A basic protease mutant and a bacillus subtilis strain with high yield of the enzyme

Abstract

The present application relates to the technical field of genetic engineering and protein engineering, and particularly relates to a kind of alkaline protease mutant and high-yield strain of bacillus subtilis of the enzyme.The alkaline protease mutant includes three mutation sites K59P / H168A / G231S, and significant improvement of specific activity can be widely used in washing and other industrial fields, and has wide market prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and protein engineering technology, specifically relating to an alkaline protease mutant and a Bacillus subtilis strain that produces the enzyme at high yield. Background Technology

[0002] Alkaline proteases are a class of enzymes that catalyze the hydrolysis of protein peptide bonds under alkaline pH conditions (optimal pH is typically 9.0–11.0). They belong to the subtilisin subclass of the serine protease family. Based on the source microorganism, alkaline proteases are mainly divided into those derived from Bacillus licheniformis (…). Bacillus licheniformis Carlsberg protease, derived from Bacillus subtilis ( Bacillus subtilis Bacillus subtilis protease BPN' and Bacillus clausti (Bacillus subtilis) Bacillus clausii Alkaline proteases, among others, are used in detergents. Due to their highly efficient degradation of protein-based stains (such as blood, sweat, milk, and sauce stains), alkaline proteases have become the most widely used and largest-volume core enzyme component in modern detergent formulations. Furthermore, alkaline proteases are also widely used in leather depilation, silk degumming, the pharmaceutical field (such as wound cleaning), and food processing, holding approximately 60% of the global industrial enzyme market share.

[0003] In detergent applications, the specific activity (catalytic activity per unit mass of enzyme protein) of alkaline proteases is a core parameter determining their industrial economics. High specific activity means that stubborn protein stains can be removed faster and more thoroughly with the same amount of protein added, or that the amount of enzyme preparation added can be significantly reduced while achieving the same washing effect. This effectively reduces the production cost of detergent formulations, reduces product volume, and lowers transportation and storage costs. Industrially used alkaline proteases often face the inhibitory effects of detergent auxiliaries (such as surfactants and chelating agents) and enzyme inactivation problems caused by high temperature and high alkalinity environments during the washing process. To achieve ideal detergency, a high amount of enzyme is often required, which significantly increases production costs and has become a key bottleneck restricting the development of the detergent enzyme industry.

[0004] To address these issues, researchers have conducted extensive work in two main directions. First, they are screening novel enzyme-producing strains to discover natural alkaline protease resources with higher catalytic efficiency. For example, they are screening alkaliphilic or thermotolerant microorganisms from extreme environments such as deep-sea sediments, salt lakes, and hydrothermal vents to obtain enzyme molecules with superior performance. Second, they are using protein engineering and site-directed mutagenesis to directionally modify existing alkaline protease molecules. For instance, using alkaline protease from Bacillus licheniformis as the starting enzyme, specific amino acid substitutions have resulted in mutants with significantly increased specific activity, exhibiting a catalytic efficiency (kcat / Km) more than 30% higher than the wild type. Alkaline protease mutants developed by companies such as Blue Ocean Biotechnology, containing mutation sites such as Q63K, A109T, K120N, and G154W, generally show a 13%–79% increase in specific activity compared to the wild type, demonstrating a feasible pathway to improve enzyme catalytic performance through molecular modification.

[0005] Developing an alkaline protease mutant that can significantly improve specific activity, and screening a protein expression system that can efficiently secrete and express this mutant, has important industrial application value and practical significance for reducing detergent production costs and enhancing the market competitiveness of domestic alkaline protease products. Summary of the Invention

[0006] To address the problems of existing technologies, this invention provides an alkaline protease mutant and a Bacillus subtilis strain that produces the enzyme at high yield, which is beneficial for promoting the widespread application of alkaline protease in industrial fields such as washing.

[0007] This invention relates to an alkaline protease mutant, wherein the 59th amino acid of the alkaline protease with the amino acid sequence SEQ ID NO:1 is mutated from Lys to Pro, the 168th amino acid is mutated from His to Ala, and the 231st amino acid is mutated from Gly to Ser.

[0008] The present invention also relates to DNA molecules encoding the above-mentioned alkaline protease mutants.

[0009] The present invention also relates to recombinant expression plasmids comprising the above-described DNA molecules.

[0010] The present invention also relates to an engineered strain of Bacillus subtilis carrying the above-mentioned recombinant expression plasmid.

[0011] The present invention also relates to the application of the engineered strain of Bacillus subtilis in the production of alkaline protease.

[0012] The present invention also relates to the application of the alkaline protease mutant in detergents.

