Glutathione bifunctional synthetase mutant and application thereof in catalytic synthesis of glutathione

Abstract

The invention discloses a glutathione bifunctional synthetase mutant and application thereof in catalytic synthesis of glutathione. According to the glutathione bifunctional synthetase mutant, the thermal stability of glutathione bifunctional synthetase is improved through site-directed mutagenesis, and the activity of catalyzing synthesis of glutathione from L-glutamic acid, L-cysteine and glycine is improved. The relative enzyme activity of the mutant GshFST-S619T-A691V reaches 279.84% of the relative enzyme activity before mutation, 30.42% of enzyme activity is still kept after incubation for 180 min at the temperature of 35 DEG C, the pure enzyme activity reaches 3.31 U / mg, and the yield of the product glutathione reaches 82.03% after reaction is conducted for 8 h.

Description

(I) Technical field

[0001] The invention belongs to the technical field of biochemical engineering, and specifically relates to a glutathione bifunctional synthetase mutant derived from thermophilic streptococcus and application thereof in catalytic synthesis of glutathione. (II) Background technology

[0002] Glutathione (GSH) is a tripeptide compound containing a γ-amide bond and a sulfhydryl group. It is formed by the condensation of L-glutamic acid, L-cysteine, and glycine through peptide bonds. Its chemical name is γ-L-glutamyl-L-cysteinyl-glycine. There are free sulfhydryl groups in the structure of GSH, which has a strong electron-donating capacity. It has detoxification, antioxidant, anticonvulsant, antithrombotic, and anti-atherosclerotic effects, and is widely used in the fields of medicine, food, and cosmetics.

[0003] GSH was synthesized chemically in the early days, but the chemical method has disadvantages such as low product purity and cumbersome operation, and has been gradually replaced by biological methods. In eukaryotic cells and most Gram-negative bacteria, the biosynthesis of GSH is completed through a two-step ATP-dependent enzyme-catalyzed reaction. Among them, γ-glutamyl-L-cysteine ​​synthetase (γ-GCS) catalyzes L-glutamate and L-cysteine ​​to synthesize the dipeptide intermediate γ-glutamate-cysteine, and then glutathione synthetase (GS) catalyzes the dipeptide intermediate and glycine to generate the tripeptide product GSH. Studies have shown that γ-GCS is subject to feedback inhibition by the product GSH, affecting the activity of γ-GCS. In addition, the accumulation of high-concentration dipeptide intermediates during the reaction leads to a low yield of GSH synthesis and increases the cost of subsequent separation and purification.

[0004] Studies have found that some Gram-positive bacteria contain a glutathione bifunctional synthetase GshF that has both γ-GCS and GS activities, which can catalyze L-glutamate, L-cysteine, and glycine to synthesize GSH ( Figure 1 ), and most GshFs do not have product feedback inhibition. However, the natural GshF reported so far still has poor catalytic properties such as enzyme activity and cannot meet the requirements of industrial application. Therefore, the present invention transforms glutathione bifunctional synthetase through protein engineering to improve its catalytic activity and thermal stability, making it more potential for industrial application. (III) Summary of the invention

[0005] The present invention aims to provide a glutathione bifunctional synthetase mutant and its application in catalytic synthesis of glutathione, which effectively improves the catalytic activity and thermal stability of wild-type glutathione bifunctional synthetase, thereby improving the yield of glutathione and solving the problem that the existing glutathione bifunctional synthetase has poor catalytic properties in synthesizing glutathione.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides a glutathione bifunctional synthetase mutant, wherein the mutant is obtained by mutating the 619th and 691st amino acids of the amino acid sequence shown in SEQ ID NO.1.

[0008] Furthermore, it is preferred that the mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.1 is mutated into one of the following: (1) the serine at position 619 is mutated into threonine; (2) the alanine at position 691 is mutated into valine; (3) the serine at position 619 is mutated into threonine and the alanine at position 691 is mutated into valine, the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.4.

