Difunctional glutathione synthetase based on ancestor sequence reconstruction and application thereof

Abstract

The invention discloses difunctional glutathione synthetase based on ancestor sequence reconstruction and application of the difunctional glutathione synthetase, and belongs to the technical field of enzyme engineering and bioengineering. A difunctional glutathione synthetase Anc427 with an amino acid sequence shown as SEQ ID NO: 1 is obtained through an ancestral sequence reconstruction technology, and compared with St-GshF, the obtained difunctional glutathione synthetase Anc427 has the advantages that the melting temperature is increased to 56.2 DEG C from 45.4 DEG C through a stability test and an amplification reaction; at the temperature of 40 DEG C, the half-life period is prolonged from 173.29 min to 3465.74 min; the half-life period at 50 DEG C is 266.6 min, and the thermal stability is remarkably improved. Meanwhile, the activity of the bifunctional glutathione synthetase Anc427 is improved by 17% compared with that of St-GshF, and the conversion rate under the substrate concentration of 100 mM is improved by 13.6%. The industrial application value is extremely high.

Description

TECHNICAL FIELD

[0001] The present application relates to a bifunctional glutathione synthetase based on ancestral sequence reconstruction and its application, belonging to the field of enzyme engineering and biological engineering. BACKGROUND

[0002] Glutathione (GSH) is a thiol compound containing γ-peptide bond widely existing in organisms, which has clear biological activities such as antioxidant, detoxification and immunomodulation, and has realized large-scale application in the fields of medicine, food and cosmetics, etc., and the market demand continues to grow. However, its efficient synthesis is still the core bottleneck of industrial production.

[0003] The traditional production method has obvious limitations: solvent extraction method depends on biological raw materials, has low yield and high cost; chemical synthesis method has harsh reaction conditions and serious environmental pollution; microbial fermentation method is currently the mainstream, but the downstream separation and purification steps are complicated, the yield is insufficient, and there are problems of bacterial contamination and product residue, which is difficult to meet the demand of high-purity application. Enzymatic synthesis has the core advantages of high product specificity, mild reaction conditions and environmental friendliness, and has become an important direction to replace traditional process. The early two-step enzyme method relies on the cascade reaction of γ-glutamyl cysteine synthetase (GSH I) and glutathione synthetase (GSH II), but GSH I is inhibited by the final product GSH, and the production efficiency is limited. The discovery of bifunctional glutathione synthetase (referred to as "GshF") realizes a key breakthrough, which has γ-glutamyl cysteine ligase (γ-ECL) and glutathione synthetase (GS) activity, can catalyze two-step reaction at the same time, complete ATP-dependent synthesis of GSH, completely avoid product inhibition, and the reaction system is simple, easy to couple with ATP regeneration system, significantly reduce production cost, and show significant industrial application potential. However, the natural source of GshF generally has low catalytic activity and poor stability, which seriously restricts its application in industrial reaction system, and it is urgent to optimize it through enzyme molecular modification technology.

[0004] The improvement of thermal stability can not only directly improve the catalytic ability of the enzyme under high temperature conditions, but also reduce the requirement for temperature control during the storage and transportation of the enzyme, thereby reducing the cost of industrial application. The existing technology reports many enzyme molecular modification strategies for improving the thermal stability of bifunctional glutathione synthase GshF. For example, the literature "Enhancing the Thermal Stability of Glutathione Bifunctional Synthase by B-Factor Strategy and Un / Folding Free Energy Calculation" reports that the GshF derived from Streptococcus agalactiae is rationally designed, and the optimal mutant R270S is obtained, which has a half-life of 14.88 minutes at 40℃, which is 2.62 times that of the wild type (WT); the literature "High Production of Glutathione by in Vitro Enzymatic Cascade after Thermostability Enhancement" reports that the mutant has an enzyme activity of 18.16% higher than that of the chimeric parent NSS-LST-SSC, and the half-life at 45℃ is 1732.81 min; the literature "Molecular Modification of Glutathione Bifunctional Synthase and Whole Cell Catalysis" reports that the GshF derived from Streptococcus thermophilus is modified, and the optimal mutant L136K / V498 is obtained, which has a half-life of 134.24 min at 37℃. The bifunctional glutathione synthase reported in the existing technology still cannot meet the industrial demand, and the thermal stability of the bifunctional glutathione synthase needs to be further improved. SUMMARY

[0005] To solve the problems in the prior art, the first object of the present application is to provide an ancestral enzyme with higher activity and better stability than existing enzymes, which is obtained based on an ancestral sequence reconstruction method. The bifunctional glutathione synthase Anc427 provided by the present application comprises 750 amino acids, and the amino acid sequence is shown as SEQ ID NO: 1.

