阅读说明：本技术 一种碱性底物选择性提高的枯草蛋白酶e突变体及其应用 (Subtilisin E mutant with improved alkaline substrate selectivity and application thereof ) 是由 张娟 汤恒 堵国成 陈坚 于 2019-11-26 设计创作，主要内容包括：本发明公开了一种碱性底物选择性提高的枯草蛋白酶E突变体及其应用,属于基因工程和酶工程技术领域。本发明通过环替换策略,将枯草芽孢杆菌蛋白酶E催化口袋的环序列突变为富含负电氨基酸残基的环序列,以提高对带相反电荷的氨基酸底物的选择性,得到底物选择性提高的突变体。本发明的突变体的碱性底物选择性均有明显提高,经相同浓度的酶处理后,水解产物中碱性短肽的比例从原始酶的11％上升为50％。选择性提高的突变体显示出了牛奶过敏原降解等食品应用方面和体外溶栓等医疗应用方面的潜在应用价值。(The invention discloses a subtilisin E mutant with improved alkaline substrate selectivity and application thereof, belonging to the technical field of genetic engineering and enzyme engineering. According to the invention, by a loop replacement strategy, a loop sequence of a catalysis pocket of the subtilisin E is mutated into a loop sequence rich in negative amino acid residues, so as to improve the selectivity to amino acid substrates with opposite charges, and obtain a mutant with improved substrate selectivity. The mutant of the invention has obviously improved alkaline substrate selectivity, and the proportion of alkaline short peptide in the hydrolysate is increased from 11 percent of the original enzyme to 50 percent after the mutant is treated by the enzyme with the same concentration. The mutant with improved selectivity shows potential application values in food application aspects such as milk allergen degradation and medical application aspects such as in vitro thrombolysis.)

1. A subtilisin E mutant characterized by comprising the amino acid sequence shown in SEQ ID NO. 2.

2. A gene encoding the subtilisin E mutant of claim 1.

3. A recombinant plasmid vector containing the gene of claim 2.

4. The recombinant plasmid vector according to claim 3, wherein the recombinant plasmid vector is constructed on the basis of any one of the plasmids of pET series, pGEX series, pPICZ series, pAN series, or pUB series.

5. A genetically engineered bacterium expressing the subtilisin E mutant of claim 1.

6. A method for enhancing the thermal stability of subtilisin E is characterized in that, on the basis of the amino acid sequence shown in SEQ ID NO.1, the 127-th and 132-th loop sequences GGPSGS are mutated into YDADDS, and the 158-th and 162-th loop sequences SSGSS are mutated into aspartic acid QHPKEG.

7. A method for producing subtilisin E, characterized in that, the genetic engineering bacteria of claim 5 are cultured, and the subtilisin E is obtained by induction expression.

8. The method of claim 7, wherein the genetically engineered bacterium of claim 5 is cultured in LB medium at a temperature of 35-39 ℃ for 8-14h to obtain a seed solution; inoculating the seed liquid to LB liquid culture medium according to the inoculation amount of 1-10%, culturing at 35-39 deg.C and 200-220rpm to OD₆₀₀Adding IPTG, and performing induced culture at 15-17 deg.C for 12-16 hr to obtain the final product of IPTG and IPTG with concentration of 0.9-1.1.

9. Use of the subtilisin E mutant of claim 1 for degrading milk allergens or in vitro thrombolysis.

10. The subtilisin E mutant of claim 1 for use in food or pharmaceutical applications.

Technical Field

The invention relates to a subtilisin E mutant with improved alkaline substrate selectivity and application thereof, belonging to the technical field of genetic engineering and enzyme engineering.

Background

Subtilisin E (subtilisin E), called subtilisin E for short, is an alkaline serine protease produced by microorganisms and capable of degrading protein substrates. Subtilisin E is a protease with broad substrate specificity, strong hydrolysis catalytic ability, and alkaline pH preference, and has wide applications in detergent, leather, and food industries. In particular in the dairy industry, they have great potential in the production of bioactive hydrolysates. With the development of industrialization, the performance and the yield of the subtilisin E produced by wild bacteria can not meet the market demand.

