Glutathione bifunctional synthetase mutant with improved enzyme activity and thermal stability and its application

Abstract

The present invention discloses a glutathione bifunctional synthetase mutant with improved enzyme activity and thermal stability and its application, belonging to the field of genetic engineering and fermentation engineering. The glutathione bifunctional synthetase mutant is selected from any one of the following: its amino acid sequence is obtained by mutation of the sequence shown in SEQ ID NO.1, and the mutation is selected from one or more of the following amino acid residue sites: 61, 218, 485 and 722. The present application performs site-directed mutagenesis on the amino acid sequence (SEQ ID NO.1) of glutathione bifunctional synthetase GshFst, and obtains a single mutant, as well as a double mutant obtained by mutation of two sites, whose enzyme activity and thermal stability are significantly improved compared with the wild type. The enzyme mutant provided by the present application has broad application prospects in the production of glutathione and the construction of genetically engineered microorganisms for producing glutathione.

Description

Technical Field

[0001] The invention relates to a glutathione bifunctional synthetase mutant with improved enzyme activity and thermal stability and application thereof, and belongs to the field of genetic engineering and fermentation engineering. Background Art

[0002] Glutathione (GSH) is a tripeptide substance containing a γ-amide bond and a sulfhydryl group. It is widely present in various organisms and has the function of regulating intracellular redox balance and integrating detoxification. It is a white powder with a molecular weight of 307.33. It is soluble in water, dilute alcohol and liquid ammonia, but insoluble in organic solvents such as alcohol and ether. It is composed of L-cysteine ​​(L-Cys), L-glutamic acid (L-Glu) and glycine (Gly). Glutathione has two forms in the body: reduced and oxidized. The reduced form is the main active state and has a wide range of application value. Glutathione has physiological functions such as anti-oxidation, detoxification, amino acid transport and scavenging free radicals in the body. It is widely used in medicine, food, cosmetics and breeding.

[0003] The biosynthesis of glutathione is a two-step enzyme-catalyzed reaction that requires the participation of ATP. L-Glu and L-Cys generate γ-glutamylcysteine ​​(γ-Glu-Cys) under the catalysis of γ-glutamylcysteine ​​synthetase (γ-GCS), and then react with Gly under the catalysis of glutathione synthetase (GS) to generate glutathione. The activity of γ-GCS enzyme is feedback inhibited by glutathione, which is the rate-limiting step. In recent years, a glutathione bifunctional synthetase (GshF) has been discovered in Streptococcus agalactiae, which can catalyze the two-step reaction of γ-GCS and GS at the same time without feedback inhibition.

[0004] At present, GshF has been successfully expressed in Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Corynebacterium glutamicum. Qiu et al. integrated and expressed GshF from Streptococcus thermophilus in Saccharomyces cerevisiae BY4717, which increased the GSH concentration by 3 times and improved the strain's tolerance to inhibitors. Ge et al. expressed GshF from Listeria monocytogenes, Streptococcus agalactiae and Lactobacillus plantarum in Pichia pastoris GS115. The expression level of GshF from Listeria monocytogenes was significantly higher than that from Streptococcus agalactiae and Lactobacillus plantarum due to codon preference. Liu et al. used Corynebacterium glutamicum as a host to heterologously express GshF from Streptococcus agalactiae, and strengthened the supply of precursor L-cysteine ​​by modifying metabolic pathways, including increasing L-cysteine ​​biosynthesis flux, knocking out L-cysteine ​​degradation genes and deleting transcription factors. Without exogenous addition of L-cysteine ​​and L-glutamate, 756 mg·L -1 GSH.

[0005] There have been many studies on the expression of the bifunctional enzyme GshF, and great progress has been made in its use in the synthesis of glutathione. However, enzyme activity and stability are still factors that limit its efficient production. Screening for high-performance glutathione synthetase enzyme systems and obtaining mutants with good catalytic activity and stability still have important economic value. Summary of the invention

[0006] In view of the problems of low catalytic efficiency and poor stability of synthase in the current enzymatic production of glutathione, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a mutant with improved activity of glutathione bifunctional synthetase and to increase the yield of glutathione synthesized by the enzymatic method, which has a universal significance for promoting the synthesis of glutathione.

[0007] The technical solution adopted by the present invention is as follows:

[0008] The bifunctional enzyme GshF from Streptococcus thermophilus was cloned, and the protein model was established using AlaphFold 2. The protein structure was analyzed, the protein was molecularly docked with the substrate, and sites were selected for site-directed mutagenesis. The activity of the mutated enzyme was measured to determine the sites that had an important influence on the enzyme activity. Then, combined mutations were performed and the mutation advantages were superimposed to obtain mutants with improved activity and stability compared to the wild type.