[0009] The present invention also provides a coding gene of the glutathione bifunctional synthetase mutant, a recombinant expression vector containing the coding gene, and a recombinant genetic engineering bacterium constructed by the recombinant expression vector. The recombinant expression is based on plasmid pET28a, and the host cell of the recombinant genetic engineering bacterium can be various conventional host vectors in the art, and the preferred host cell is Escherichia coli BL21.

[0010] The present invention also provides an application of the glutathione bifunctional synthetase mutant in synthesizing glutathione. The application method is: using the wet bacteria obtained by fermentation and culturing the engineered bacteria containing the glutathione bifunctional synthetase mutant encoding gene, the supernatant after resuspending and crushing with a buffer solution is used as a catalyst, L-glutamic acid, L-cysteine, and glycine are used as substrates, MgSO4, ATP, and polyphosphate kinase and sodium hexametaphosphate for ATP circulation are added, a pH 6-8 buffer solution is used as a reaction medium to form a reaction system, and the reaction is carried out at 20-40° C. and 300-600 rpm (preferably 25° C. and 500 rpm) to obtain glutathione.

[0011] Furthermore, in the reaction system, the amount of catalyst added is 10-50 g / L (preferably 25 g / L) based on the weight of wet bacteria before crushing; the final concentration of L-glutamic acid added is 20-120 mM (preferably 70 mM); the final concentration of L-cysteine ​​added is 20-120 mM (preferably 70 mM); the final concentration of glycine added is 20-120 mM (preferably 70 mM); the final concentration of MgSO4 added is 20-80 mM (preferably 60 mM); the final concentration of ATP added is 5-25 mM (preferably 10 mM); the final concentration of polyphosphate kinase added is 1-10 g / L (preferably 5 g / L); and the final concentration of sodium hexametaphosphate added is 30-50 mM (preferably 35 mM).

[0012] The method for determining the activity of the glutathione bifunctional synthetase mutant of the present invention is as follows: the pure enzyme liquid extracted from the supernatant after the wet bacteria obtained by fermentation culture of the engineered bacteria containing the glutathione bifunctional synthetase mutant encoding gene are resuspended in a buffer solution and crushed is used as a catalyst, L-glutamic acid, L-cysteine, and glycine are used as substrates, MgSO4 and ATP are added, and a pH 8 buffer solution is used as a reaction medium to form a reaction system, and the reaction is carried out at 35° C. and 500 rpm for 5 minutes. Enzyme activity definition: At 35° C. and pH 8.0, the amount of enzyme required to generate 1 μmol of glutathione per minute is defined as one enzyme activity unit (1U).

[0013] Further, the catalyst was prepared as follows: the recombinant genetically engineered bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 150 rpm overnight; then inoculated into LB medium containing 50 μg / mL kanamycin at a volume concentration of 2%, and cultured at 37°C and 150 rpm until the bacterial concentration OD 600 =0.6, add 0.1 mM IPTG at a final concentration, induce culture at 28°C for 12-16 h, and finally centrifuge at 4°C, 8000 rpm for 10 min to collect wet cells;

[0014] The collected wet cells were resuspended in Tris-HCl buffer (100 mM, pH 8.0), mixed evenly, and then freeze-thawed (-80°C for 30 min, 37°C for 30 min), and after 4 cycles, centrifuged at 4000 rpm for 10 min to obtain the supernatant, which was the crude enzyme solution.

[0015] After the crude enzyme solution is filtered through a 0.22 μm microporous filter membrane, the enzyme protein is bound to a Ni-NTA column, and the impurity protein is first eluted with a 20 mM PBS buffer solution at pH 8.0 containing 50 mM imidazole and 500 mM NaCl, eluting for 2-5 (preferably 3) column volumes at an elution rate of 1-3 mL / min (preferably 2 mL / min); then, the target protein is collected using a 20 mM PBS buffer solution at pH 8.0 containing 500 mM imidazole and 500 mM NaCl, eluting for 2-5 (preferably 3) column volumes at an elution rate of 1-3 mL / min (preferably 1 mL / min); SDS-PAGE is used to verify the target protein and its purity, and finally, Tris-HCl buffer (100 mM, pH 8.0) is used as a dialyzate to perform dialysis in a 20 kD dialysis bag, and the permeate is collected to obtain a pure enzyme solution.