[0013] Compared to the alkaline protease AprE, the K59P / H168A / G231S three-point mutant provided by this invention exhibits a 123.4% increase in specific activity, reaching 10369 U / mg, achieving unexpected technical results. The recombinant Bacillus subtilis engineered strain expressing this enzyme mutant, as provided by this invention, achieves an enzyme activity of 23434 U / mL during shake-flask fermentation, which is beneficial for reducing production costs and promoting its application in industrial fields such as detergents, indicating a broad market prospect. Detailed Implementation

[0014] The method of the present invention will be further illustrated below with reference to examples. Experimental methods not specified in the examples can generally 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 the present invention with the help of these examples. However, the methods for implementing the present invention should not be limited to the specific method steps described in the embodiments of the present invention.

[0015] The present invention will now be described in detail with reference to specific embodiments.

[0016] Example 1: Screening of alkaline protease mutants The amino acid sequence of the alkaline protease AprE is SEQ ID NO: 1, and its encoding nucleotide sequence is SEQ ID NO: 2.

[0017] To improve the specific activity of the alkaline protease AprE, the applicant analyzed its protein structure and, without destroying the protein's secondary structure and active site, further mutated the gene.

[0018] Using the alkaline protease AprE gene as a template, PCR amplification was performed using the designed primers and the GeneMorph II random mutation PCR kit (Bomais). The PCR products were recovered from the gel, double-digested with KpnI and MluI, and ligated into the similarly digested pSZX101 vector. The resulting vector was transformed into *E. coli* DH5α, plated on LB+Amp plates, and incubated upside down at 37°C. After transformants appeared, plasmids were extracted and transformed into *Bacillus subtilis*. Once transformants had grown, they were individually transferred to 48-well plates using a toothpick. 20 μg / ml kanamycin was added to each well, and the plates were incubated at 37°C and 500 rpm for approximately 48 hours. The supernatant was centrifuged and used for high-throughput analysis. The alkaline protease activity and protein content were measured at 37°C, and the specific activity of different mutants was calculated.

[0019] Experimental results showed that some mutants exhibited increased specific activity, while others showed decreased specific activity. Additionally, some mutations, while increasing the specific activity of alkaline protease, resulted in significant changes in the enzymatic properties, all of which did not meet the requirements. Ultimately, the applicant selected the mutant site combination K59P / H168A / G231S, which significantly increased the specific activity of alkaline protease without significantly affecting its original enzymatic properties.

[0020] Based on the alkaline protease AprE, this invention provides a mutant containing three mutation sites: K59P, H168A, and G231S. Referring to the amino acid sequence of the mutant, the coding nucleotide sequences of the alkaline protease mutant were obtained.

[0021] Example 2: Expression of alkaline protease mutant in Bacillus subtilis Based on the codon preference of Bacillus, the gene sequences of alkaline protease AprE and its mutants were optimized and synthesized, and two restriction sites, KpnI and MluI, were added to the 5' and 3' ends of the synthesized sequences, respectively.

[0022] 1. Carrier Construction The synthesized alkaline protease gene fragment was obtained by PCR. The obtained gene fragment and the expression vector pSZX101 were double-digested with KpnI and MluI, respectively. The target fragment was recovered by gel electrophoresis and ligated overnight with T4 ligase. The ligation product was transformed into E. coli DH5α competent cells, plated on LB+Amp plates, and incubated overnight at 37°C. Single colonies grew. The transformed cells were verified by colony PCR to be ligated correctly. Plasmids were extracted from the transformed cells and sequenced for analysis.

[0023] Plasmids were purified from correctly sequenced E. coli clones using a plasmid medium-quantity preparation kit (Axygen).

[0024] 2. Preparation and transformation of competent Bacillus subtilis cells The conversion solution was prepared as follows: 1× Minimum salt solution: K2HPO4 7g, KH2PO4 3g, (NH4)2SO4 1g, trisodium citrate Na2C6H5O7·2H2O 0.5g, MgSO4·7H2O 0.1g, add water to 500 mL.

[0025] GMⅠ solution: 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.

[0026] GMⅡ solution: 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.

[0027] Transformation procedure: Inoculate the bacteria onto LB agar plates and incubate overnight at 37°C. Inoculate a loopful of bacterial growth into 5 mL of GMⅠ solution and incubate overnight at 30°C with gentle shaking (125 rpm). The next day, transfer 2 mL of the culture medium to 18 mL of GMⅠ and incubate at 37°C with rapid shaking (220 rpm) for 3.5 h. Then, transfer 5 mL of the culture medium from the previous step to 45 mL of GMⅡ and incubate at 37°C with gentle shaking (125 rpm) for 90 min. Centrifuge at 8000g for 10 min to collect the bacterial cells. Gently resuspend the bacterial cells in 5 mL of the original culture supernatant. The resuspended cells are competent cells and can be used for transformation. Preservation of competent cells: Add 30% sterile glycerol to a final concentration of 10%, mix well, aliquot into centrifuge tubes, and store immediately at -70°C.