[0006] The second object of the present application is to provide a gene encoding the bifunctional glutathione synthase Anc427.

[0007] Further, the gene sequence encoding the bifunctional glutathione synthase Anc427 is shown as SEQ ID NO: 2.

[0008] The third technical solution provided by the present application is a recombinant plasmid carrying the gene of the second technical solution.

[0009] In some embodiments, the vector for the recombinant plasmid is the pET-28a(+) plasmid.

[0010] The fourth technical solution provided by the present invention is a microbial cell that expresses the bifunctional glutathione synthase described in the first technical solution, or carries the gene described in the second technical solution, or is transformed with the recombinant plasmid described in the third technical solution.

[0011] In some embodiments, the host cell in the microbial cell is Escherichia coli.

[0012] The fifth technical solution provided by the present invention is a method for preparing the above-mentioned bifunctional glutathione synthase Anc427. The method involves inoculating host cells expressing the mutant described in the first technical solution into a fermentation medium for fermentation to obtain a fermentation broth; centrifuging the fermentation broth to collect the cells; rupturing the cells and centrifuging them to obtain the supernatant containing the bifunctional glutathione synthase Anc427, which is a crude enzyme solution. The target protein is then purified using nickel column affinity chromatography in an AKTA system.

[0013] In some implementations, the target protein is eluted at an imidazole concentration of 300-500 mM.

[0014] The sixth technical solution provided by this invention is the application of bifunctional glutathione synthase Anc427 in the catalytic synthesis of glutathione. Compared with ST-GshF, bifunctional glutathione synthase Anc427 has better thermal stability at higher temperatures, making it more suitable for industrial applications.

[0015] The sixth technical solution provided by the present invention is a method for efficient synthesis of glutathione, wherein the method involves adding the bifunctional glutathione synthase Anc427 described in the first technical solution or adding the lysate of the recombinant Escherichia coli to a reaction system containing L-cysteine, glycine, L-glutamic acid, ATP and MgCl2 for reaction.

[0016] In some embodiments, L-cysteine, glycine, L-glutamic acid, ATP, and MgCl2 are added in equal amounts in the reaction system; the molar ratio of L-cysteine ​​to the bifunctional glutathione synthase Anc427 is 20-24.

[0017] In some embodiments, the concentration of all substrates in the reaction system is 95-105 mmol / L.

[0018] In some embodiments, the amount of the bifunctional glutathione synthase Anc427 added to the reaction system is 4.5~5 mmol / L.

[0019] In some embodiments, the reaction temperature is 20~50°C.

[0020] The present invention also provides the use of the bifunctional glutathione synthase Anc427 or the recombinant Escherichia coli in the preparation of glutathione or glutathione-containing products.

[0021] The beneficial effects of this invention are as follows: This invention utilizes ancestral sequence reconstruction technology to screen and obtain the bifunctional glutathione synthase Anc427, which exhibits significantly superior thermostability and enzyme activity compared to St-GshF. Compared to the bifunctional glutathione synthase St-GshF, the bifunctional glutathione synthase Anc427 of this invention shows an increased melting temperature from 45.4℃ to 56.2℃; an increased half-life at 40℃ from 173.29 min to 3465.74 min; and a half-life of 266.6 min at 50℃, while St-GshF exhibits no activity at 50℃, demonstrating a significant improvement in thermostability. Attached Figure Description

[0022] Figure 1 SDS-PAGE electrophoresis analysis results of expression products obtained from shake-flask fermentation of recombinant *E. coli*; a: SDS-PAGE analysis of protein expression at different IPTG concentrations. ①-④ represent final IPTG concentrations as follows (①: 0.05 mM, ②: 0.1 mM, ③: 0.15 mM, ④: 0.2 mM); Lanes: M: molecular weight label; S: protein expression in the lysed supernatant; P: protein expression in the lysed precipitate. b: Protein purification curve of the AKTA system. c: SDS-PAGE gel image corresponding to the AKTA collector.