[0016] The glutathione bifunctional synthetase mutant of the present invention has a variety of feasible forms to be selected as a catalyst, including use in the form of whole cells, use in the form of unpurified crude enzymes, and use in the form of partially or completely purified enzymes. In addition, the glutathione bifunctional synthetase mutant of the present invention can be made into a biocatalyst in the form of immobilized enzymes or immobilized cells by using known enzyme immobilization technology, thereby further improving the stability and reusability of the enzyme, which is conducive to its application in industrial production.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0018] The present invention improves the thermal stability of glutathione bifunctional synthetase and the activity of catalyzing L-glutamate, L-cysteine ​​and glycine to synthesize glutathione through site-directed mutagenesis. The relative enzyme activity of the mutant GshFST-S619T-A691V reaches 279.84% of that before mutation, and still retains 30.42% of the enzyme activity after incubation at 35°C for 180 minutes. The pure enzyme activity reaches 3.31U / mg, and after 8 hours of reaction, the yield of product glutathione reaches 82.03%. (IV) Description of the drawings

[0019] Figure 1 , the reaction formula for the synthesis of glutathione catalyzed by glutathione bifunctional synthetase.

[0020] Figure 2 , bar graph of relative enzyme activities of WT and mutants in Example 2.

[0021] Figure 3 HPLC spectra of GSH synthesis catalyzed by glutathione standards, GshFST and double mutant GshFST-S619T-A691V.

[0022] Figure 4 Comparison of enzyme activities of GshFST and its mutants GshFST-S619T, GshFST-A691V, and GshFST-S619T-A691V.

[0023] Figure 5 Comparison of thermal stability of GshFST and its mutants GshFST-S619T, GshFST-A691V, and GshFST-S619T-A691V.

[0024] Figure 6 , GshFST and mutant GshFST-S619T-A691V catalyzed the reaction process of GSH synthesis.

[0025] Figure 7 , the reaction process of GSH synthesis catalyzed by mutant GshFST-S619T-A691V under different substrate concentrations.

[0026] Figure 8 , the effect of different ATP concentrations on the specific enzyme activity of mutant GshFST-S619T-A691V.

[0027] Fig. 9 , the effect of different temperatures on the specific enzyme activity of mutant GshFST-S619T-A691V. (V) Specific implementation methods

[0028] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0029] The LB solid culture medium of the present invention is composed of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, the solvent is water, and the pH value is 7.0.

[0030] The LB liquid culture medium is composed of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, the solvent is water, and the pH is 7.0.

[0031] The GSH concentration was detected by high performance liquid chromatography (HPLC): the liquid column was a C18 column, the mobile phase was an aqueous solution containing 10 mM sodium heptane sulfonate and 50 mM potassium dihydrogen phosphate: methanol = 95:5, v:v, flow rate 1 mL / min, injection volume 10 μL, detection wavelength 210 nm, and column temperature 30 °C.

[0032] Example 1: Construction of wild-type GshFST recombinant engineering bacteria

[0033] The glutathione bifunctional synthetase gene from Streptococcus thermophilus (ACCESSION NO: QCT24552.1, amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.3) was connected to the NcoI and XhoI restriction sites of plasmid pET-28a, and transferred into E. coli DH5 competent cells, spread on LB plates containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37°C overnight, and positive transformants were selected for sequencing verification. The positive clones were inoculated into 5 mL of LB liquid culture medium containing kanamycin at a final concentration of 50 μg / mL, cultured at 37°C, 150 rpm overnight, and the plasmids were extracted and verified. After verification, the recombinant expression vector was transferred into E. coli BL21 (DE3) to obtain the recombinant bacteria E. coli BL21 (DE3) / pET28b-GshFST (denoted as WT), which was then inoculated on an LB plate containing a final concentration of 50 μg / mL kanamycin and cultured overnight at 37 ° C. A single clone was picked and inoculated into 5 mL of LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37 ° C at 150 rpm overnight, and then the bacterial solution and 30% glycerol (v / v = 1: 1) were mixed in a glycerol tube and stored in a -80 ° C refrigerator.

