High-enzyme-activity alkaline protease mutant and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and discloses an alkaline protease mutant obtained through an error-prone PCR technology and a saturated mutation technology and application of the alkaline protease mutant. The alkaline protease mutant is obtained by carrying out random mutation and saturated mutation screening on an alkaline protease gene hapR from bacillus velezensis by utilizing an error-prone PCR (Polymerase Chain Reaction) technology, the 303th leucine of the HapR protein is mutated into valine, and the enzymatic activity of the alkaline protease mutant is improved by 34.02%.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme engineering, and particularly relates to a high-enzyme-activity alkaline protease mutant obtained through error-prone PCR technology and saturation mutation and application thereof. BACKGROUND

[0002] Protease is an enzyme that catalyzes protein hydrolysis, can hydrolyze peptide bonds, and generate peptides and amino acids. It has important applications in many fields such as medicine, food, cosmetics, etc. With the continuous development of science and technology, the application prospect of protease is also more broad. In the past few decades, error-prone PCR technology has been increasingly mature in the field of protease molecular modification. This method can randomly mutate selected residues without understanding the corresponding relationship between protein structure and function, thereby creating a large mutant library to increase the possibility of obtaining positive variants. Error-prone PCR generally includes two steps: 1. Constructing a high-quality mutant library: obtaining multiple mutants by random mutation or gene recombination; 2. High-quality screening: obtaining positive mutants with desired traits through specific high-throughput analysis methods. Therefore, using error-prone PCR technology to accurately edit enzyme molecules and realize the directional modification of protein properties has been proven to be a reasonable and practical strategy. This method not only allows us to accurately control the structure and function of proteins, but also effectively enhances their performance in specific industrial or biological processes, opening up new avenues for the development of biotechnology and protein engineering fields.

[0003] Therefore, the application provides a method for molecular modification of alkaline protease HapR derived from B. velezensis by using error-prone PCR technology. By optimizing the error-prone PCR reaction conditions, introducing base mutations, constructing a gene diversity mutant library, and preliminarily screening mutant strains with obvious changes in enzyme activity on skimmed milk powder-containing nutrient agar plates, and then using B. amyloliquefaciens expression system for precise screening, alkaline protease mutants with improved enzyme activity are obtained. SUMMARY

[0004] The purpose of the application is to mutate alkaline protease HapR to obtain alkaline protease HapR mutants with significantly improved enzyme activity, and to improve the activity of protease.

[0005] In order to achieve the above purpose, the following technical measures are adopted in the application:

[0006] The alkaline protease HapR mutant is a protein with the amino acid sequence shown in SEQ ID NO. 2, which is obtained by mutating the 303rd leucine of alkaline protease HapR derived from B. velezensis to valine.

[0007] A polynucleotide encoding the alkaline protease HapR mutant, the nucleotide sequence of which is shown in SEQ ID NO. 3. An expression vector containing the polynucleotide.

[0008] A host cell or genetically engineered bacteria expressing the alkaline protease mutant. Preferably, the alkaline protease HapR mutant-expressing engineered bacteria is constructed using B. amyloliquefaciens HZ-12 as the starting strain.

[0009] Application of the alkaline protease HapR mutant in improving protease activity: In specific embodiments of the present application, the expression plasmids pHY-hapR, pHY-hapR L303V (303rd mutation to valine) were respectively electroporated into B. amyloliquefaciens HZ-12, and after fermentation, the alkaline protease activity in the fermentation supernatant was determined. The results showed that compared with the control strain, the alkaline protease activity of the mutant strain was increased by 34.02%. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 : hapR gene PCR amplification electrophoresis map.

[0011] Figure 2 : Free expression vector pHY-hapR PCR amplification electrophoresis map.

[0012] Figure 3 : Comparison of alkaline protease enzyme activity of mutant L303H and wild-type recombinant strain.

[0013] Figure 4 : Comparison of alkaline protease enzyme activity of different HapR protein 303 site mutants. DETAILED DESCRIPTION

[0014] The present application is described in detail below through examples.

[0015] Biological material source explanation: B. amyloliquefaciens HZ-12 has been reported in a paper (Min Y. Analysis of spermidine metabolism and key gene mining of B. amyloliquefaciens [D]. Central China Agricultural University, 2020. DOI: 10.27158 / d.cnki.ghznu.2020.001154.), and is currently preserved in the Microbial Engineering Laboratory of Central China Agricultural University.

[0016] Example 1 Construction of free expression vector

[0017] 1. Extraction of target strain genomic DNA

[0018] Remove the Bacillus belye WH-7 seed culture glycerol tubes from the -80℃ freezer and inoculate them onto nutrient agar plates using the streak method. Incubate at 37℃ until single colonies appear, then inoculate them into 50 mL of LB liquid medium and culture at 37℃ with shaking at 200 rpm for 8 h as seed culture. Inoculate the seed culture at a 3% inoculation rate into 50 mL of fermentation medium (10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract), and incubate at 37℃ with shaking at 180 rpm for 8 h. Centrifuge at 4000×g for 10 min (4℃), discard the supernatant, collect the bacterial cells, and extract total bacterial DNA according to the instructions of the Vazyme Bacterial DNA Kit. Assess the concentration and quality of the DNA using the NanoDrop 2500 system (OD260 / OD280 = 1.8-2.0, ≥10 μg).

