Oxidized glutathione synthetase, application thereof and preparation method of oxidized glutathione

Abstract

The invention discloses oxidized glutathione synthetase, application of the oxidized glutathione synthetase and a preparation method of oxidized glutathione. Mainly relates to application of oxidized glutathione synthetase in direct catalytic synthesis of GSSG by adopting a whole-enzyme method, wherein the oxidized glutathione synthetase has a wild amino acid sequence as shown in SEQ ID NO.1 and / or at least one mutant amino acid sequence of S534Q, Y185N and S380L in the wild amino acid sequence as shown in SEQ ID NO.1. Cystine, sodium glutamate and glycine are used as substrates for the first time, and the oxidized glutathione is synthesized through enzymatic catalysis in the presence of ATP and Mg < 2 + >. No chemical oxidation reagent is needed, the specificity is good, the process is simple, the reaction condition is mild, and no impurity is generated. Compared with a traditional chemical oxidation synthesis method, the separation problem of the GSH intermediate is effectively avoided, meanwhile, an enzyme method is adopted for coupling ATP regeneration, and the conversion rate is larger than or equal to 90%.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to oxidized glutathione synthetase and its application, and a preparation method of oxidized glutathione. Background Art

[0002] Oxidized glutathione (GSSG), molecular formula: C 20 H 32 N6O 12 S2, is formed by linking two molecules of reduced glutathione (GSH) through a disulfide bond (-S-S-). GSSG is a stable storage form of glutathione. Compared with reduced glutathione GSH, GSSG has better stability, and GSSG has a variety of physiological functions. For example, it can be used as an oxidation-reduction buffer. The ratio of GSH / GSSG in cells (usually >90%:10%) is an important indicator for measuring oxidative stress; the accumulation of GSSG in the body usually indicates oxidative damage (such as excessive free radicals); GSSG can also combine with toxins to form excretable complexes (such as heavy metals, drug metabolites), having a detoxifying effect; in addition, as a biological signal regulatory molecule, GSSG affects cell apoptosis, inflammation and other pathways through protein disulfide modification. Based on the fact that the stability of GSSG is better than that of GSH, it can also be used in the fields of health foods, medicines, cosmetics, etc.

[0003] The existing production methods of GSSG mainly use enzymatically catalyzed synthetic GSH as a raw material and are chemically oxidized. So far, there have been no relevant research and literature reports on using amino acids (such as cystine) as raw materials to enzymatically catalyze the synthesis of GSSG.

[0004] The chemical oxidation method for preparing GSSG, such as using chemical oxidants: hydrogen peroxide, oxygen, sodium hydroxide, diethyl bromomalonate, etc., oxidizes reduced glutathione in an aqueous solution under appropriate pH conditions to prepare GSSG. For example, in the process of preparing GSSG by the chemical oxidation method in the patent application with the publication number CN116120393A, the oxidation rate is fast and the reaction is violent. It is necessary to strictly control the addition amount of the oxidant and the condition parameters of the reaction system during the reaction, such as temperature, pH, control nodes, etc. Its main problem is that more by-product impurities (impurity A, impurity B, impurity C, etc.) will be generated during the chemical oxidation process, and there is still uncompletely oxidized GSH in the system, increasing the subsequent extraction and purification process steps and affecting the product yield; at the same time, during the use of chemical oxidants, there are also certain safety and environmental pollution problems. Summary of the Invention

[0005] In view of the above problems, the object of the present application is to provide an oxidized glutathione synthetase, its application, and a preparation method of oxidized glutathione. Firstly, mutants of oxidized glutathione synthetase are provided to improve the catalytic efficiency. Secondly, for the first time, GSSG synthetase and its mutants are applied to the enzymatic catalytic synthesis of GSSG. Thirdly, a preparation method of GSSG with low cost, mild reaction conditions, few reaction impurities, high product purity, and being green and environmentally friendly is provided, which is of great significance and meets the production requirements of industrial scale-up.

[0006] The technical solutions involved in the present invention are as follows:

[0007] The first aspect of the present invention is to provide an oxidized glutathione synthetase, the amino acid sequence of which is an amino acid sequence with at least one mutation among S534Q, Y185N, and S380L in the wild amino acid sequence shown in SEQ ID NO.1.

[0008] The mutations are at least one of: S534Q + S380L, S534Q + Y185N, Y185N + S380L, S534Q + Y185N + S380L, S534Q, S380L, Y185N.