[0034] Example 2: Construction of glutathione bifunctional synthetase mutants GshFST-S619T and GshFST-A691V

[0035] 1. Selection of mutation sites

[0036] The protein structure of wild-type GshFST was modeled, and the complex structure of substrate and protein binding was obtained by molecular docking calculation. The amino acids in the range were virtually mutated, and the mutation sites with ΔΔG ≤ -0.5 kcal / mol were predicted by FoldX software, namely G81, Q162, E241, T297, N18, G510, S536, Q548, E582, V588, S619, D673, and A691. After random mutation of each site, the enzyme activity was tested using the method of Example 5 (the enzyme activity of some mutants is shown in Figure 2 ), with the enzyme activity of WT crude enzyme solution as 100%, and finally the dominant mutants S619T and A691V were screened and verified by experiments.

[0037] 2. Construction of dominant single mutants

[0038] The recombinant expression vector pET28a-GshFST obtained in Example 1 was used as a template, and S619T-Forward and S619T-Reverse in Table 1 were used as primers. The template was amplified according to the overlap extension PCR method, and the serine at position 619 and the alanine at position 691 in the parent amino acid sequence were site-directedly mutated.

[0039] Table 1. Primer sequences

[0040]

[0041] Note: Lowercase letters represent mutated bases

[0042] PCR amplification system (50 μL): template DNA 0.5 ng, 2×Phanta Max Buffer 25 μL, dNTPs (10 mM each) 1 μL, upstream and downstream mutation primers 1 μL each, Phanta Max Super-Fidelity DNA Polymerase 1U, ddH2O to make up the total volume.

[0043] The PCR reaction conditions were as follows: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 4 min 30 s, step (2) for 30 cycles; (3) re-extension at 72°C for 10 min. Storage at 16°C.

[0044] After the PCR product was positive after 0.9% agarose gel electrophoresis analysis, 20 μL of the PCR reaction solution was taken, and 1 μL of endonuclease Dpn I was added to remove the template plasmid DNA at 37°C for 3h, and inactivated at 65°C for 10min. Heat shock transformation was performed into E. coli BL21 (DE3) competent cells, and after recovery, it was spread on LB plates containing 50 μg / mL kanamycin and cultured overnight. About 300 clones were obtained on each plate.

[0045] Then, 4-5 clones were picked into LB medium, cultured at 37°C for 8 h, and then the bacterial solution was sequenced to obtain the recombinant engineered bacteria E. coli BL21 (DE3) / pET28b-GshFST-S619T and E. coli BL21 (DE3) / pET28b-GshFST-A691V of glutathione bifunctional synthetase mutants.

[0046] Example 3: Construction of glutathione bifunctional synthetase mutant GshFST-S619T-A691V

[0047] In order to construct the glutathione bifunctional synthetase double mutant GshFST-S619T-A691V, corresponding primers were designed (Table 1).

[0048] The recombinant plasmid pET28b-GshFST-S619T constructed in Example 2 was used as a template, and the primers A691V-Forward and A691V-Reverse in Table 1 were used. The construction method was similar to that in Example 2 to obtain the recombinant engineered bacteria E. coli BL21 (DE3) / pET28b-GshFST-S619T-A691V.