Most of wild bacteria for producing the subtilisin E obtained by screening at present are concentrated in the bacillus subtilis, and the wild bacteria have the defects of multiple extracellular secreted enzymes, poor substrate action specificity, unstable enzyme production and no contribution to industrial production. The gene engineering bacteria are adopted to strengthen gene transcription and translation, so that high-efficiency expression and active secretion are achieved, and the production strength of the subtilisin E can be effectively improved. The recombinant subtilisin E is generally subjected to performance modification, the extracellular enzyme activity is single, and the purification work of the downstream fermentation is simplified. In addition, the industrial application has strict requirements on the enzyme, such as high temperature environment which can greatly influence the activity of the subtilisin E. In order to reduce the production cost, the repeated utilization of the subtilisin E is required, and the fine chemical catalysis of the subtilisin E with high catalytic activity and strong substrate specificity is also required. The Bacillus subtilis protease E screened and separated from the nature is far from meeting the industrial demand, so that the search for novel Bacillus subtilis protease E through a molecular modification technology is a new research means.

Disclosure of Invention

The present inventors previously expressed subtilisin E (SES7) derived from Bacillus subtilis S7 in a heterologous manner (patent publication No.: CN109837219A), and obtained subtilisin E with high solubility expression. However, in intracellular expression in E.coli, the substrate selectivity is not specific. In order to further improve the performance of the enzyme in use, it is necessary to further improve the substrate selectivity of subtilisin E.

The first purpose of the invention is to provide a subtilisin E mutant, which contains the amino acid sequence shown in SEQ ID NO. 2.

The amino acid sequence of the subtilisin E mutant is that on the basis of the amino acid sequence shown in SEQ ID NO.1, the 127-th and 132-th loop sequences GGPSGS are mutated into YDADDS, and the 158-th and 162-th loop sequences SSGSS are mutated into QHPKEG. The resulting subtilisin E mutant was designated L1-2& L2-5.

It is a second object of the present invention to provide a gene encoding the above subtilisin E mutant.

In one embodiment of the invention, the nucleotide sequence of the gene encoding subtilisin E mutant L1-2& L2-5 is the sequence shown in SEQ ID NO. 3.

The third purpose of the invention is to provide a recombinant plasmid vector containing the gene.

In one embodiment of the present invention, the recombinant plasmid vector is constructed on the basis of any one of the plasmids of pET series, pGEX series, pPICZ series, pAN series, or pUB series.

The fourth purpose of the invention is to provide a genetically engineered bacterium for expressing the subtilisin E mutant.

In one embodiment of the present invention, the genetically engineered bacterium is a bacterium, a yeast, or other fungus.

The fifth objective of the invention is to provide a method for enhancing the thermal stability of subtilisin E, which comprises mutating the loop sequence GGPSGS at position 127 and 132 to YDADDS and the loop sequence SSGSS at position 158 and 162 to aspartic acid QHPKEG on the basis of the amino acid sequence shown in SEQ ID NO. 1.

The sixth purpose of the invention is to provide a method for producing the subtilisin E mutant protein, which comprises culturing the recombinant expression transformant and inducing to obtain the subtilisin E mutant protein.

In one embodiment of the invention, the method comprises the steps of culturing a monoclonal of the genetically engineered bacteria in an LB culture medium at the temperature of 35-39 ℃ for 8-14h to obtain a seed solution; inoculating the seed liquid to LB liquid culture medium according to the inoculation amount of 1-10%, culturing at 35-39 deg.C and 200-220rpm to OD₆₀₀0.9-1.1, isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to the medium at a final concentration of 0.1-1.0mM, and induction-cultured at 15-17 ℃ for 12-16 h.

In one embodiment of the invention, the LB medium contains peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH 7.2.

The seventh purpose of the invention is to provide the application of the subtilisin E mutant in degrading milk allergens, such as alpha-lactalbumin and casein.

The seventh purpose of the invention is to provide the application of the subtilisin E mutant in vitro thrombolysis.

The eighth purpose of the invention is to provide the application of the subtilisin E mutant in the food or pharmaceutical field.

The invention has the beneficial effects that:

the invention provides a subtilisin E mutant L1-2&L2-5, kcat/Km 0.032min^-1μM^-10.034min with wild type subtilisin E^-1μM^-1Similarly. The proportion of alkaline short peptides in the hydrolysate of the mutant increased from 11% to 50% of the original enzyme. The subtilisin E mutant L1-2 of the present invention&L2-5 has more application value and potential than wild type subtilisin E.

Drawings

FIG. 1: schematic construction of recombinant plasmids.

FIG. 2: carrying out SDS-PAGE gel electrophoresis on bacillus subtilis protease E and escherichia coli broken liquid supernatant of mutants L1-2& L2-5; where M represents the protein molecular weight standard, the different lane names represent the different muteins, and the arrow indicates the position of the protein band of interest.