[0009] Further preferably:

[0010] The mutations are at least one of: S534Q + S380L, S534Q + Y185N, Y185N + S380L, S534Q + Y185N + S380L, S534Q.

[0011] Even more preferably:

[0012] The mutations are at least one of: S534Q + S380L, S534Q + Y185N, Y185N + S380L.

[0013] Most preferably:

[0014] The mutation is: S534Q + S380L.

[0015] The second aspect of the present invention is to provide the application of the above-mentioned oxidized glutathione synthetase for catalytic synthesis of oxidized glutathione.

[0016] The third aspect of the present invention is to provide a preparation method of oxidized glutathione, which uses the above-mentioned oxidized glutathione synthetase to catalytically prepare oxidized glutathione.

[0017] Furthermore,

[0018] In the reaction system, using cystine, sodium glutamate, and glycine as substrates, in the presence of ATP, Mg 2+In the presence of [conditions], oxidized glutathione is synthesized by enzymatic catalysis.

[0019] Furthermore,

[0020] In the reaction system, ATP cycle regeneration is achieved by using acetyl phosphate and acetate kinase, or sodium hexametaphosphate and polyphosphate kinase.

[0021] The acetyl phosphate and acetate kinase system is more significant than the sodium hexametaphosphate and polyphosphate kinase system in achieving ATP cycle regeneration. However, this invention is the first to realize the enzymatic preparation of GSSG, and any ATP cycle regeneration system is within the protection scope of this invention.

[0022] Further,

[0023] In the reaction system, the reaction temperature is 20 - 40 °C, and the reaction pH is 5.0 - 9.0.

[0024] Preferably: the reaction temperature is 25 - 35 °C, and the reaction pH is 6.5 - 8.0.

[0025] In this application, the dosage ratio of amino acids can be calculated according to Figure 1 the reaction equation.

[0026] For example: in the reaction system, the molar ratio of cystine, glycine, and sodium glutamate is 1:1:1 ~ 1:2:2.

[0027] The amount of GSSG synthase used in every 100 mL of the reaction system is: 297 - 500 U; the ratio of GSSG synthase activity to cystine weight: 297 - 500 U / g cystine.

[0028] In every 100 mL of the reaction system: 300 - 500 U of acetate kinase; 40 - 60 mL of 40% - concentration acetyl phosphate, 3.8 - 5 g of sodium hexametaphosphate, 1000 U - 2000 U of polyphosphate kinase; 0.12 - 3.0 g of ATP, 1.5 - 2.0 g of magnesium ions.

[0029] The dosages of ATP, magnesium ions, and the raw materials and enzymes required for ATP cycle regeneration can refer to the enzymatic reaction of GSH.

[0030] Advantages of this invention

[0031] (1) This invention first uses GSSG synthase to directly catalyze the synthesis of GSSG by the whole - enzyme method. Using three amino acids, cystine, glycine, and sodium glutamate, as substrates, it does not require chemical oxidation reagents, has good specificity, a simple process, and mild reaction conditions; it is environmentally friendly and more suitable for continuous large - scale production.

[0032] (2) Compared with the traditional chemical oxidation synthesis method, there is no reduced glutathione in the reaction system, effectively avoiding the separation problem of GSH, which is beneficial to subsequent extraction and purification. The purity of the purified product is ≥99% (detected by HPLC), and it is more suitable for the large-scale production of high-purity GSSG in the pharmaceutical, food, and cosmetic industries. At the same time, enzyme-coupled ATP regeneration is adopted, and the conversion rate is up to ≥90% at most. Description of the Drawings

[0033] Figure 1 : Reaction equation diagram of the enzymatic synthesis of GSSG in the present invention;

[0034] Figure 2 : HPLC detection GSSG spectrum of the present invention;

[0035] Figure 3 : Purity spectrum of purified GSSG of the present invention;

[0036] Figure 4 : Control of the spectrum of the reduced glutathione GSH standard product. Detailed Embodiments

[0037] Example 1 Construction of GSSG synthase and verification of GSSG synthesis:

[0038] Screening of GSSG synthase gene, downloading the amino acid sequence of Bifunctional gamma-glutamate-cysteine ligase from Actinobacillus succinogenes (Sequence ID: WP_012073672.1) from the NCBI database. After optimizing the E. coli expression codons for the amino acid sequence, it was submitted to General Biology (Anhui) Co., Ltd. for synthesis. The 5' end of the gene carried an NdeI restriction site, the 3' end carried an XhoI restriction site, and the 3' end of the gene carried a nucleotide sequence with 6 His tags. The synthesized gene sequence was cloned into the prokaryotic expression plasmid pET30a(+), and the recombinant expression plasmid pET30a-GSSG was constructed. After the recombinant plasmid was verified by sequencing to be correct, it was transferred into the E. coli expression host E. coli BL21(DE3), and the E. coli recombinant strain containing the GSSG gene was obtained.