[0049] Example 4: Inducible expression and purification of glutathione bifunctional synthetase and its mutants

[0050] The engineered bacteria E. coli BL21 (DE3) / pET28b-GshFST, E. coli BL21 (DE3) / pET28b-GshFST-S619T and E. coli BL21 (DE3) / pET28b-GshFST-S619T-A691V containing wild-type and mutant glutathione bifunctional synthetase were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 ° C, 150 rpm overnight. Then, the inoculum was inoculated into LB medium containing 50 μg / mL kanamycin at a 2% (v / v) inoculum and cultured at 37 ° C, 150 rpm until the bacterial concentration OD 600 =0.6, add IPTG with a final concentration of 0.1 mM, induce culture at 28°C for 12-16 h, and finally collect wet cells by centrifugation at 4°C and 8000 rpm for 10 min.

[0051] 1.5 g of the collected wet cells were resuspended in 30 mL of Tris-HCl buffer (100 mM, pH 8.0), mixed evenly, and then freeze-thawed (-80°C for 30 min, 37°C for 30 min). After 4 cycles, the cells were centrifuged at 4000 rpm for 10 min and the supernatant was obtained, which was the crude enzyme solution of glutathione bifunctional synthetase and mutants.

[0052] After the crude enzyme solution was filtered with a 0.22 μm microporous filter membrane, the enzyme protein was bound to a Ni-NTA column, and the impurity protein was first eluted with a 20 mM PBS buffer solution at pH 8.0 containing 50 mM imidazole + 500 mM NaCl, eluted for 3 column volumes, and the elution rate was 2 mL / min; then, the target protein was collected with a 20 mM PBS buffer solution at pH 8.0 containing 500 mM imidazole + 500 mM NaCl, eluted for 3 column volumes, and the elution rate was 1 mL / min; SDS-PAGE was used to analyze and verify the target protein and its purity, and finally Tris-HCl buffer (100 mM, pH 8.0) was used as a dialysate to dialyze in a 20 kD dialysis bag, and the permeate was collected to obtain a pure enzyme solution of glutathione bifunctional synthetase and its mutants.

[0053] Example 5: Activity determination of glutathione synthesis catalyzed by glutathione bifunctional synthetase and its mutants

[0054] The pure enzyme solution containing glutathione bifunctional synthetase GshFST, GshFST-S619T, and GshFST-S619T-A691V prepared in Example 4 was used as a catalyst, and the catalytic reaction was carried out at 35° C. and 500 rpm.

[0055] 1mL reaction system composition: 500μL Tris-HCl buffer (100mM, pH 8.0), 20mM L-glutamic acid, 20mML-cysteine, 20mM glycine, 20mM MgSO4, 10mM ATP, 500μL (add final concentration of 0.3g / L based on protein content) pure enzyme solution, 35℃, 500rpm reaction for 5min. Add 100μL 2M HCl to terminate the reaction and centrifuge at 12000rpm for 2min. After collecting the supernatant, HPLC analysis was performed. The test results showed that the enzyme activity of the double mutant GshFST-S619T-A691V was 279.84% compared with the enzyme activity of the wild-type glutathione bifunctional synthase GshFST. The results are shown in Table 2, Figure 3 , Figure 4 shown.

[0056] Enzyme activity definition: At 35°C and pH 8.0, the amount of enzyme required to generate 1 μmol of glutathione per minute is defined as one enzyme activity unit (1U).

[0057] Specific enzyme activity refers to the enzyme activity U per mg of protein.

[0058] Table 2. Specific enzyme activities of wild-type and mutant glutathione bifunctional synthetase

[0059]

[0060] Example 6: Determination of thermal stability of glutathione bifunctional synthetase and its mutants

[0061] The pure enzyme solution containing glutathione bifunctional synthetase GshFST, GshFST-S619T, GshFST-A691V, and GshFST-S619T-A691V prepared in Example 4 was used as a catalyst. The pure enzyme solution was incubated in a 35° C. water bath, and samples were taken every 30 min. The enzyme activity was determined using the conditions and methods of Example 5, and the relative enzyme activity was calculated with the enzyme activity at 0 min as 100%.

