Glutathione bifunctional synthetase mutants and their applications in catalytic glutathione synthesis

Abstract

The present invention discloses a glutathione bifunctional synthetase mutant and its use in catalyzing glutathione synthesis. Through site-directed mutagenesis, the present invention improves the thermostability of the glutathione bifunctional synthetase and its activity in catalyzing the synthesis of glutathione from L-glutamate, L-cysteine, and glycine. The mutant GshFST-S619T-A691V achieved a relative enzyme activity of 279.84% of its premutation level, retained 30.42% of its activity after incubation at 35°C for 180 minutes, and achieved a pure enzyme activity of 3.31 U / mg. After an 8-hour reaction, the yield of the product glutathione reached 82.03%.

Description

(1) Technical field

[0001] The invention belongs to the technical field of biochemical engineering, and particularly relates to a glutathione bifunctional synthetase mutant derived from Streptococcus thermophilus and application thereof in catalyzing the synthesis of glutathione. (2) Background technology

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

[0003] GSH was initially synthesized chemically, but this method has been gradually replaced by biological methods due to drawbacks such as low product purity and cumbersome procedures. In eukaryotic cells and most Gram-negative bacteria, GSH biosynthesis occurs through a two-step, ATP-dependent, enzyme-catalyzed reaction. γ-Glutamyl-L-cysteine ​​synthetase (γ-GCS) catalyzes the synthesis of the dipeptide intermediate γ-glutamate-cysteine ​​from L-glutamate and L-cysteine. Glutathione synthetase (GS) then catalyzes the reaction of the dipeptide intermediate with glycine to form the tripeptide product GSH. Studies have shown that γ-GCS is subject to feedback inhibition by the product GSH, which affects its activity. Furthermore, the accumulation of high concentrations of the dipeptide intermediate during the reaction results in low GSH synthesis yields and increases the cost of subsequent separation and purification.

[0004] Studies have found that some Gram-positive bacteria have a glutathione bifunctional synthetase GshF that has both γ-GCS and GS activities and can catalyze the synthesis of GSH from L-glutamate, L-cysteine, and glycine. Figure 1 ), and most GshFs lack product feedback inhibition. However, currently reported natural GshFs still lack optimal catalytic properties, such as enzyme activity, and thus fail to meet the requirements for industrial application. Therefore, the present invention utilizes protein engineering to modify the bifunctional glutathione synthetase to enhance its catalytic activity and thermal stability, thus increasing its potential for industrial application. (3) Summary of the invention

[0005] The present invention aims to provide a glutathione bifunctional synthetase mutant and its use in catalytic synthesis of glutathione, which effectively improves the catalytic activity and thermal stability of the wild-type glutathione bifunctional synthetase, thereby increasing 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 in the present invention is:

[0007] The present invention provides a glutathione bifunctional synthetase mutant, which 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) serine at position 619 is mutated into threonine; (2) alanine at position 691 is mutated into valine; (3) serine at position 619 is mutated into threonine and 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 gene encoding the glutathione bifunctional synthetase mutant, a recombinant expression vector containing the gene, and a recombinant genetically engineered bacterium constructed using the recombinant expression vector. The recombinant expression vector uses the plasmid pET28a as a base vector, and the host cell of the recombinant genetically engineered bacterium can be any conventional host vector in the art, with Escherichia coli BL21 being a preferred host cell.

[0010] The present invention also provides an application of the glutathione bifunctional synthetase mutant in glutathione synthesis. The application method comprises the following steps: using a supernatant obtained by fermenting and culturing an engineered bacterium containing a gene encoding the glutathione bifunctional synthetase mutant and resuspending and crushing wet cells in a buffer solution as a catalyst; using L-glutamate, L-cysteine, and glycine as substrates; adding MgSO4, ATP, and polyphosphate kinase and sodium hexametaphosphate for ATP circulation; and using a pH 6-8 buffer solution as a reaction medium to form a reaction system. 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 the wet cells 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 a mutant glutathione synthetase is as follows: A pure enzyme solution extracted from the supernatant of wet cells obtained by fermentation of an engineered bacterium containing the gene encoding the mutant glutathione synthetase is used as a catalyst. L-glutamate, L-cysteine, and glycine are used as substrates. MgSO4 and ATP are added to a pH 8 buffer solution to form a reaction system. The reaction is carried out at 35°C and 500 rpm for 5 minutes. One unit (1U) of enzyme activity is defined as the amount of enzyme required to produce 1 μmol of glutathione per minute at 35°C and pH 8.0.