[0039] Using a sterilized pipette tip, carefully pick a single colony of the recombinant strain containing the GSSG gene from the above LB solid medium plate (containing 50 μg / mL kanamycin) and inoculate it into a triangular flask containing 20 mL of LB liquid medium. Incubate overnight at 37 °C with shaking at 200 r / min. The next day, inoculate the flask culture into a triangular flask containing 100 mL of TB liquid medium at an inoculation amount of 1%, and incubate at 37 °C with shaking at 220 r / min. Measure the cell concentration (OD 600 value) of the culture broth every 1 h. When the OD value of the culture broth 600 = 1.5, add lactose with a final concentration of 1% (m / v), and continue to incubate at 25 °C with shaking at 220 rpm for 4 h - 6 h. Stop the incubation, collect the cells, and break them to obtain the crude enzyme solution containing GSSG recombinase.

[0040] The synthesis of GSSG is carried out according to the following steps:

[0041] (1) Preparation of reaction substrates: Weigh 2.37 g of sodium glutamate, 1.22 g of glycine, 1.68 g of cystine, and 1.55 g of magnesium chloride in the reaction system, add 30 mL of water, stir, and adjust the pH to about 6.8 for dissolution;

[0042] (2) Add 3.0 g of ATP to the reaction system, dissolve it thoroughly, and adjust the pH to about 6.8;

[0043] (3) Add 500 U of GSSG recombinant enzyme solution to the reaction system (the activity assay is carried out according to the method in Food and Fermentation Industries, 2025, 51(06): 91 - 96), make up the volume to 100 mL, control the pH at 6.8, turn on the stirrer, control the rotation speed at 150 r / min, and react at 25 °C;

[0044] (4) The reaction time is 2 - 10 h, and the generation amount of GSSG is monitored during the process.

[0045]

[0046] As can be seen from Table 1, when cystine, sodium glutamate, and glycine are selected as substrates, it is unexpectedly found that GSSG products can be obtained. The concentration of GSSG in the reaction system is 14.87 mg / mL, and the yield is 34.78%. To further improve the specificity of GSSG synthesis, it is necessary to further screen and modify GSSG synthase.

[0047] Calculation of transformation reaction yield = (molar amount of target product generated ÷ molar amount of substrate cystine) × 100%.

[0048] The molar generation amount of GSSG in Table 1 system is: 0.0024 mol;

[0049] The addition amount of cystine in the reaction system is: 0.0069 mol;

[0050] Conversion yield = 34.78%.

[0051] Example 2 Construction and screening of GSSG synthase mutants and verification of GSSG synthesis:

[0052] Extract the plasmid pET30a-GSSG of GSSG synthase. After being detected and analyzed by 0.8% agarose gel electrophoresis without error, using this plasmid as a template, perform a site-directed saturation mutagenesis PCR reaction.

[0053] After the PCR reaction product is used for the construction of recombinant plasmid DNA, it is transformed into Escherichia coli cells to obtain an error-prone mutant library containing different GSSG synthase mutants. High-throughput screening is carried out on this mutant library. Use a sterilized pipette tip to pick single colonies of the mutant library into a 96-well culture plate (the culture plate is added with LB liquid medium). Place the 96-well cell culture plate in a constant temperature shaker at 37 °C and 700 rpm for 6 hours. Then use an 8-channel pipette to take 50 µL and save it in a new 96-well plate as the seed solution. Then add lactose with a final concentration of 1% (m / v) to each well, and induce culture at 25 °C and 250 rpm for 8 hours. After the induction culture is completed, put the 96-well cell culture plate into an ultra-low temperature refrigerator at -86 °C and freeze for 2 hours. Take it out and place it at room temperature for half an hour. Centrifuge at 4000 r / min and 4 °C for 20 minutes and take 50 μL of the supernatant from each well. For the 96-well plate containing 50 μL of the supernatant, then add 100 μL of the substrate solution (pH 6.8 40 mM Tris-HCl substrate solution: 14 mM sodium glutamate, 14 mM glycine, 7 mM cystine, 14 mM magnesium chloride, 6 mM ATP) to each well. After the reaction temperature is 25 °C and the reaction time is 10 h, add 20% trichloroacetic acid to terminate the reaction. Place the 96-well plate in a centrifuge, centrifuge at 4000 rpm for 5 minutes, take 1 - 5 µL of the supernatant, and perform UPLC detection and analysis to quantitatively determine the product formation amount. Select the sample with a large peak area of the target product for subsequent re-screening and sequencing analysis of the sample.