[0019] Using the extracted genome of Bacillus belye as a template, a pair of primers was designed upstream and downstream of the ORF frame of the alkaline protease gene hapR to introduce the restriction enzyme sites BamHI and XbaI, respectively. The amplification primers are as follows:

[0020] Upstream primer P1: 5'-ATGAGAGGCAAAAAGGTATGGA-3'

[0021] Downstream primer P2: 5'-TTACTGAGCTGCCGCCT-3'

[0022] Using P1 and P2 as upstream and downstream primers, amplification was performed using Bacillus belye genomic DNA as a template.

[0023] Table 1 Amplification reaction system

[0024]

[0025] The amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles; 95℃ denaturation for 40 s, 55℃ annealing for 45 s, 72℃ extension for 1.5 min, 30 cycles; 72℃ extension for 5 min, 10℃ hold for 5 min. The PCR amplification products were subjected to 0.8% agarose gel electrophoresis, yielding a 1049 bp band. Figure 1 The PCR product was recovered using a small-volume DNA recovery kit to obtain the wild-type alkaline protease zymogen region gene hapR (SEQ ID NO.1). hapR and the pHY300(PLK) plasmid were double-digested with restriction endonucleases BamHI and XbaI, respectively. The recovered hapR was then ligated into the pHY300(PLK) vector to obtain the recombinant plasmid pHY-hapR. Enzyme digestion verification was performed as follows. Figure 2The plasmid was transformed into E. coli DH5a and B. amyloliquefaciens HZ-12, respectively.

[0026] Example 2 Screening of Mutants Based on Alkaline Protease Mutant Library Constructed by E. coli Expression System

[0027] With hapR as the reporter gene, the effects of two different E. coli expression vectors (pTrc99a, pET28a), two different host bacteria (BL21(DE3), MG1655), and three different signal peptides (OmpA signal peptide, Pelb signal peptide, and hapR endogenous signal peptide) on the in vitro expression of HapR were compared. The diameter of protease diffusion on a nutrient agar plate containing 0.8% skimmed milk powder was used to characterize the secretion effect. The results showed that the strains MG1655 / pTrc99a-OmpA-hapR and BL21(DE3) / pET28a-OmpA-hapR had the best secretion effect. When the vector was pTrc99a, the most suitable host bacteria was MG1655, and the most suitable signal peptide was OmpA signal peptide; when the vector was pET28a, the most suitable host bacteria was BL21(DE3), and the most suitable signal peptide was OmpA signal peptide.

[0028] Since pET28a is an inducible expression, IPTG low-temperature induction is needed when the fermentation broth is 0.6-0.8. Under the premise of the same secretion effect and ensuring experimental efficiency, we selected pTrc99a as the most suitable vector for the construction of the mutant library (vector pTrc99A, signal peptide OmpA, host bacteria MG1655), and used the transparent circle method on the nutrient agar plate containing skimmed milk powder to screen the mutants at high throughput and select beneficial mutants from them.

[0029] 2. Establishment of High-Quality Mutant Library

[0030] To construct a high-quality mutant library, we used error-prone PCR technology to achieve directional mutation of the target gene by optimizing the reaction conditions. The specific method is as follows: linearization of the vector and elimination of empty vectors To reduce the empty vector background, the vector pTrc99A was amplified by reverse amplification, and the unmutated template was digested with DPNI enzyme. By investigating the effects of different enzyme digestion times (1 h, 1.5 h, 2 h, 2.5 h, 3 h) on the transformation efficiency, it was found that 3 h treatment could completely linearize the vector, and there were no empty vector clones after transformation, ensuring a high insertion rate of the library. Precise control of mutation frequency: the key of error-prone PCR is to control the mutation frequency to maintain it within the ideal range of 1-3 amino acid mutations per gene. By adjusting the concentration of Mn 2+ (0.5 M, 1.0 M, 1.5 M, 2.0 M, 2.5 M), it was found that 1.0 M Mn 2+Under the condition, the mutation frequency was stabilized at 1-3 amino acid changes, which met the requirements of high-quality mutation library. In summary, we successfully constructed a high-diversity, low-background, and mutation frequency-controllable mutation library by optimizing the DPNI enzyme digestion time and precisely regulating the Mn 2+ concentration.

[0031] 3. Screening of precise model

[0032] By giving the form of protein substrate, with the help of hydrolysis transparent circle method, using skimmed milk containing nutrient agar plate to screen the mutants, try different concentrations of skimmed milk powder (8 g / L, 10 g / L, 12 g / L), it is found that when the concentration of skimmed milk powder is 8 g / L, the transparent circle can be observed more obviously, the circle time is about 20 h, and the contrast effect is obvious. In order to avoid the growth of mixed bacteria during the culture of the plate, the addition of tetracycline with a final concentration of 20 ng / mL can effectively inhibit the contamination of mixed bacteria. The transparent circle formed by the mutants on the nutrient agar plate containing 8 g / L skimmed milk powder is used to preliminarily characterize the alkaline protease activity of the mutants.