[0013] Furthermore, 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 the recombinant genetically engineered bacteria were inoculated into LB liquid 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 to a final concentration, induce culture at 28°C for 12-16 h, and finally collect wet cells by centrifugation at 8000 rpm for 10 min at 4°C;

[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). After four cycles, the cells were centrifuged at 4000 rpm for 10 min and the supernatant was obtained as the crude enzyme solution.

[0015] The crude enzyme solution was filtered through a 0.22 μm microporous filter membrane, and then bound to the enzyme protein using a Ni-NTA column. The enzyme was first eluted with 20 mM PBS buffer (pH 8.0) containing 50 mM imidazole and 500 mM NaCl to remove impurities, with an elution rate of 1-3 mL / min (preferably 2 mL / min). The target protein was then collected using 20 mM PBS buffer (pH 8.0) containing 500 mM imidazole and 500 mM NaCl, with an elution rate of 1-3 mL / min (preferably 1 mL / min). The target protein and its purity were verified using SDS-PAGE, and finally, dialyzed using Tris-HCl buffer (100 mM, pH 8.0) as the dialysate in a 20 kD dialysis bag, and the permeate was collected to obtain a pure enzyme solution.

[0016] The glutathione bifunctional synthetase mutants of the present invention can be used as catalysts in a variety of feasible forms, including whole-cell form, unpurified crude enzyme form, and partially or completely purified enzyme form. Furthermore, the glutathione bifunctional synthetase mutants of the present invention can be prepared into biocatalysts in the form of immobilized enzymes or immobilized cells using known enzyme immobilization techniques, thereby further improving the enzyme's stability and reusability, facilitating its application in industrial production.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention uses site-directed mutagenesis to improve the thermostability of glutathione bifunctional synthase and its activity in catalyzing the synthesis of glutathione from L-glutamate, L-cysteine, and glycine. The mutant, GshFST-S619T-A691V, achieved a relative enzyme activity of 279.84% of its pre-mutation level. After incubation at 35°C for 180 minutes, it retained 30.42% of its activity. The pure enzyme activity reached 3.31 U / mg, and after an 8-hour reaction, the yield of the product glutathione reached 82.03%. (IV) Description of the accompanying 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 , 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 the 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] Figure 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 with reference to 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 is 7.0.

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

[0031] 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 heptanesulfonate and 50 mM potassium dihydrogen phosphate: methanol = 95:5, v:v, the flow rate was 1 mL / min, the injection volume was 10 μL, the detection wavelength was 210 nm, and the column temperature was 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 shown in SEQ ID NO. 1, nucleotide sequence shown in SEQ ID NO. 3) was ligated to the NcoI and XhoI restriction sites of the plasmid pET-28a and transformed into E. coli DH5 competent cells. The cells were plated onto LB plates containing a final concentration of 50 μg / mL kanamycin and cultured overnight at 37°C. Positive transformants were selected for sequencing verification. Positive clones were inoculated into 5 mL of LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured overnight at 37°C at 150 rpm. The plasmids were then extracted and verified. After verification, the recombinant expression vector was transformed into E. coli BL21(DE3) to obtain recombinant E. coli BL21(DE3) / pET28b-GshFST (denoted as WT). The recombinant strain was then inoculated onto LB plates containing kanamycin at a final concentration of 50 μg / mL and cultured overnight at 37°C. A single colony was selected and inoculated into 5 mL of LB liquid medium containing kanamycin at a final concentration of 50 μg / mL. After incubation at 150 rpm at 37°C overnight, the bacterial suspension was mixed with 30% glycerol (v / v = 1:1) in a glycerol tube and stored in a -80°C freezer.

