Method for preparing glutathione by immobilized enzyme and application thereof

Abstract

The application specifically relates to a method for preparing glutathione by immobilized enzyme and application. The method for preparing glutathione provided by the application comprises synthesizing glutathione by using saccharomyces cerevisiae haploid ascospores as a fixed enzyme catalyst to catalyze substrate reaction, using an ATP in-situ regeneration reaction system of phosphoenolpyruvate as an energy supply system, the saccharomyces cerevisiae haploid ascospores are knocked out or inactivated in combination of one or more than two of cell wall beta-glucan synthesis genes, and GSH1 genes, GSH2 genes and PYK1 genes are co-expressed. The saccharomyces cerevisiae haploid ascospores for simultaneously producing GSH1, GSH2 and pyruvate kinase are used as a fixed enzyme catalyst to catalyze synthesis of glutathione, three enzymes no longer need to be added externally, raw material cost is greatly reduced, the obtained product is easy to purify, and the prepared glutathione is high in purity.

Description

Technical Field

[0001] This invention relates to the field of enzyme-catalyzed biosynthesis technology, specifically to a method and application of immobilized enzymes for preparing glutathione. Background Technology

[0002] Glutathione (GSH) is widely found in animals, plants, and microorganisms. It is an important non-protein thiol compound present in living organisms, synthesized from three precursor amino acids—glutamate, cysteine, and glycine—through the sequential catalysis of glutamylcysteine ​​synthase and glutathione synthase. Glutathione possesses various physiological functions, such as antioxidant activity, detoxification, and immune enhancement. It is primarily used in the preparation of powder injections for clinical pharmaceutical applications and in the cosmetics manufacturing industry. In China, the European Union, Japan, and other countries, glutathione is listed as a pharmaceutical raw material and preparation.

[0003] Currently, there are two main technologies for glutathione production: The first is fermentation, using natural yeast strains as the starting material. Sugars, starches, or molasses are used as the carbon source, and ammonia, ammonium sulfate, ammonium carbonate, or urea are used as the nitrogen source. In a culture medium supplemented with cysteine, glutamic acid, and glycine, the yeast cells' own enzyme system metabolizes and synthesizes glutathione. The second is enzymatic synthesis, using γ-glutamylcysteine ​​synthase and glutathione synthase as catalysts. These catalysts react with a solution of glutamic acid, cysteine, and glycine, with the addition of ATP, acetate kinase, and Mg2+. 2+ Energy-assisted systems such as acetyl phosphate are used to catalyze the synthesis of glutathione.

[0004] Existing technology CN101575578A discloses a yeast strain, dry yeast rich in reduced glutathione, and its preparation method, including the following steps: *Saccharomyces cerevisiae* strain → slant culture → liquid culture in culture bottles → liquid culture in a Pond's tank → seed culture → seed culture in a fermenter → fermentation culture in a fermenter → separation → drying → packaging. The fermentation process is as follows: pH 5.5–6.5, fermentation temperature 28–32℃, oxygenation rate 0.5–1.5 g O2 / 1 g yeast*min, fermentation time 20–22 hours, and yeast concentration 170–250 g / L. The glutathione-rich yeast obtained by this invention contains 3–5% reduced glutathione.

[0005] The prior art CN 106086126A discloses a method for enzyme-catalyzed synthesis of glutathione, comprising the following steps: S1, mixing γ-glutamylcysteine ​​synthase solution and glutathione synthase solution to obtain mixture A, and then mixing mixture A with acetate kinase solution to obtain mixture B; S2, adding an immobilization carrier to mixture B, stirring for immobilization, and filtering to obtain immobilized enzyme; S3, preparing a reaction solution with glutamic acid, L-cysteine, glycine, magnesium sulfate, ATP, and acetyl phosphate, adding the immobilized enzyme, and stirring for reaction; S4, after the reaction is completed, filtering the reaction solution, and extracting and purifying the filtrate to obtain glutathione. Summary of the Invention

[0006] The technical problem this invention aims to solve is as follows: Existing fermentation followed by extraction methods for producing high-purity glutathione suffers from low glutathione content in yeast cells (typically around 6% total glutathione), long fermentation times, high energy consumption, and complex extraction processes that require the use of organic solvents and toxic hydrogen sulfide gas. While existing enzyme-catalyzed synthesis methods offer significantly higher production efficiency compared to fermentation extraction, they involve the introduction of three enzyme preparations and a fixative. Due to the extreme difficulty in purifying these enzyme preparations, the resulting crude enzyme product from microbial fermentation has a complex composition and contains a large amount of non-target enzyme proteins. When used as an injectable drug, these constitute unknown impurities and allergens, leading to non-compliance with drug regulatory requirements and posing significant technical challenges for manufacturers in registering drug approvals and obtaining drug filing certificates.

[0007] To address the problems of low glutathione content and low purity obtained by existing preparation methods, this invention provides a glutathione synthesis method with high conversion rate and easy purification.