[0009] The present invention provides a glutathione bifunctional synthetase mutant, which is subjected to any of the following mutations based on the amino acid sequence as shown in SEQ ID NO.1:

[0010] (1) mutating phenylalanine at position 61 to serine to obtain the mutant F61S;

[0011] (2) mutating phenylalanine at position 61 to serine and histidine at position 218 to tyrosine to obtain the mutant F61S / H218Y;

[0012] (3) mutating the phenylalanine at position 61 to serine and the threonine at position 485 to isoleucine to obtain the mutant F61S / T485I;

[0013] (4) mutating the phenylalanine at position 61 to serine and the serine at position 722 to alanine to obtain the mutant F61S / S722A;

[0014] (5) mutating the phenylalanine at position 61 to serine, the histidine at position 218 to tyrosine, and the threonine at position 485 to isoleucine to obtain the mutant F61S / H218Y / T485I;

[0015] (6) mutating the phenylalanine at position 61 to serine, the histidine at position 218 to tyrosine, and the serine at position 722 to alanine to obtain the mutant F61S / H218Y / S722A;

[0016] (7) mutating the phenylalanine at position 61 to serine, the threonine at position 485 to isoleucine, and the serine at position 722 to alanine to obtain the mutant F61S / T485I / S722A;

[0017] (8) The phenylalanine at position 61 was mutated to serine, the histidine at position 218 was mutated to tyrosine, the threonine at position 485 was mutated to isoleucine, and the serine at position 722 was mutated to alanine to obtain the mutant F61S / H218Y / T485I / S722A.

[0018] In one embodiment, the wild-type glutathione bifunctional synthetase GshF is derived from Streptococcus thermophilus, and the nucleotide sequence encoding the glutathione bifunctional synthetase is shown in SEQ NO.2.

[0019] The present invention also provides a gene encoding the mutant.

[0020] The invention also provides an expression vector carrying the gene.

[0021] The present invention also provides a microbial cell expressing the mutant or carrying the expression vector.

[0022] In one embodiment of the present invention, the microbial cell uses Escherichia coli BL21 (DE3) as a host and pET-28a (+) as a vector.

[0023] The invention also provides a method for producing glutathione by using the mutant or the microbial cell as a catalyst.

[0024] In one embodiment of the present invention, glutathione is produced by converting a substrate using a microbial cell expressing the above-mentioned glutathione bifunctional synthetase mutant, wherein the substrate is a combination of L-glutamate, L-cysteine ​​and glycine.

[0025] In one embodiment of the present invention, the microbial cells are cultured in a culture system until OD 600 The pH value is 0.6-0.8, the inducer is added to induce at 25°C for 10-12 hours, and the bacteria are collected; the bacteria are added to the reaction system containing the substrate and reacted for 1-10 hours.

[0026] In one embodiment of the present invention, the recombinant monoclonal clone is inoculated into 10 mL LB liquid culture medium containing kanamycin, cultured at 37°C 220 rpm for 10-12 h, and then 2% of the culture solution is transferred to a new 50 mL LB liquid culture medium containing kanamycin and cultured for about 2-3 h to achieve OD = 0.6-0.8, and the filter-sterilized inducer IPTG is added to a final concentration of 0.2 mM, and then cultured at 25°C 220 rpm for 10-12 h, and the bacteria are collected by centrifugation.

[0027] In one embodiment of the present invention, the substrate concentration in the reaction system is 20-60 mM, the strain OD 600 =(4-60)±0.5, react at pH 8±1, 30~45℃.

[0028] The invention also provides glutathione obtained by the method.

[0029] The present invention also provides the use of the mutant, expression vector or microbial cell in producing glutathione and products containing glutathione in the fields of food, cosmetics, and drug preparation.

[0030] Beneficial effects:

[0031] The present invention performs site-directed combined mutagenesis on the 61st, 218th, 485th and 722nd positions of the glutathione bifunctional synthetase GshFst, and obtains mutants F61S / S722A, F61S / H218Y / T485I, F61S / H218Y / S722A, F61S / T485I / S722A and F61S / H218Y / T485I / S722A, which have significantly improved enzyme activities of 1.52, 1.55, 1.76, 1.86 and 1.51 U·mL, respectively. -1 Compared with the wild-type enzyme activity, the enzyme activity increased by 113.93%, 118.54%, 147.27%, 161.81% and 113.29%, respectively, indicating that the enzyme activity of the mutant was greatly improved. DETAILED DESCRIPTION

[0032] The present invention aims to provide several bifunctional glutathione synthetase mutants and their applications in glutathione synthesis. For further detailed description, the present invention is further described below in conjunction with examples.

[0033] Example 1: Construction of wild-type GshF expression vector

[0034] The glutathione bifunctional synthetase gene gshFst derived from Streptococcus thermophilus and codon-optimized for Escherichia coli was artificially synthesized by GENEWIZ (Suzhou GENEWIZ Biotechnology Co., Ltd.). The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.1.

