Oxidized glutathione synthetase and its application and preparation method of oxidized glutathione

Abstract

The present invention discloses an oxidized glutathione synthetase, its application and a method for preparing oxidized glutathione. It mainly relates to the application of an oxidized glutathione synthetase having a wild amino acid sequence shown in SEQ ID NO.1 and / or an amino acid sequence having at least one mutation of S534Q, Y185N and S380L in the wild amino acid sequence shown in SEQ ID NO.1, directly catalyzing the synthesis of GSSG using a holoenzyme method. For the first time, cystine, sodium glutamate and glycine are used as substrates in the presence of ATP, Mg 2+ Under the presence of ATP, oxidized glutathione is enzymatically synthesized. This method eliminates the need for chemical oxidizing agents, offers excellent specificity, a simple process, mild reaction conditions, and no impurities. Compared to traditional chemical oxidation synthesis methods, this method effectively avoids the problem of separating GSH intermediates. Furthermore, it utilizes an enzymatically coupled ATP regeneration method, achieving a conversion rate of ≥90%.

Description

Technical Field

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

[0002] Oxidized glutathione (GSSG), molecular formula: C 20 H 32 N6O 12 S2 is composed of two molecules of reduced glutathione (GSH) linked by a disulfide bond (-SS-). GSSG is a stable storage form of glutathione and is more stable than reduced glutathione (GSH). GSSG also has multiple physiological functions, including acting as a redox buffer. The intracellular GSH / GSSG ratio (typically >90%:10%) is a key indicator of oxidative stress. GSSG accumulation in the body often indicates oxidative damage (such as excessive free radicals). GSSG can also bind to toxins (such as heavy metals and drug metabolites) to form excretable complexes, exerting a detoxifying effect. Furthermore, as a biological signaling regulator, GSSG influences pathways such as apoptosis and inflammation through protein disulfide bond modification. Because GSSG is more stable than GSH, it is also suitable for use in health foods, pharmaceuticals, cosmetics, and other fields.

[0003] Existing methods for producing GSSG primarily utilize enzymatically synthesized GSH as a raw material, followed by chemical oxidation. To date, there are no studies or reports on the enzymatic synthesis of GSSG using amino acids (such as cystine) as a raw material.

[0004] Chemical oxidation methods for preparing GSSG involve oxidizing reduced glutathione in an aqueous solution at an appropriate pH using chemical oxidants such as hydrogen peroxide, oxygen, sodium hydroxide, and diethyl bromomalonate. For example, the chemical oxidation method for preparing GSSG, as described in patent application publication number CN116120393A, exhibits rapid and intense oxidation, requiring strict control of the amount of oxidant added and reaction parameters such as temperature, pH, and control points. A major challenge is the generation of numerous byproduct impurities (impurities A, B, and C), along with the presence of incompletely oxidized GSH in the system. This increases the number of subsequent extraction and purification steps, impacting product yield. Furthermore, the use of chemical oxidants poses safety and environmental concerns. Summary of the Invention

[0005] To address the above issues, the present application aims to provide an oxidized glutathione synthetase, its application, and a method for preparing oxidized glutathione. First, a mutant of oxidized glutathione synthetase is provided to improve catalytic efficiency. Second, GSSG synthase and its mutants are applied to the enzymatic synthesis of GSSG for the first time. Third, a method for preparing GSSG that is low-cost, has mild reaction conditions, few reaction impurities, and a high-purity product is provided. These methods are environmentally friendly and of great significance, meeting the needs of industrial-scale production.

[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 in which at least one mutation among S534Q, Y185N and S380L exists in the wild amino acid sequence shown in SEQ ID NO.1.

[0008] The mutation is at least one of S534Q+S380L, S534Q+Y185N, Y185N+S380L, S534Q+Y185N+S380L, S534Q, S380L, and Y185N.

[0009] Further preferred:

[0010] The mutation is at least one of S534Q+S380L, S534Q+Y185N, Y185N+S380L, S534Q+Y185N+S380L, and S534Q.

[0011] Further optimization:

[0012] The mutation is at least one of S534Q+S380L, S534Q+Y185N, and Y185N+S380L.

[0013] Most preferred:

[0014] The mutation is: S534Q+S380L.

[0015] The second aspect of the present invention is to provide the use of the oxidized glutathione synthetase for catalyzing the synthesis of oxidized glutathione.

[0016] The third aspect of the present invention is to provide a method for preparing oxidized glutathione, which utilizes the oxidized glutathione synthetase to catalyze the preparation of oxidized glutathione.