FIG. 3: Lineweaver-Burk curves for subtilisin E and its mutants L1-2& L2-5, A: SES 7; b: l1-2& L2-5.

FIG. 4: ratio of alkaline short peptides in hydrolysates of subtilisin E and mutant L1-2& L2-5, A: SES 7; b: l1-2& L2-5.

Detailed Description

Enzyme activity determination method

The activity of the subtilisin E enzyme is determined by a p-nitrophenol short peptide analogue (Suc-AAPF-pNA) color development method. The subtilisin E hydrolyzes the p-nitrophenol short peptide analogue to release p-nitrophenol under a certain condition, and generates a color reaction, the color depth of the subtilisin E can be in direct proportion to the release amount of the p-nitrophenol within a certain range, so that the color comparison can be carried out at the wavelength of 405nm, and the enzyme activity can be calculated.

Definition of enzyme activity unit: under the above conditions, the amount of enzyme per ml which catalyzes the decomposition of the short peptide analog paranitrophenol per minute was defined as one enzyme activity unit, with the unreacted sample as a blank.

(II) an enzymatic kinetic constant determination step:

A. and (3) carrying out enzymolysis reaction: mu.L of purified subtilisin E (0.02 mg/mL) and 900. mu.L of Tris-HCl buffer (50mM, pH 9.0) containing Suc-AAPF-pNA at substrate concentration steps of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5mg/mL were mixed well, reacted at 37 ℃ for 30 minutes, then 10. mu.L of 10mM PMSF was added rapidly to terminate the reaction, and the absorbance at 405nm was measured.

B. Calculation of enzymatic kinetic constants: calculated from the absorbance values, under these conditions only a small fraction (< 10%) of the substrate is converted to the product by subtilisin E, so kinetic constants can be calculated by linear regression using the Lineweaver-Burk plot.

(III) adopting an AKTA avant protein purifier to purify the recombinant protein

The subtilisin E and the mutant thereof both contain histidine tags, so the subtilisin E can be separated and purified by a nickel ion affinity chromatography purification column, and the specific steps are as follows:

(1) balancing: equilibrating the purification column with 5 volumes of 50mmol/L, pH 7.2, Tris-HCl buffer;

(2) loading: loading the pretreated sample at a flow rate of 0.5mL/min, wherein the loading volume is generally not more than 5 times of the column volume;

(3) and (3) elution: comprises eluting unadsorbed substances, heteroproteins and target proteins at the flow rate of 1.0mL/min, carrying out gradient elution on 50mmol/L eluent containing 300mM imidazole, pH 7.2 and Tris-HCl buffer solution, wherein the detection wavelength is 280nm, and collecting the eluent containing the enzymatic activity of subtilisin E in batches.

Example 1: construction of bacillus subtilis protease E alkaline substrate selectivity improving mutant

Bacillus subtilis S7-derived subtilisin E (SES7) is used as a parent enzyme to construct a loop replacement mutant L1-2& L2-5. The primary sequence of the parent enzyme is shown in SEQ ID NO. 1. Replacement of Loop I with a Loop region rich in negatively charged side chain amino acid residues is designed to act synergistically with the negatively charged Glu156 to preferentially recognize oppositely charged amino acid residues in the substrate pocket. Therefore, the invention obtains the mutant L1-2& L2-5 (FIG. 1) by mutating the loop sequence GGPSGS at positions 127 and 132 to YDADDS and by mutating the loop sequence SSGSS at positions 158 and 162 to QHPKEG on the basis of the amino acid sequence shown in SEQ ID NO. 1.

Construction of subtilisin E mutant L1-2& L2-5: connecting the gene for coding the subtilisin E with pET-24a (+) through enzyme digestion to obtain a plasmid SES7/pET-24a (+); the plasmid SES7/pET-24a (+) is used as a template to design a PCR primer (Table 1), the PCR primer is adopted for amplification to obtain a mutant plasmid L1-2& L2-5/pET-24a (+) for nucleic acid electrophoresis and gel recovery.

PCR condition steps: firstly, pre-denaturation is carried out for 5min at 95 ℃; then 25 cycles were entered: denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 10s, and extension at 72 deg.C for 8 min; finally, extension is carried out for 10min at 72 ℃, and heat preservation is carried out at 4 ℃.