[0023] Figure 2 Figure showing the results of the depolymerization temperature test.

[0024] Figure 3 Figure 1: Half-life test results; a: Residual activity curves of St-GshF and Anc427 at 40℃; b: Residual activity curve of Anc427 at 50℃.

[0025] Figure 4 Chromatogram of glutathione synthesis by bifunctional glutathione synthase; HPLC curve of the enzymatic reaction. Red, orange, and blue curves correspond to GSH standard, St-GshF, and Anc427, respectively.

[0026] Figure 5: Yield graph of glutathione synthesis by bifunctional glutathione synthase; a: Blue (St-GshF) and orange (Anc427) represent the yield of GSH synthesis in a 50 mL reaction system containing 100 mM cysteine ​​and insufficient ATP; Pink (St-GshF) and green (Anc427) represent the yield of GSH synthesis in a 50 mL reaction system containing 100 mM cysteine ​​and sufficient ATP; b: Total yield of GSH synthesis in a 50 mL reaction system containing 100 mM cysteine ​​and sufficient ATP. Detailed Implementation

[0027] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0028] Test method: Fluorescence colorimetric method conditions: This method is based on the reaction of DTNB with thiol groups to generate a yellow product with characteristic absorption at 412 nm, and uses formaldehyde to eliminate interference from substances such as free cysteine. The specific steps are as follows: First, prepare a 0.01 mol / L DTNB standard stock solution with disodium hydrogen phosphate buffer (0.05 mmol / L, pH 7.0), and then mix it with Tris-HCl buffer (0.25 mol / L) at a volume ratio of 1:99 to make a 0.1 mmol / L DTNB analytical solution. Then, add the sample, NaOH (0.15 mol / L), and formaldehyde solution (3%) (volume ratio 1:3:1) to a 96-well plate in sequence. After standing for 2 min, add 5 times the volume of DTNB analytical solution, react at 25℃ for 5 min, and then measure the absorbance at 412 nm.

[0029] HPLC analysis conditions were as follows: Diamonsil C column 18 (250 mm × 4.6 mm, 5 μm), the mobile phase was 0.1 mol / L dipotassium hydrogen phosphate and 0.01 mol / L sodium heptanesulfonate aqueous solution (pH 2.65) and acetonitrile (95:5, v / v), the column temperature was 30 ℃, the detection wavelength was 210 nm, and the flow rate was 0.8 mL / min.

[0030] Materials used in the examples: The Escherichia coli involved in the following examples E. coliBL21(DE3) was purchased from Sangon Biotech (Shanghai) Co., Ltd., and pET-28a(+) plasmid was purchased from Sangon Biotech (Shanghai) Co., Ltd.; GSH used in the following examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and isopropyl-β-D-thiogalactoside and kanamycin were purchased from Shanghai Jierui Biotechnology Co., Ltd. Other chemical reagents were purchased from Sinopharm Industrial Co., Ltd.

[0031] LB liquid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, kanamycin 0.2 mmol / L.

[0032] LB solid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 20 g / L, kanamycin 0.2 mmol / L.

[0033] TB liquid medium: yeast extract 24 g / L, tryptone 12 g / L, 0.017 M KH2PO4 and 0.072 M K2HPO4·3H2O, 0.4% (v / v) glycerol, kanamycin 0.2 mmol / L.

[0034] Example 1: Preparation, expression and purification of bifunctional glutathione synthase Anc427 1. Obtaining the ancestral enzyme Anc427 S1. Using GshF (St-GshF) from Streptococcus thermophilus, which is reported in the literature to have high stability and activity, as a probe, the probe sequence (its amino acid sequence is shown in SEQ ID NO:3) was submitted to the UniProt database, and UniRef90 was used to search for homologous sequences with sequence identity between 40% and 90%.

[0035] S2. The obtained sequences are deredundant using MMseqs2, and the sequence similarity threshold is set to 0.8. Representative sequences are obtained after two rounds of screening.

[0036] S3. Multiple sequence alignment was performed using Clustal Omega and the ClustalW algorithm. Based on the alignment results, a phylogenetic tree was constructed using the IQ-TREE maximum likelihood method, and the reliability of the topology was evaluated by performing 1000 bootstrapping tests.

[0037] S4. Import the phylogenetic tree and alignment results into PAMLX to reconstruct and obtain the ancestral protein sequence.