[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 5). Figure 2 ), with the enzyme activity of WT crude enzyme solution as 100%, and finally the dominant mutants S619T and A691V were obtained through experimental verification and screening.

[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 by overlap extension PCR, and site-directed mutations were performed on serine at position 619 and alanine at position 691 in the parent amino acid sequence, respectively.

[0039] Table 1. Primer sequences

[0040]

[0041] Note: Lowercase letters indicate mutated bases

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

[0043] 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, and extension at 72°C for 4 min 30 s, for a total of 30 cycles of step (2); (3) re-extension at 72°C for 10 min. Storage at 16°C.

[0044] If the PCR product is positive by 0.9% agarose gel electrophoresis, 20 μL of the PCR reaction mixture is digested with 1 μL of the endonuclease Dpn I at 37°C for 3 hours to remove the template plasmid DNA, followed by inactivation at 65°C for 10 minutes. Heat shock transformation is then performed into E. coli BL21(DE3) competent cells. After recovery, the cells are plated on LB plates containing 50 μg / mL kanamycin and cultured overnight. Approximately 300 colonies are obtained per plate.

[0045] Then, 4-5 clones were picked and transferred to LB medium. After culturing at 37°C for 8 hours, 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, respectively.

[0046] Example 3: Construction of the 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 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, a 2% (v / v) inoculum was inoculated into LB medium containing 50 μg / mL kanamycin 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 four cycles, the cells were centrifuged at 4000 rpm for 10 min and the supernatant was obtained as the crude enzyme solution of glutathione bifunctional synthetase and mutants.

[0052] The crude enzyme solution was filtered through a 0.22 μm microporous filter membrane and then bound to the enzyme protein using a Ni-NTA column. Impurities were first eluted with 20 mM PBS buffer containing 50 mM imidazole + 500 mM NaCl, pH 8.0, for 3 column volumes at a rate of 2 mL / min. The target protein was then collected using 20 mM PBS buffer containing 500 mM imidazole + 500 mM NaCl, pH 8.0, for 3 column volumes at a rate of 1 mL / min. The target protein and its purity were analyzed and verified using SDS-PAGE. Finally, the protein was dialyzed in a 20 kD dialysis bag using Tris-HCl buffer (100 mM, pH 8.0) as the dialysate, and the permeate was collected to obtain the 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 catalytic reaction was carried out at 35° C. and 500 rpm using the pure enzyme solution containing glutathione bifunctional synthetase GshFST, GshFST-S619T, and GshFST-S619T-A691V prepared in Example 4 as catalyst.

[0055] The 1mL reaction system consists of: 500μL Tris-HCl buffer (100mM, pH 8.0), 20mM L-glutamic acid, 20mML-cysteine, 20mM glycine, 20mM MgSO4, 10mM ATP, 500μL (added to a final concentration of 0.3g / L based on protein content) pure enzyme solution, 35°C, 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 to 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: One enzyme activity unit (1U) is the amount of enzyme required to generate 1 μmol of glutathione per minute at 35°C and pH 8.0.

[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. Samples were taken every 30 minutes and the enzyme activity was determined using the conditions and method of Example 5. The relative enzyme activity was calculated with the enzyme activity at 0 minutes as 100%.

[0062] The results are as follows Figure 5As shown, the assay results showed that the residual enzyme activity of the single-point mutant GshFST-S619T was consistently higher than that of the wild-type initial enzyme GshFST. The combined mutant GshFST-S619T-A691V retained 30.42% of its activity after 180 minutes of incubation, exceeding that of the wild-type 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 used was based on the weight of the wet cells before disruption. The crude polyphosphate kinase enzyme solution was prepared as in Example 1, and the amount used was based on the weight of the wet cells before disruption. The corresponding engineered bacteria were constructed as 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 40 mL reaction system consists of: 20 mL Tris-HCl buffer (100 mM, pH 7.5), 25 g / L catalyst, 5 g / L crude polyphosphate kinase enzyme solution, and the substrates are: 70 mM L-glutamate, 70 mM L-cysteine, 70 mM glycine, 60 mM MgSO4, 2 mM ATP, and 35 mM sodium hexametaphosphate.