[0028] 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 on the appropriate resistant medium and incubate overnight at 37°C. The single colony that grows overnight is the engineered strain containing alkaline protease and its mutant.

[0029] Example 3 Fermentation Verification The recombinant Bacillus subtilis strains expressing alkaline protease and its mutants were inoculated into 5 mL LB medium (0.5% yeast extract, 1% tryptone, 1% sodium chloride) and cultured at 37℃ and 220 rpm for 6-8 h. The cultures were then transferred to 50 mL liquid fermentation medium (0.5% yeast extract, 0.5% tryptone, 1% glucose, 1.8% K₂HPO₄) and fermented in shake flasks at 37℃ and 220 rpm for 72 h. The supernatant was collected by centrifugation at 5000 g for 10 min, yielding the fermentation supernatant containing alkaline protease and its mutants. The alkaline protease activity and protein content of the fermentation supernatant were determined.

[0030] (a) Alkaline protease activity detection 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 reduces these amino acids 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. The definition of protease activity, expressed in units, is as follows: 1 unit of enzyme activity is defined as the amount of 1 μg of tyrosine produced by hydrolyzing casein in 1 minute under specific temperature and pH conditions, expressed as μ / g (μ / ml).

[0031] 1. 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 μg / mL and 1 mg / mL L-tyrosine standard solutions; (7) 6.54% trichloroacetic acid.

[0032] 2. Enzyme activity assay (1) Preparation of standard curve: Prepare L-tyrosine standard solutions with concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL and 50 μg / 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 the tyrosine-free tube 0 as a blank, and measure the absorbance of each. Plot the standard curve with absorbance A as the ordinate and the tyrosine concentration C as the abscissa (this line should pass through the zero point).

[0033] 3. Enzyme activity assay Take 1 ml of pre-diluted enzyme solution, then add 1 ml of 10% casein preheated at 40°C, and react at 40°C for 10 min. Then add 2 ml of trichloroacetic acid (6.54%), 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 incubate at 40°C for 20 min. Finally, measure the OD. 680 value.

[0034] 4. Calculation Read the enzyme activity of the final diluted sample from the standard curve, in μ / mL. The enzyme activity of the sample is calculated using the following formula: X=A×K×4 / 10×n=2 / 5×A×K×n.

[0035] Where: X — enzyme activity of the sample (μ / g or μ / 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.

[0036] (ii) Protein content determination 1. Measurement method: The Coomassie Brilliant Blue (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 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.

[0037] The enzyme solution and Coomassie Brilliant Blue solution were mixed at a volume ratio of 1:5, allowed to stand for 10 minutes, and the protein content was determined by the Coomassie Brilliant Blue (Bradford) binding method.

[0038] (III) Calculation of specific vitality Specific activity refers to the number of enzyme activity units per unit weight of protein, usually expressed as U / mg protein. Generally speaking, the higher the specific activity of an enzyme, the purer the enzyme.

[0039] Specific activity calculation formula: Specific activity (U / mg) = enzyme activity (U / mL) / protein content (mg / mL).

[0040] The specific activity of the fermentation supernatant of the recombinant Bacillus subtilis engineered bacteria expressing alkaline protease and its mutants constructed in this invention is shown in Table 1.

[0041] Table 1. Specific activities of alkaline proteases and their mutants Alkaline protease AprE 9050 U / mL 1.95 mg / mL 4641 U / mg K59P / H168A / G231S mutant 23434 U / mL 2.26 mg / mL 10369 U / mg As can be seen from the data in Table 1, compared with the alkaline protease AprE, the specific activity of the K59P / H168A / G231S three-point mutant provided by the present invention is increased by 123.4%, achieving unexpected technical results.

[0042] In summary, the alkaline protease mutant provided by this invention has significantly improved specific activity and can be widely used in industrial fields such as washing, with broad market prospects.

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 amino acid 59 is mutated from Lys to Pro, amino acid 168 is mutated from His to Ala, and amino acid 231 is mutated from Gly to Ser.

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

3. A recombinant expression plasmid comprising the DNA molecule of claim 2.

4. An engineered strain of Bacillus subtilis, characterized in that, The Bacillus subtilis carries the recombinant expression plasmid as described in claim 3.

5. The application of the engineered Bacillus subtilis strain of claim 4 in the production of alkaline protease.

6. The use of the alkaline protease mutant of claim 1 in detergents.