The gene is recovered, a template chain is digested by DpnI at 37 ℃ for 2 hours, then T4 DNA ligase and T4 phosphokinase are used for room temperature ligation overnight, and finally the recombinant Escherichia coli is obtained by expression in transduction competent Escherichia coli E.coli BL21(DE 3).

TABLE 1 primer sequences for subtilisin E mutants

Example 2: expression and purification of subtilisin E mutants

Selecting the recombinant Escherichia coli positive monoclonal obtained in example 1, and growing the recombinant Escherichia coli positive monoclonal in an LB liquid culture medium (containing 50 mug/mL ampicillin) overnight to obtain seed fermentation liquid; inoculating the seed fermentation liquid to LB liquid culture medium (containing 50 mug/mL ampicillin) according to 10% inoculation amount, and shake-culturing at 37 deg.C for 2.5h until OD600 is about 1.0; adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.05mM to induce the cells to express the subtilisin E extracellularly, and continuously culturing and fermenting overnight at 18 ℃ by a shaking table; the fermentation broth was centrifuged at 6000rpm for 1 hour at 4 ℃ to collect the cells, after disruption, the disrupted supernatant was collected and analyzed by SDS-PAGE, and the molecular weight of L1-2& L2-5 was 28kDa (FIG. 2). And purifying the recombinant protein by adopting an AKTA avant protein purifier.

Example 3: enzyme activity assay

The results of the Lineweaver-Burk plot calculation of kinetic constants by linear regression are shown in FIG. 3, from which the relevant kinetic parameters were calculated.

The results of determination of the enzymatic kinetic parameters of the recombinant enzyme after heterologous expression of subtilisin E and the truncated mutant L1-2& L2-5 in E.coli are shown in Table 2.

TABLE 2 determination of the enzymatic kinetic parameters of subtilisin E and mutants L1-2& L2-5

Example 4: substrate selectivity assays for subtilisin E mutants

Purified subtilisin E and the loop replacement mutant L1-2&L2-5 was diluted with 50mM Tris-HCl buffer to a protein content of 0.02mg/mL and a pH of 9.0, mixed with 10% w/V skim milk powder, incubated in a 37 ℃ constant temperature water bath for 48 hours, and then quenched with 10mM PMSF. The reaction supernatants were all filtered using a 3kDa ultrafiltration membrane and only the filtrates were collected for LC-MS analysis. Two-step sample treatment was performed on 20 μ g hydrolysate filtrate using Empore SDB-XC reverse phase material and extraction disc material (Sigma, st. Capillary LC-MS analysis of the hydrolysate was performed using an Orbitrap vessels mass spectrometer (Thermo Fisher Scientific), and tandem mass spectrometry results were usedStudio 518.5 (Bioinformatics Solutions, inc., Waterloo, Ontario, Canada) identified the peptide fragment sequence and the protein to which it belongs, and classified the hydrolysate for analysis of the amino acid distribution at position P1 and the proportion of basic peptide fragments, as shown in fig. 4, the proportion of H in the hydrolysate of the mutant increased from 0% to 1%, the proportion of R increased from 4% to 20%, the proportion of K increased from 7% to 29%, and the proportion of total basic short peptides increased from 11% to 50% of the original enzyme.

Comparative example

In the rest steps, on the basis of the amino acid sequence shown in SEQ ID NO.1, only the 127-plus 132-th loop sequence GGPSGS is mutated into PCTES and YDADDAI respectively, and the obtained subtilisin E mutants completely lose enzyme activity.

In the rest steps, on the basis of the amino acid sequence shown in SEQ ID NO.1, the loop sequence GGPSGS at the 127-th and 132-th positions is mutated into PCTES and YDADDAI, and the loop sequence SSGSS at the 158-th and 162-th positions is mutated into QHPKEG, so that the obtained subtilisin E mutants completely lose enzyme activity.

In the rest steps, on the basis of the amino acid sequence shown in SEQ ID NO.1, the loop sequences GGPSGS at the 127-fold and 132-fold positions are respectively mutated into YDADDS, the enzyme activity of the obtained subtilisin E mutant is reduced by more than 1000 times compared with that of the original enzyme, but the proportion of the alkaline short peptide in the hydrolysate of the mutant is improved to 16 percent.

The rest steps are the same as the example, only the SSGSS of the loop sequence at the 158 th-162 th site is mutated into QHPKEG on the basis of the amino acid sequence shown in SEQ ID NO.1, and the kcat/Km of the obtained subtilisin E mutant is 0.036min^-1μM^-1The proportion of the basic short peptide in the hydrolysate of the mutant was 36%.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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