[0017] Furthermore,

[0018] In the reaction system, cystine, sodium glutamate and glycine are used as substrates, and in the presence of ATP and Mg 2+Under the presence of glutathione, the enzyme catalyzes the synthesis of oxidized glutathione.

[0019] Furthermore,

[0020] In the reaction system, ATP cycle regeneration is achieved by using acetyl phosphate and acetate kinase, or sodium 6-metaphosphate and polyphosphate kinase.

[0021] The acetyl phosphate and acetate kinase system is more effective in achieving ATP cycle regeneration than the 6-sodium metaphosphate and polyphosphate kinase system. However, the present invention is the first to achieve enzymatic preparation of GSSG. Regardless of which ATP cycle regeneration system is used, it is within the scope of protection of the present invention.

[0022] Furthermore,

[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] The ratio of amino acids used in this application can be based on Figure 1 The reaction equation was calculated.

[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 each 100 mL reaction system is: 297-500 U; the GSSG synthase activity / cystine weight ratio is: 297-500 U / g cystine.

[0028] Per 100 mL reaction system: 300-500 U of acetate kinase; 40-60 mL of 40% acetyl phosphate, 3.8-5 g of sodium 6-metaphosphate, 1000 U-2000 U of polyphosphate kinase; 0.12-3.0 g of ATP, and 1.5-2.0 g of magnesium ion.

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

[0030] Beneficial effects of the present invention

[0031] (1) The present invention adopts GSSG synthase for the first time, and directly catalyzes the synthesis of GSSG by the whole enzyme method, using three amino acids, cystine, glycine and sodium glutamate, as substrates. No chemical oxidizing reagents are required, and the process is simple and the reaction conditions are mild. It is environmentally friendly and more suitable for continuous large-scale production.

[0032] (2) Compared with traditional chemical oxidation synthesis methods, the reaction system does not contain reduced glutathione, which effectively avoids the problem of GSH separation and facilitates subsequent extraction and purification. The purity of the purified product is ≥99% (HPLC detection), making it more suitable for large-scale production of high-purity GSSG in the pharmaceutical, food and cosmetics industries. At the same time, the enzyme-coupled ATP regeneration is used, with a conversion rate of up to ≥90%. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0036] Figure 4 :Comparison of the spectrum of reduced glutathione GSH standard. DETAILED DESCRIPTION

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

[0038] To screen for the GSSG synthase gene, the amino acid sequence of the bifunctional gamma-glutamate-cysteine ligase from Actinobacillus succinogenes (Sequence ID: WP_012073672.1) was downloaded from the NCBI database. After codon optimization for E. coli expression, the amino acid sequence was submitted to General Biotechnology (Anhui) Co., Ltd. for synthesis. The gene contained an NdeI restriction site at the 5' end and an XhoI restriction site at the 3' end. The 3' end of the gene also contained a six-mer His-tag nucleotide sequence. The synthesized gene sequence was cloned into the prokaryotic expression plasmid pET30a(+) to construct the recombinant expression plasmid pET30a-GSSG. The recombinant plasmid was verified by sequencing and transformed into the E. coli expression host strain BL21(DE3), resulting in a recombinant E. coli strain containing the GSSG gene.

[0039] 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) using a sterilized pipette tip and inoculate it into a flask containing 20 mL of LB liquid medium. Incubate at 37°C, 200 rpm, and shake overnight. The next day, inoculate the shake flask culture solution into a flask containing 100 mL of TB liquid medium at a 1% inoculum volume. Incubate at 37°C, 220 rpm, and measure the bacterial cell concentration (OD) of the culture solution every 1 h. 600 value), the OD value of the culture medium 600 =1.5, add lactose with a final concentration of 1% (m / v), continue to culture at 25°C and 220 rpm for 4 h-6 h, stop the culture, collect the bacteria and crush them to obtain the crude enzyme solution containing GSSG recombinant enzyme.

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

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

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

[0043] (3) Add 500 U of GSSG recombinant enzyme solution to the reaction system (activity determination was carried out according to the method of Food and Fermentation Industries, 2025, 51(06): 91-96), adjust the volume to 100 mL, control the pH to 6.8, start stirring and control the speed to 150 r / min, and react at 25 °C;

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

[0045]

[0046] As shown in Table 1, using cystine, sodium glutamate, and glycine as substrates, the unexpected discovery of GSSG production was achieved. The GSSG concentration in the reaction system was 14.87 mg / mL, and the yield was 34.78%. To further enhance the specificity of GSSG synthesis, further screening and modification of GSSG synthases are required.