[0054]

[0055] As can be seen from Table 2, the GSSG mutants obtained by screening can significantly improve the yield of the GSSG synthesis reaction. To further improve the reaction yield, the above mutants are stacked to study the effect of the stacked mutants on the yield of the GSSG synthesis reaction.

[0056]

[0057] As can be seen from Table 3, through the superimposed mutations, the content of GSSG in the reaction system can be significantly increased. Among them, for the mutant GSSG-5, the concentration of GSSG synthesized in the reaction system can reach 37.63 mg / mL, and the yield is 88.01%. It can be used as the optimal mutant enzyme for subsequent optimization experiments.

[0058] Example 3 Optimization of the GSSG synthesis reaction system:

[0059] (1) Optimization of the ATP energy supply system:

[0060] To better meet the requirements of industrial scale production and reduce costs, ATP is recycled. The reaction system and conditions are shown in Table 4 below:

[0061]

[0062] It can be clearly seen from Table 4 that the amount of GSSG synthesized in reaction system 2 is significantly higher than that in reaction system 1. Therefore, using the acetate kinase and acetyl phosphate route to achieve the recycling of ATP has better yield and economy, and is more suitable for industrial scale production applications.

[0063] (2) Optimization of reaction conditions:

[0064] To further improve the yield of the reaction and reduce costs, the reaction parameters were optimized. The reaction system and conditions are shown in Table 5 below:

[0065]

[0066] Figure 2 This is the HPLC detection spectrum of the product GSSG prepared in the present invention. The corresponding peaks, retention times, peak areas, relative percentages and compound results are shown in Table 6. It can be seen that GSH is not present in the product, which is beneficial for the subsequent separation and purification of the product.

[0067]

[0068] Figure 3 This is the spectrum of the product GSSG prepared in the present invention with a purity of ≥99% after purification.

Claims

1. Glutathione synthetase (oxidized form), characterized in that, Its amino acid sequence is an amino acid sequence in which there is at least one mutation among S534Q, Y185N, and S380L in the wild-type amino acid sequence shown in SEQ ID NO.

1.

2. The oxidized glutathione synthetase according to claim 1, characterized in that, The mutations are at least one of: S534Q + S380L, S534Q + Y185N, Y185N + S380L, S534Q + Y185N + S380L, S534Q, S380L, Y185N.

3. The oxidized glutathione synthetase according to claim 2, wherein The mutations are at least one of: S534Q + S380L, S534Q + Y185N, Y185N + S380L, S534Q + Y185N + S380L, S534Q.

4. Use of the oxidized glutathione synthetase according to any one of claims 1-3, characterized in that, It is used to catalyze the synthesis of oxidized glutathione.

5. A preparation method of oxidized glutathione, characterized in that, The oxidized glutathione is catalytically prepared by using the oxidized glutathione synthetase described in any one of claims 1-3.

6. The preparation method according to claim 5, wherein In the reaction system, cystine, sodium glutamate, and glycine are used as substrates, and in the presence of ATP and Mg 2+ oxidized glutathione is synthesized by enzymatic catalysis under existing conditions.

7. The preparation method according to claim 5, characterized in that: In the reaction system, ATP cycle regeneration is achieved by using acetyl phosphate and acetate kinase, or sodium hexametaphosphate and polyphosphate kinase.

8. The preparation method according to claim 5, characterized in that, In the reaction system, the reaction temperature is 20-40 °C and the reaction pH is 5.0-9.

0.

9. The preparation method according to claim 8, wherein In the reaction system, the reaction temperature is 25-35 °C and the reaction pH is 6.5-8.0.

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