[0035] The target fragment gshFst was amplified according to the primers in Table 1, and then connected with the plasmid pET-28a cut with restriction endonuclease (Hind III) using the homologous recombinase ClonExpress IIOne Step Cloning Kit in a 37°C metal bath for 30 minutes to obtain the recombinant plasmid pET28a-gshFst. After that, the recombinant plasmid pET28a-gshFst was transformed into Escherichia coli BL21 (DE3), spread on the LB plate containing Kan, and when a single colony grew, the transformant was picked for colony PCR verification, and the bands were sent to Tianlin Biotechnology (Shanghai) Co., Ltd. for sequencing.

[0036] Table 1 Primers used for construction of recombinant strains

[0037]

[0038] Example 2: Determination and construction of site-directed mutagenesis sites

[0039] The site-directed mutation site was introduced by the inverse PCR method, and the recombinant plasmid pET28a-gshFst constructed above was used as a template for single-point mutation. The primers are shown in Table 1. The product recovered by inverse PCR amplification was used to recognize the methylation site and digest the template with Dpn I enzyme, and then transformed into Escherichia coli BL21 (DE3) by chemical transformation, and spread on LB plates containing Kan, and incubated at 37°C, 220 r·min -1 After culturing for 12 h, single colonies were picked for colony PCR verification, and the agarose gel electrophoresis results were analyzed. Transformants that were consistent with the theoretical values ​​were sent to Tianlin Biotechnology Co., Ltd. for sequencing to obtain recombinant Escherichia coli expressing the mutant.

[0040] Table 2 Primers used for site-directed mutagenesis

[0041]

[0042]

[0043] Note: Lowercase bases are mutation sites

[0044] The transformants with correct sequencing results were selected, three lines were drawn on LB solid medium, and cultured in a 37°C incubator for 12-14 hours; a single colony was inoculated into a 10 mL LB vial containing antibiotics and incubated at 37°C, 220 r·min-1 After culturing for 12 h, the cells were transferred to 50 mL LB liquid medium containing antibiotics at a 2% inoculum and cultured at 37°C until OD 600 =0.6-0.8, add IPTG with a final concentration of 0.2mM and then incubate at 25℃, 220r·min -1 Induce expression for 12 h. Take 2 mL of fermentation broth and incubate at 4 °C and 8000 r / min -1 Centrifuge for 5 minutes, resuspend and wash the cells with 20mM Tris-HCl (pH 8.0) buffer, centrifuge for 5 minutes, and repeat the washing twice. Take 1mL of buffer to suspend the cells, perform ultrasonic disruption, and then spin at 4℃ and 12000r·min. -1 Centrifuge for 10 minutes and the supernatant is the crude enzyme solution, and its crude enzyme activity is determined.

[0045] The enzyme activity (U) of GshFst was defined as the amount of GshFst required to produce 1 μmol of GSH per minute, and the volume ratio (U·mL -1 ) to measure. Enzyme activity reaction system (based on final concentration): 40mM L-glutamic acid, 20mM L-cysteine, 40mM glycine, 20mM ATP, 20mM magnesium chloride, 100mM Tris-HCl buffer (pH 8.0), take 100μL crude enzyme solution, react in a 37℃ water bath for 20min, add an equal volume of 10% (w / v) trichloroacetic acid solution to the reaction solution to terminate the reaction, ice bath for 30min, and rotate at 4℃, 12000r·min -1 Centrifuge for 10 min and dilute the supernatant to an appropriate multiple to determine the GSH concentration.

[0046] The detection method of GSH is high performance liquid chromatography, and the supernatant of the reaction solution (diluted to the corresponding multiple) is filtered with a 0.22 μm water filter membrane. HPLC analysis: the mobile phase is V (0.01M sodium heptane sulfonate and 0.05M potassium dihydrogen phosphate solution): V (methanol) = 95:5, and the pH is adjusted to 3.0 with phosphoric acid; the chromatographic column is Diamonsil C18 (250x 4.6mm, 5μm), the flow rate is 0.6mL / min, the column temperature is 30°C, the ultraviolet detection wavelength is 210nm, and the injection volume is 10μL.

[0047] Under the same conditions, the relative enzyme activity of the mutant and the wild type was calculated with the crude enzyme activity of the wild type as 100%, as shown in Table 3. The enzyme activity of the wild type was 0.71 U·mL -1 The enzyme activities of mutants F61S, H218Y, A482E and T485I were 1.17, 0.82 and 1.18 U·mL -1 , among which the enzyme activities were increased by 64.29%, 27.89%, 15.48% and 65.51% respectively compared with the wild type.