[0038] S5. To ensure the potential functional activity of the reconstructed protein, a threshold of at least 50% identity between the ancestral sequence and the probe enzyme sequence was set during the screening process. Based on this standard and phylogenetic analysis, sequence 427 was found to be the most evolutionarily close to the probe enzyme and was therefore selected as the representative sequence (abbreviated as "Anc427").

[0039] The amino acid sequence of the bifunctional glutathione synthase (ancestral enzyme) Anc427 was obtained through the above screening (as shown in SEQ ID NO:1).

[0040] 2. Expression and purification of the progenitor enzyme Anc427 (1) Construction of plasmid for ancestral enzyme Anc427: The nucleotide sequence encoding the ancestral enzyme Anc427 was chemically synthesized (as shown in SEQ ID NO:2, with 6 nucleotides at each end of the gene serving as restriction sites); the obtained gene was ligated with plasmid pET-28a(+) digested with BamHI and XhoI, and the ligation product was transformed into Escherichia coli. E. coli BL21(DE3) transformation products were plated on Kan-resistant LB solid medium and cultured at 37 ℃ for 10–12 h. Five transformants were picked from the LB solid medium and inoculated into LB liquid medium. After culturing at 37 ℃ for 12 h, plasmids were extracted. The extracted plasmids were verified by restriction enzyme digestion and sequencing. If the verification was correct, the recombinant plasmid pET28a- containing the gene encoding the ancestral enzyme Anc427 was obtained. EcAnc427 And recombinant bacteria containing the gene encoding the ancestral enzyme Anc427 E. coli BL21 / pET28a- E c Anc427 500 μL of the recombinant bacterial culture was added to 500 μL of 30% glycerol and then preserved at -80 °C.

[0041] (2) Protein expression To achieve better soluble expression of Anc427, 20 μL of the glycerol bacteria preserved in step (1) was inoculated into a 5 mL shaker tube and cultured at 37 ℃ and 220 rpm for 10-12 h to obtain a seed culture. The seed culture was then inoculated into a 50 mL shaker flask at an inoculation rate of 1% (v / v) and cultured at 37 ℃ and 220 rpm until the OD600 value reached 0.8. At this point, different final concentrations of IPTG (such as...) were added. Figure 1As shown in Figure a, ①: 0.05mM, ②: 0.1mM, ③: 0.15mM, ④: 0.2mM) were induced in a shaker at 25℃ and 220 rpm for 12 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 5 min at 4℃, the supernatant was discarded, and 7 mL of pH 7.5 PBS buffer was added for 10 min of sonication (350 W, on for 2 s, off for 3 s) to obtain the cell lysate. After centrifugation at 8000 rpm for 30 min, the supernatant and precipitate were collected and their soluble expression was verified using SDS-PAGE. The results are shown in the figure. Figure 1 a.

[0042] Depend on Figure 1 As can be seen from the presence of a band at 85 kDa in the cell lysis supernatant, which corresponds to the theoretical molecular weight, the ancestral enzyme Anc427 was successfully expressed, and the expression level was optimal at a final IPTG concentration of 0.2 mM.

[0043] (3) Protein purification Take 20 μL of the glycerol bacteria preserved in step (1) and inoculate it into a 5 mL shaking tube. Incubate at 37 ℃ and 220 rpm for 10-12 h to obtain seed culture. Inoculate the seed culture into a 700 mL shaking flask at an inoculation rate of 1% (v / v) and incubate at 37 ℃ and 220 rpm until the OD600 value reaches 0.8. Add IPTG to a final concentration of 0.2 mM and induce fermentation at 25 ℃ and 220 rpm for 12 h to obtain fermentation broth. Centrifuge the fermentation broth at 4 ℃ and 8000 rpm for 10 min, discard the supernatant, and resuspend the bacterial cells in Tris-HCl buffer (pH 7.4) containing 20 mM imidazole to 0.1 g / mL. Cyclate the cells three times using a high-pressure homogenizer at 4 ℃. After lysis, centrifuge the bacterial broth at 8000 × g for 30 min, collect the supernatant, and filter it through a 0.22 μm filter membrane to obtain crude enzyme solution. Purification was performed using the ÄKTA pure system. The mobile phase consisted of three solutions: Solution A (20 mM imidazole in Tris-HCl, pH 7.4), Solution B (500 mM imidazole in Tris-HCl, pH 7.4), and Solution C (100 mM Tris-HCl, pH 7.4). The nickel column was equilibrated sequentially with ultrapure water and Solution A. After loading the crude enzyme solution, two column volumes were washed with Solution A, followed by linear gradient elution with Solution B. The eluted fractions were automatically collected with a collection threshold of 50 mAU. After SDS-PAGE analysis, fractions with a single target band were combined and concentrated to approximately 2 mL using a 30 kDa ultrafiltration tube. The solution was then passed through a pre-equilibrated desalting column to remove imidazole. The final protein solution was concentrated to 2 mg / mL, aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C. Protein concentration was determined using a NanoDrop 2000.