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

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

[0049] The amount of cystine added to the reaction system was: 0.0069 mol;

[0050] Conversion yield = 34.78%.

[0051] Example 2 Construction and Screening of GSSG Synthetase Mutants and Verification of GSSG Synthesis:

[0052] The plasmid pET30a-GSSG containing GSSG synthase was extracted and analyzed by 0.8% agarose gel electrophoresis. Then, the plasmid was used as a template for site-directed saturation mutagenesis PCR reaction.

[0053] After constructing recombinant plasmid DNA from the PCR reaction products, they were transformed into Escherichia coli cells to generate an error-prone mutant library containing different GSSG synthase mutants. This mutant library was subjected to high-throughput screening. Single colonies from the mutant library were picked with a sterile pipette tip and transferred to a 96-well culture plate (containing LB liquid medium). The 96-well cell culture plate was incubated in a constant temperature shaker at 37°C, 700 rpm for 6 hours. Then, 50 µL of the culture medium was transferred using an 8-channel pipette and stored as seed solution in a new 96-well plate. Lactose was then added to each well to a final concentration of 1% (m / v) and induced at 25°C, 250 rpm for 8 hours. After induction, the 96-well cell culture plate was placed in a -86°C ultra-low temperature freezer for 2 hours, then removed and allowed to stand at room temperature for half an hour. The plate was then centrifuged at 4000 rpm for 20 minutes at 4°C, and 50 μL of supernatant was collected from each well. The 96-well plate containing 50 μL of supernatant was then added to each well of the plate. 100 μL of substrate solution (40 mM Tris-HCl, pH 6.8: 14 mM sodium glutamate, 14 mM glycine, 7 mM cystine, 14 mM magnesium chloride, 6 mM ATP) was added. The reaction was incubated at 25°C for 10 hours, and then 20% trichloroacetic acid was added to terminate the reaction. The 96-well plate was then centrifuged at 4000 rpm for 5 minutes. 1-5 μL of the supernatant was collected and analyzed by UPLC to quantify the amount of product produced. Samples with high peak areas of the target product were selected for subsequent rescreening and sequencing analysis.

[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. In order to further improve the reaction yield, the above mutants were superimposed to study the effect of the superimposed mutants on the yield of the GSSG synthesis reaction.

[0056]

[0057] As shown in Table 3, the superimposed mutations significantly increased the GSSG content in the reaction system. Mutant GSSG-5 achieved a GSSG synthesis concentration of 37.63 mg / mL in the reaction system, with a yield of 88.01%. This mutant enzyme can be selected as the optimal mutant for subsequent optimization experiments.

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

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

[0060] In order to better meet the needs of industrial-scale production and reduce costs, ATP can be 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, the use of acetate kinase and acetyl phosphate route to achieve ATP recycling has better yield and economy and is more suitable for industrial-scale production applications.

[0063] (2) Optimization of reaction conditions:

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

[0065]

[0066] Figure 2 The HPLC detection spectrum of the product GSSG prepared in the present invention is shown in Table 6. 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 found in the product, which is beneficial for subsequent product separation and purification.

[0067]

[0068] Figure 3 This is a spectrum showing that the purity of the purified product GSSG prepared in the present invention is ≥99%.

Claims

1. Oxidative glutathione synthetase, characterized in that The amino acid sequence thereof is an amino acid sequence in which at least one mutation among S534Q, Y185N and S380L exists in the wild amino acid sequence shown in SEQ ID NO.

1.

2. The oxidized glutathione synthetase according to claim 1, wherein The mutation is at least one of S534Q+S380L, S534Q+Y185N, Y185N+S380L, S534Q+Y185N+S380L, S534Q, S380L, and Y185N.

3. The oxidized glutathione synthetase according to claim 2, characterized in that The mutation is at least one of S534Q+S380L, S534Q+Y185N, Y185N+S380L, S534Q+Y185N+S380L, and S534Q.

4. The use of the oxidized glutathione synthetase according to any one of claims 1 to 3, characterized in that Used to catalyze the synthesis of oxidized glutathione.

5. A method for preparing oxidized glutathione, characterized in that: The oxidized glutathione synthetase according to any one of claims 1 to 3 is used to catalyze the preparation of oxidized glutathione.

6. The preparation method according to claim 5, characterized in that In the reaction system, cystine, sodium glutamate and glycine are used as substrates, and in the presence of ATP and Mg 2+ Under the presence of glutathione, the enzyme catalyzes the synthesis of oxidized glutathione.

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 6-metaphosphate 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, characterized in that In the reaction system, the reaction temperature is 25-35°C, and the reaction pH is 6.5-8.0.

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