[0048] Table 3 Relative enzyme activities of mutants

[0049]

[0050]

[0051] The mutant F61S was combined with the mutants T485I, H218Y and S722A (enzyme activity increased by 48.93%). The construction method of the combined mutant was as follows: Taking the construction of the combined mutant T485I / S722A as an example, the recombinant plasmid pET28a-gshFst was used as a template, T485I-F and T485I-R in Table 1 were used as primers to construct the mutant plasmid pET28a-gshFst T485I ; Then plasmid pET28a-gshFst T485I As a template, S722A-F and S722A-R in Table 1 were used as primers to construct the mutant plasmid pET28a-gshFst T485I / S722A The obtained mutant plasmid was transformed into Escherichia coli BL21 (DE3) and verified to obtain the recombinant E. coli E. coli BL21-pET28a-gshFst expressing the mutant T485I / S722A T485I / S722A .

[0052] Cultivate recombinant E. coli BL21-pET28a-gshFst according to the above method T485I / S722A The crude enzyme solution was collected and the enzyme activity was determined. As shown in Table 4, the relative enzyme activities of the combined mutations were higher than those of the single point mutations. The enzyme activities of the mutants H218Y / T485I / S722A, F61S / H218Y / S722A and F61S / T485I / S722A were significantly improved, reaching 1.69, 1.76 and 1.86 U·mL, respectively. -1 , compared with the wild-type enzyme activity increased by 137.61%, 147.27% and 161.81%, respectively.

[0053] Table 4 Relative enzyme activities of combined mutations

[0054]

[0055] Definition of specific enzyme activity: the number of enzyme activity units per unit weight (mg) of GshF.

[0056] The calculation formula of specific enzyme activity is as follows:

[0057]

[0058] The wild type and combined mutants were purified by nickel column affinity chromatography, and their specific enzyme activities and half-lives were determined under optimal conditions. The specific enzyme activity of the double mutant T485I / S722A was 18.33 U·mg -1 , which was 1.85 times higher than that of the wild type.

[0059] Table 5 Specific enzyme activities of mutants

[0060]

[0061] To investigate the thermal stability of the mutants, the crude enzyme solution was incubated in a 37°C water bath for 30 min and placed on ice for 5 min. The residual enzyme activity at 37°C was measured and compared with that of the enzyme solution without heat treatment. The results are shown in Table 6. The residual enzyme activity of the wild type was 56.75%, and the residual enzyme activities of the mutants F61S, H218Y, T485I and S722A were 77.36%, 51.73%, 62.89% and 87.41%, respectively. The residual enzyme activity of the combined mutations after incubation at 37°C for 30 min was higher than that of the wild type, among which the residual enzyme activities of the mutants F61S / T485I, T485I / S722A and F61S / T485I / S722A were all greater than 80%, and their thermal stability at 37°C was significantly increased.

[0062] Table 6 Residual enzyme activity of mutants after heat treatment at 37°C

[0063]

[0064] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A glutathione bifunctional synthetase mutant, characterized in that: Based on the amino acid sequence as shown in SEQ ID NO.1, any of the following mutations is performed: (1) mutate phenylalanine at position 61 to serine; (2) mutating phenylalanine at position 61 to serine and histidine at position 218 to tyrosine; (3) mutating phenylalanine at position 61 to serine and threonine at position 485 to isoleucine; (4) mutating phenylalanine at position 61 to serine and mutating serine at position 722 to alanine; (5) mutating phenylalanine at position 61 to serine, histidine at position 218 to tyrosine, and threonine at position 485 to isoleucine; (6) mutating phenylalanine at position 61 to serine, histidine at position 218 to tyrosine, and serine at position 722 to alanine; (7) mutating the phenylalanine at position 61 to serine, the threonine at position 485 to isoleucine, and the serine at position 722 to alanine; (8) The phenylalanine at position 61 was mutated to serine, the histidine at position 218 was mutated to tyrosine, the threonine at position 485 was mutated to isoleucine, and the serine at position 722 was mutated to alanine.

2. A gene encoding the mutant according to claim 1.

3. An expression vector carrying the gene according to claim 2.

4. A microbial cell expressing the mutant according to claim 1 or carrying the expression vector according to claim 3.

5. The microbial cell according to claim 4, characterized in that Escherichia coli BL21 (DE3) was used as the host and pET-28a (+) was used as the vector.

6. A method for producing glutathione, characterized in that The mutant according to claim 1 or the lysate of the microbial cells according to claim 4 or 5 is used as a catalyst to react in a reaction system containing L-glutamic acid, L-cysteine ​​and glycine.

7. The method according to claim 6, characterized in that In the reaction system, the concentrations of L-glutamic acid, L-cysteine ​​and glycine are 20-80 mM respectively.

8. The method according to claim 7, characterized in that The reaction was carried out at pH 7-9 and 30-45°C.

9. Use of the mutant according to claim 1 or the microbial cell according to claim 4 or 5 in the production of glutathione and products containing glutathione in the fields of food, medicine and cosmetics.

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