[0044] The collected solutions obtained during the purification process were analyzed by SDS-PAGE. The results are shown in the figure below. Figure 1 b, Figure 1 c.

[0045] Depend on Figure 1 b and Figure 1 c indicates that the enzyme solution has a band at 85 kDa that matches the theoretical molecular weight, indicating that the pure enzyme solution of the ancestral enzyme Anc427 shows a single band at 85 kDa and has few impurities, indicating that the AKTA purification effect is good.

[0046] Following the same method described above, a recombinant plasmid pET28a-St-GshF containing the gene encoding the ancestral enzyme St-GshF (nucleotide sequence as shown in SEQ ID NO:4, with BamHI and XhoI restriction sites identical to those in SEQ ID NO:2 added to both ends of the gene) and a recombinant bacterium containing the gene encoding the ancestral enzyme St-GshF were constructed. E. coli BL21 / pET28a-St-GshF was used to culture recombinant bacteria and express and purify the protein. The crude enzyme activity, purified enzyme activity, thermostability, and catalytic activity of the bifunctional glutathione synthase St-GshF and the bifunctional glutathione synthase Anc427 were analyzed and compared.

[0047] Example 2: Stability determination of bifunctional glutathione synthase Anc427 (1) Determination of melting temperature Differential scanning calorimetry (DSC) was used for determination. The enzyme solution purified in step (3) of Example 1 was diluted to 3 mg / mL (using Tris-HCl buffer at pH 8.0). A Nano-DSC instrument was used to scan from 20°C to 80°C at a heating rate of 1°C / min, and the heat flow changes were recorded in real time. The thermal denaturation curve was fitted using a Two-State Scaled model with Nano Analyze™ software, and the temperature corresponding to the peak value was taken as the thermal denaturation temperature (Tm). The detection results are shown in […]. Figure 2 .

[0048] Depend on Figure 2 It can be seen that the melting temperature of the ancestral enzyme Anc427 is 56.21℃, which is 10.83℃ higher than that of St-GshF (45.38℃).

[0049] (2) Half-life determination The purified enzyme solution obtained in step (3) of Example 1 was diluted to 1 mg / mL and incubated in water baths at 40℃ and 50℃ respectively. Samples were taken at different time points and rapidly cooled in an ice bath. The treated enzyme solution was added to a standard reaction system and reacted at 37℃ for 10 minutes. The residual enzyme activity was determined using the DTNB method in the detection method. The enzyme activity detection reaction was carried out in a 100 μL standard reaction system containing 100 mM Tris-HCl (pH 7.0), 20 mM magnesium chloride, 20 mM L-glycine, 20 mM L-glutamate, 20 mM L-cysteine, and 20 mM ATP. The enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the generation of 1 micromolar (μmol) GSH per minute under the above reaction conditions.

[0050] Enzyme activity (U) = × V ; In the formula: A GSH V represents the molar concentration of GSH in the sample after the reaction is terminated; V represents the volume of the sample after the reaction is terminated; and t represents the reaction time.

[0051] Using the enzyme activity of the unincubated sample as 100%, calculate the percentage of residual enzyme activity at each time point. The thermal half-life (t1 / 2) is defined as the time required for the enzyme activity to decrease to 50%. The enzyme half-life is calculated using a first-order kinetic equation, as follows: t1 / 2= ;ln A = k d ×t; In the formula, k d t is the inactivation rate constant, A is the residual activity, t is the incubation time, and t1 / 2 is the half-life.