[0066] The reaction was carried out at 25°C and 500 rpm for 8 h under nitrogen atmosphere. 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 the table. Figure 6 As shown, the concentration of product GSH of the mutant GshFST-S619T-A691V reached 17.65 g / L after 8 h of reaction, and the product yield was increased from 74.27% to 82.03% compared with the 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 based on the weight of the wet cells before crushing.

[0069] 40mL reaction system composition: 20mL Tris-HCl buffer (100mM, pH 7.5), catalyst 25g / L, polyphosphate kinase crude enzyme solution (same as Example 7) 5g / L, substrates are: 20-120mM (20, 45, 70, 95, 120mM) L-glutamic acid, L-cysteine, glycine, the added concentration of L-cysteine ​​and glycine is 1:1 with the concentration of L-glutamic acid, 60mM MgSO4, 2mM ATP, 35mM sodium hexametaphosphate. Under nitrogen environment, react at 25 ° C, 500rpm for 8h. Take 500μL 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. After collecting the supernatant, dilute 10 times and detect the reaction progress by HPLC. The results are shown in Figure 3. 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 protein content.

[0072] The reaction system composition of 1mL is as follows: 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), and the reaction is carried out at 35℃ and 500rpm for 5min. The reaction is terminated by adding 100μL 2M HCl and centrifuged at 12000rpm for 2min. The supernatant is collected and analyzed by HPLC. The ATP concentration is changed to 5, 15, 20, and 25mM. The enzyme activity results are shown in the figure. Figure 8 As shown, the results showed that the best specific enzyme activity reached 3.31 U / mg when the added amount was 10 mM ATP.

[0073] Example 10: Effect of different temperatures on the enzymatic 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 protein content.

[0075] The 1ml reaction system consists of: 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), and reacted at 35℃, 500rpm for 5min. Add 100μL 2M HCl to terminate the reaction, and centrifuge at 12000rpm for 2min. Collect the supernatant and perform HPLC analysis. The reaction temperature was changed to 25℃, 30℃, 35℃, 40℃, and 45℃. The results are as follows: Figure 9 As shown, the results showed that the best enzyme activity reached 3.31 U / mg when the reaction temperature was 35°C.

Claims

1. A glutathione bifunctional synthetase mutant, 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) serine at position 619 is mutated into threonine; (2) alanine at position 691 is mutated into valine; (3) serine at position 619 is mutated into threonine and alanine at position 691 is mutated into valine.

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

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

4. The use according to claim 3, characterized in that The application method is as follows: the wet bacteria obtained by fermentation and culture of engineered bacteria containing a glutathione bifunctional synthetase mutant encoding gene are resuspended in a buffer solution and then crushed, and the supernatant is used as a catalyst. L -Glutamate, L -Cysteine ​​and glycine are used as substrates, MgSO4, ATP, polyphosphate kinase and sodium hexametaphosphate are added, a pH 6-8 buffer 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 to obtain glutathione.

5. The use according to claim 4, characterized in that In the reaction system, the amount of catalyst added is 10-50 g / L based on the weight of wet cells before crushing; L -Glutamate is added to a final concentration of 20-120 mM; L -Cysteine ​​is added to a final concentration of 20-120 mM; glycine is added to a final concentration of 20-120 mM; MgSO4 is added to a final concentration of 20-80 mM; ATP is added to a final concentration of 5-25 mM; polyphosphate kinase is added to a final concentration of 1-10 g / L; sodium hexametaphosphate is added to a final concentration of 30-50 mM.

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

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