[0062] The results are as follows Figure 5As shown, the results show that the residual enzyme activity of the single point mutant GshFST-S619T is always higher than that of the wild-type initial enzyme GshFST, and the combined mutant GshFST-S619T-A691V still retains 30.42% of the enzyme activity after incubation for 180 minutes, which is higher than that of the wild enzyme. Combined with Example 5, GshFST-S619T-A691V was selected as the optimal mutant.

[0063] Example 7: Reaction process of glutathione synthesis catalyzed by glutathione bifunctional synthetase and its mutants

[0064] The crude enzyme solution containing glutathione bifunctional synthetase GshFST (WT) and GshFST-S619T-A691V prepared in Example 4 was used as a catalyst, and the amount was calculated based on the weight of wet cells before crushing. The preparation method of the crude enzyme solution of polyphosphate kinase was the same as that in Example 1, and the amount was calculated based on the weight of wet cells before crushing. The construction method of the corresponding engineered bacteria was the same as that in Example 1. The amino acid sequence of polyphosphate kinase is shown in SEQ ID NO.5; the nucleotide sequence is shown in SEQ ID NO.6.

[0065] The 40mL reaction system consists of: 20mL Tris-HCl buffer (100mM, pH 7.5), 25g / L catalyst, 5g / L polyphosphate kinase crude enzyme solution, and the substrates are: 70mM L-glutamate, 70mM L-cysteine, 70mM glycine, 60mM MgSO4, 2mM ATP, and 35mM sodium hexametaphosphate.

[0066] In a nitrogen atmosphere, the reaction was carried out at 25°C and 500 rpm for 8 h. 500 μL of the reaction solution was taken at 0.5 h, 1 h, 2 h, 4 h, and 8 h, and 100 μL of 2M HCl was added to terminate the reaction. The reaction was centrifuged at 12000 rpm for 2 min. The supernatant was collected and diluted 10 times. The reaction progress was detected by HPLC. The results are shown in Figure 6 As shown, the concentration of product GSH of mutant GshFST-S619T-A691V reached 17.65 g / L after 8 hours of reaction, and the product yield was increased from 74.27% to 82.03% compared with wild-type GshFST.

[0067] Example 8: Reaction process of glutathione synthesis catalyzed by glutathione bifunctional synthetase mutants at different substrate concentrations

[0068] The crude enzyme solution containing the glutathione bifunctional synthetase mutant GshFST-S619T-A691V prepared in Example 4 was used as a catalyst, and the amount used was calculated based on the weight of the wet cells before crushing.

[0069] The 40mL reaction system consists of: 20mL Tris-HCl buffer (100mM, pH 7.5), 25g / L catalyst, 5g / L polyphosphate kinase crude enzyme solution (same as Example 7), substrates are: 20-120mM (20, 45, 70, 95, 120mM) L-glutamate, L-cysteine, glycine, the added concentration of L-cysteine ​​and glycine is 1:1 with the concentration of L-glutamate, 60mM MgSO4, 2mM ATP, 35mM sodium hexametaphosphate. In a nitrogen environment, react at 25°C and 500rpm for 8h. Take 500μL of the reaction solution at 0.5h, 1h, 2h, 4h, and 8h, respectively, and add 100μL 2M HCl to terminate the reaction, and centrifuge at 12000rpm for 2min. Collect the supernatant and dilute it 10 times. Use HPLC to detect the reaction progress. The results are shown in Figure 7 As shown, the results showed that the product yield was the highest at 70 mM, with a yield of 82.03%.

[0070] Example 9: Effect of different ATP concentrations on the activity of glutathione bifunctional synthetase mutants

[0071] The pure enzyme solution containing the glutathione bifunctional synthetase mutant GshFST-S619T-A691V prepared in Example 4 was used as a catalyst, and the amount used was calculated based on the protein content.

[0072] 1mL reaction system composition: 500μL Tris-HCl buffer (100mM, pH 8.0), 20mM L-glutamic acid, 20mM L-cysteine, 20mM glycine, 20mM MgSO4, 10mM ATP, 500μL catalyst (added to a final concentration of 0.3g / L based on protein content), 35℃, 500rpm for 5min. Add 100μL 2M HCl to terminate the reaction, centrifuge at 12000rpm for 2min, collect the supernatant and perform HPLC analysis. Change the ATP concentration to 5, 15, 20, 25mM, and the enzyme activity results are as follows Figure 8 As shown, the results showed that the best specific enzyme activity reached 3.31U / mg when the added amount was 10mM ATP.