[0052] The detection system is shown in Table 1: Table 1 DTNB Method Detection System

[0053] Test results as follows Figure 3 As shown in the analysis, the ancestral enzyme Anc427 has a half-life of 3465.74 min at 40℃, which is 20 times that of St-GshF; at 50℃, its half-life is 266.6 min, while St-GshF is inactive at 50℃, indicating that the ancestral enzyme Anc427 has significantly improved thermostability. Therefore, the bifunctional glutathione synthase Anc427 can be applied more efficiently in industrial production, avoiding enzyme inactivation due to high temperatures; it can also increase the number of enzyme batches under immobilized enzyme conditions, saving production costs.

[0054] Table 2. Stability parameters of St-GshF and ancestral enzyme Anc427

[0055] Example 3: Preparation of glutathione catalyzed by bifunctional glutathione synthase Anc427 To evaluate the scale-up effect and catalytic performance of the reaction system, the reaction volume was increased to 50 mL, and the substrate concentration was increased to 100 mM. Since this enzyme is ATP-dependent, according to the reaction mechanism, the synthesis of one molecule of ATP requires the consumption of two molecules of ATP. To better evaluate the application value of the bifunctional glutathione synthase Anc427, two experimental groups were set up: Group a: 100 mM L-cysteine, 100 mM L-glycine, 100 mM L-glutamate, 100 mM ATP, and 100 mM magnesium chloride; Group b: 100 mM L-cysteine, 100 mM L-glycine, 100 mM L-glutamate, 200 mM ATP, and 100 mM magnesium chloride. Given that ATP hydrolysis during the reaction is accompanied by the release of phosphate groups (leading to a decrease in system pH), real-time acid-base titration with NaOH solution was used to maintain the system pH stable at 7.0–7.2; a constant temperature incubator was used to control the reaction temperature at 37℃.

[0056] 1. Determination of catalytic enzyme activity To compare enzyme activity under different conditions, a sample from group a with a reaction time of 10 minutes was taken, and an equal volume of 25% trichloroacetic acid was immediately added to terminate the reaction. The mixture was then centrifuged at 12000 r / min for 10 min. One mL of the supernatant was placed in a 50 mL volumetric flask and diluted to the mark with pure water to obtain a 50-fold diluted test solution. This solution was filtered through a 0.22 μm aqueous filter and analyzed by HPLC. The concentration of glutathione (GSH) generated was calculated based on the chromatographic results, and the corresponding enzyme activity was calculated accordingly. In industrial applications, crude enzyme solutions are typically used for rapid evaluation. To verify its application potential in an industrial environment, this experiment further determined the crude enzyme activities of bifunctional glutathione synthase St-GshF and bifunctional glutathione synthase Anc427 in the cell lysate prepared in Example 1 under the same conditions. The relevant results are summarized in Table 3.

[0057] Enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 micromolar (μmol) of GSH per minute under the above reaction conditions.

[0058] Enzyme activity (U) = × V ; In the formula: A GSHV represents the molar concentration of GSH in the sample after the reaction is terminated; V represents the volume of the sample after the reaction is terminated; and t represents the reaction time.

[0059] Enzyme specific activity is defined as the number of enzyme activity units per milligram of protein.

[0060] Crude enzyme activity is defined as the number of enzyme activity units per milliliter of protein.

[0061] enzyme activity (U·mg) -1 ) = ; Crude enzyme activity (U·mL) -1 ) = .

[0062] Table 3. Enzyme activity and conversion rate of St-GshF and ancestral enzyme Anc427.

[0063] The specific activity of Anc427 was (3.3 ± 0.02) U / mg, higher than that of the control enzyme St-GshF (2.81 ± 0.03) U / mg; its crude enzyme activity was (19.1 ± 0.03) U / mL, higher than that of the control enzyme St-GshF (17.2 ± 0.05) U / mL. This significant increase in activity at an industrial scale not only confirms the high efficiency of Anc427 but also indicates its great potential in large-scale biocatalytic applications. The high activity maintained in a scale-up system highlights the robustness and practical value of the enzyme engineering strategy employed in this study.

[0064] 2. Determination of reaction yield During the reaction, 1 mL of sample was taken every hour, and an equal volume of 25% trichloroacetic acid was immediately added to terminate the reaction. The mixture was then centrifuged at 12000 r / min for 10 min. 1 mL of the supernatant was transferred to a 50 mL volumetric flask and diluted to the mark with pure water to obtain a 50-fold diluted test solution. This solution was filtered through a 0.22 μm aqueous filter membrane and analyzed by HPLC. The concentration of glutathione (GSH) and the reaction yield were calculated based on the chromatographic results. A comparison of the yields of groups a and b is provided. Figure 5 As shown. Using GSH standard as a reference, the peak time in the chromatogram was determined. Figure 4 ).