[0033] By screening the mutant library under the condition of 1.0M Mn 2+ concentration, one E. coli recombinant mutant with significantly changed protease activity was finally obtained by skimmed milk plate transparent circle method. DNA sequencing showed that the mutant had an amino acid substitution of leucine (L) to histidine (H) at position 303 (L303H).

[0034] Example 3 Verification of mutants based on Bacillus expression system

[0035] The expression activity of the mutants in B. amyloliquefaciens HZ12 was explored by heterologous expression. Therefore, the Bacillus expression vector PHY-hapR containing the L303H mutant site was constructed. L303H Then it was transformed into B. amyloliquefaciens HZ-12, and the mutant recombinant strain HZ12 / pHY-hapR L303H The mutant recombinant strain and the wild-type recombinant strain were subjected to liquid fermentation at 37℃ and 180 r / min for 48 h. The alkaline protease activity in the fermentation supernatant was detected by Folin-phenol method, and the results are shown in Figure 3 When the protease HapR 303 site was mutated from leucine to histidine, the overall alkaline protease activity was significantly decreased.

[0036] In view of the significance and specificity of the above experimental results, it is reasonably speculated that this site may constitute a core node in the functional regulation network of alkaline protease HapR. The stability, charge properties or interaction mode with other molecules of this site may directly or indirectly affect the catalytic efficiency, stability or substrate recognition ability of HapR, and then manifest as the reduction of the overall enzyme activity of the strain at the macro level. In order to verify this hypothesis, a more refined amino acid saturation mutation is used for this site to clarify the range and degree of its influence. The experimental results are shown in Table 1. Figure 4 As shown in Table 1, when the 303 site is mutated from leucine to valine, the alkaline protease activity is the highest, reaching 260.82 U / mL, which is increased by 34.02% compared with the wild-type recombinant strain (HZ12 / pHY-hapR).

[0037] Example 4 Expression and preparation of alkaline protease mutants in Bacillus amyloliquefaciens HZ-12

[0038] The wild-type alkaline protease encoding gene hapR and the mutant encoding gene hapR L303V and the mutant recombinant bacteria HZ12 / pHY-hapR were obtained by ligation of the alkaline protease wild-type encoding gene hapR and the mutant encoding gene hapR L303V and the mutant recombinant bacteria HZ12 / pHY-hapR were obtained by ligation of the alkaline protease wild-type encoding gene hapR and the mutant encoding gene hapR L303V The mutant recombinant bacteria HZ12 / pHY-hapR L303V and the wild-type recombinant bacteria HZ12 / pHY-hapR were inoculated in 50 mL of fermentation medium (containing 50 μg / mL kanamycin) at 37°C and 220 r / min overnight, and then transferred to 50 mL of fresh fermentation medium at an inoculation amount of 3%, and continued to be cultured at 37°C and 220 r / min for 48 h. The preparation method of the fermentation medium is as follows: tryptone 80 g / L, yeast powder 25 g / L, KH2PO4 5 g / L, NH4Cl 6 g / L, and the rest is water, sterilized at 121°C for 30 min.

[0039] The fermentation supernatant was collected by centrifugation, and the alkaline protease activity was tested by the Folin-phenol method. The alkaline protease activity of the wild-type recombinant strain HZ12 / pHY-hapR was 194.64 U / mL, and the mutant recombinant bacteria HZ12 / pHY-hapR L303VThe enzyme activity of the alkaline protease is 260.82 U / mL. The prepared mutant crude enzyme solution has wide application potential and can be applied in various industries and consumer product manufacturing across fields, specifically including but not limited to the detergent industry to improve cleaning efficiency and environmental friendliness; in the leather industry, as a biological treatment agent, to promote the greening and efficiency of the leather processing process; in the food industry, it can be used as a food processing aid to enhance the taste, color or shelf life of the product; in addition, in the feed industry, the enzyme solution can also be used as a biological additive to promote animal digestion and absorption and improve feed utilization, thereby promoting the sustainable development and product innovation of these industries.

Claims

1. An alkaline protease HapR mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

2.

2. A polynucleotide encoding the alkaline protease HapR mutant of claim 1.

3. An expression vector containing the polynucleotide of claim 2.

4. A host cell expressing the alkaline protease HapR mutant of claim 1 or containing the expression vector of claim 3.

5. Genetically engineered bacteria expressing the alkaline protease mutant of claim 1.

6. The genetically engineered bacterium according to claim 5, characterized in that, An engineered bacterium expressing the alkaline protease HapR mutant of claim 1 was constructed using Bacillus amyloliquefaciens HZ-12 as the starting bacterium.

7. The use of the genetically engineered bacteria according to claim 5 or 6 in the production of alkaline protease with high enzyme activity.

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