[0073] Example 10: Effect of different temperatures on the activity of glutathione bifunctional synthetase mutants

[0074] The pure enzyme solution containing the glutathione bifunctional synthetase mutant GshFST-S619T-A691V prepared in Example 4 was used as a catalyst, and the amount used was calculated based on the protein content.

[0075] 1ml reaction system composition: 500μL Tris-HCl buffer (100mM, pH 8.0), 20mM L-glutamic acid, 20mM L-cysteine, 20mM glycine, 20mM MgSO4, 10mM ATP, 500μL catalyst (added to a final concentration of 0.3g / L based on protein content), 35℃, 500rpm for 5min. Add 100μL 2M HCl to terminate the reaction, centrifuge at 12000rpm for 2min, collect the supernatant and perform HPLC analysis. Change the reaction temperature to 25℃, 30℃, 35℃, 40℃, 45℃, the results are as follows Fig. 9 As shown, the results showed that the enzyme activity was best at 3.31 U / mg when the reaction temperature was 35°C.

Claims

1. A glutathione bifunctional synthetase mutant, characterized in that: The mutant is obtained by mutating the 619th and 691st amino acids of the amino acid sequence shown in SEQ ID NO.

1.

2. The glutathione bifunctional synthetase mutant according to claim 1, characterized in that The mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.1 is mutated into one of the following: (1) the serine at position 619 is mutated into threonine; (2) the alanine at position 691 is mutated into valine; (3) the serine at position 619 is mutated into threonine and the alanine at position 691 is mutated into valine.

3. A recombinant genetically engineered bacterium containing a gene encoding the glutathione bifunctional synthetase mutant according to claim 1.

4. Use of the glutathione bifunctional synthetase mutant according to claim 1 in synthesizing glutathione.

5. The use according to claim 4, characterized in that The application method comprises the following steps: using wet bacteria obtained by fermentation and culturing an engineered bacterium containing a gene encoding a mutant of a glutathione bifunctional synthetase, resuspending and crushing the wet bacteria in a buffer solution as a catalyst, using L-glutamic acid, L-cysteine ​​and glycine as substrates, adding MgSO4, ATP, polyphosphate kinase and sodium hexametaphosphate, and using a pH 6-8 buffer solution as a reaction medium to form a reaction system, reacting at 20-40° C. and 300-600 rpm to obtain glutathione.

6. The use according to claim 5, characterized in that In the reaction system, the amount of catalyst added is 10-50 g / L based on the weight of wet bacteria before crushing; the final concentration of L-glutamic acid added is 20-120 mM; the final concentration of L-cysteine ​​added is 20-120 mM; the final concentration of glycine added is 20-120 mM; the final concentration of MgSO4 added is 20-80 mM; the final concentration of ATP added is 5-25 mM; the final concentration of polyphosphate kinase added is 1-10 g / L; and the final concentration of sodium hexametaphosphate added is 30-50 mM.

7. The use according to claim 5, characterized in that The catalyst was prepared as follows: the recombinant genetically engineered bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 150 rpm overnight; then inoculated into LB medium containing 50 μg / mL kanamycin at a volume concentration of 2%, and cultured at 37°C and 150 rpm until the bacterial concentration OD 600 =0.6, add IPTG with a final concentration of 0.1mM, induce culture at 28℃ for 12-16h, and finally collect wet cells by centrifugation at 4℃, 8000rpm for 10min; the collected wet cells are resuspended in 100mM, pH 8.0 Tris-HCl buffer, mixed evenly, and repeatedly frozen and thawed at -80℃ for 30min and 37℃ for 30min, then centrifuged and the supernatant is taken.

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