[0065] The formula for calculating yield is: Yield / % = ×100%; In the formula: Mp represents the molar concentration of GSH generated (mmol / L); Ms represents the molar concentration of the substrate L-cysteine ​​(mmol / L).

[0066] Formula for calculating total yield: Total yield / % = ×100%; In the formula: M p1 Indicates the molar concentration (mmol / L) of GSH generated. M p2 The molar concentration (mmol / L) of GSSH generated is indicated. M This indicates the molar concentration (mmol / L) of the substrate L-cysteine.

[0067] Analysis of the conversion reaction over time revealed the superior performance of the ancestral enzyme. Under ATP deficiency, the reaction catalyzed by Anc427 achieved a 54.4% yield within 6 hours, with a final product concentration of 16.7 g / L. In contrast, when St-GshF was used, only a maximum yield of 53.1% was achieved after 5 hours. More importantly, the ancestral enzyme Anc427 continued to catalyze product formation after 5 hours, while the activity of St-GshF essentially ceased. Similarly, with sufficient ATP, the catalytic reaction involving Anc427 achieved a yield of 79.54% within 3 hours. In contrast, when St-GshF was used for catalysis, the highest yield was only 71.45% after 2 hours, and product formation ceased. Since GSH is easily oxidized (two molecules of GSH are oxidized to one molecule of GSSH), antioxidants such as ascorbic acid are typically added industrially to prevent oxidation. Therefore, to more accurately compare their performance, this study statistically analyzed the amount of oxidized product and the total product yield when ATP was sufficient. Figure 5 As shown in b, the comparison revealed that when using Anc427 as a catalyst, the total yield after 3 hours was 81.19%, significantly higher than the 71.49% total yield after 2 hours using St-GshF as a catalyst, representing an improvement of approximately 10%. With a reaction time of 3 hours, the total yield of the Anc427-catalyzed reaction was 81.19%, while the total yield of the St-GshF-catalyzed reaction remained around 71%, showing no significant improvement, similar to the 2-hour reaction.

[0068] In summary, the newly discovered bifunctional glutathione synthase Anc427 exhibits significantly improved thermostability, with a 20-fold increase in half-life at 40°C and an approximately 13.6% increase in overall yield at high substrate concentrations. This sustained activity confirms the superior operational stability of the ancestral enzyme under scale-up reaction conditions. The combination of higher product yield and strong stability makes Anc427 an ideal candidate enzyme for industrial biocatalytic processes.

[0069] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A bifunctional glutathione synthase Anc427, characterized in that, The amino acid sequence of the bifunctional glutathione synthase Anc427 is shown in SEQ ID NO:

1.

2. The gene encoding the bifunctional glutathione synthase Anc427 of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. Recombinant microbial cells containing the recombinant vector of claim 3 or expressing the bifunctional glutathione synthase Anc427 of claim 1.

5. A recombinant Escherichia coli, characterized in that, Using pET-28a(+) plasmid as a vector, the bifunctional glutathione synthase Anc427 described in claim 1 was expressed.

6. A method for catalytic synthesis of glutathione, characterized in that, The bifunctional glutathione synthase Anc427 of claim 1 or the lysate of the recombinant Escherichia coli of claim 5 is added to a reaction system containing L-cysteine, glycine, L-glutamate, ATP and MgCl2 for reaction.

7. The method as described in claim 6, characterized in that, The reaction temperature is 20~50℃.

8. The method as described in claim 7, characterized in that, In the reaction system, L-cysteine, glycine, L-glutamic acid, ATP, and MgCl2 are added in equal amounts; the molar ratio of L-cysteine ​​to bifunctional glutathione synthase Anc427 is 20-24.

9. The method as described in claim 7, characterized in that, The reaction system contains 95-105 mM L-cysteine, 95-105 mM L-glycine, 95-105 mM L-glutamate, 95-105 mM ATP, 95-105 mM magnesium chloride, and 4.5-5 mM glutathione synthase.

10. The use of the bifunctional glutathione synthase Anc427 of claim 1 or the recombinant Escherichia coli of claim 5 in the preparation of glutathione or glutathione-containing products.

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