to claim 1 or 2.

4. A recombinant expression plasmid, characterized in that: The recombinant expression plasmid comprises the DNA molecule according to claim 3.

5. A host cell, characterized in that The host cell comprises the recombinant expression plasmid according to claim 4.

6. The host cell according to claim 5, characterized in that The host cell is Bacillus subtilis ( Bacillus subtilis ).

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