[0008] This invention utilizes yeast spores with high cell wall permeability as immobilized enzyme catalysts. By leveraging the yeast spore wall to prevent or block the exudation of soluble secretory proteins from within the spore, it is the first time that three enzymes used in glutathione synthesis have been expressed and immobilized on the yeast cell wall in the same haploid yeast spore. This significantly reduces the problem of numerous unknown impurities present in enzymatic glutathione synthesis and improves enzyme reusability. Furthermore, this invention employs an overexpression system of yeast's own endogenous enzymes, using PEP for energy, and constructs an in-situ ATP regeneration system, substantially reducing production costs.

[0009] The technical solution of this invention:

[0010] This invention provides a method for preparing glutathione, comprising: using haploid ascospores of *Saccharomyces cerevisiae* as an immobilized enzyme catalyst to catalyze the substrate reaction to synthesize glutathione; and using a phosphoenolpyruvate ATP in-situ regeneration reaction system as an energy supply system.

[0011] The haploid ascospores of *Saccharomyces cerevisiae* are characterized by the knockout or inactivation of one or more of the cell wall β-glucan synthesis genes, and the co-expression of the GSH1, GSH2, and PYK1 genes.

[0012] Preferably, the synthesis method includes the following steps:

[0013] (1) Prepare a reaction solution by mixing glutamic acid, L-cysteine, glycine, magnesium chloride, potassium chloride, ATP and PEP, add yeast spore microcapsules to fix the enzyme, and stir to react;

[0014] (2) After the reaction is completed, the reaction solution is separated, the fixed enzyme is recovered, and the supernatant is extracted and purified to produce glutathione.

[0015] Preferably, in step (1), the concentrations of glutamic acid, L-cysteine, glycine, magnesium chloride, potassium chloride, ATP, and PEP are 0.1-0.3 wt%, 0.2-0.6 wt%, and the amount of immobilized enzyme catalyst added is 5-10 wt%.

[0016] Preferably, in step (1), the catalytic reaction temperature is 20-30℃;

[0017] Alternatively, preferably, the pH value of the catalytic reaction is 6.5-7.5;

[0018] Alternatively, preferably, the catalytic reaction time is 2-5 hours.

[0019] Preferably, in step (2), the refining process includes:

[0020] 1) Adjust the pH to 2.5-3.0;

[0021] 2) Cation exchange resins adsorb glutathione through exchange;

[0022] 3) Gradient elution with 0-3% sodium chloride to collect glutathione in fractions;

[0023] 4) Concentrate glutathione using nanofiltration membrane to achieve a glutathione concentration of 80-100 g / L, and wash with purified water after concentration.

[0024] 5) Reduced pressure evaporation and concentration to achieve a glutathione concentration of 300-450 g / L;

[0025] 6) Crystallize and dry to obtain reduced glutathione.

[0026] Preferably, in step 4), the nanofiltration membrane has a pore size of 100-200D, more preferably 150D;

[0027] Preferably, in step 5), the temperature for reduced pressure evaporation is 45-55°C, and the vacuum degree is 0.08-0.09 MPa;

[0028] Preferably, in step 6), an equal volume of 75-80% ethanol is added, and crystallization is carried out in a low-temperature, oxygen-free environment at 4-8°C;

[0029] Preferably, in step 6), the obtained crystals are vacuum dried at 50-60°C.

[0030] Preferably, the haploid ascospore walls of the *Saccharomyces cerevisiae* are immobilized with γ-glutamylcysteine ​​synthase, glutathione synthase, and pyruvate kinase.

[0031] Preferably, the haploid ascospores of Saccharomyces cerevisiae contain a recombinant vector expressing γ-glutamylcysteine ​​synthase, glutathione synthase and pyruvate kinase;

[0032] Preferably, the recombinant vector contains the base sequences described in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24;

[0033] Preferably, the recombinant vector contains the base sequence described in SEQ ID NO. 21. Preferably, the haploid ascospores of *Saccharomyces cerevisiae* are obtained from wild-type *Saccharomyces cerevisiae* strains with one or more combinations of cell wall β-glucan synthesis genes knocked out or inactivated, and capable of co-expressing the GSH1 gene, GSH2 gene, and PYK1 gene.

[0034] Preferably, the wild yeast strain is Saccharomyces cerevisiae FX-2, with the preservation number CCTCC NO:M2016418;

[0035] Preferably, the cell wall β-glucan synthesis gene is one or a combination of two or more of KRE1, KRE6 and KRE9; preferably KRE1.

[0036] Preferably, the method for preparing haploid ascospores of Saccharomyces cerevisiae includes the following steps:

[0037] (1) Obtain haploid yeast spores of wild-type Saccharomyces cerevisiae strain;

[0038] (2) Knock out or inactivate one or more of the genes that synthesize cell wall glucans in haploid yeast;

[0039] (3) The recombinant vector containing genes expressing γ-glutamylcysteine ​​synthase, glutathione synthase and pyruvate kinase was transferred into the haploid yeast obtained in step (2).

[0040] (4) Hybridize the haploid yeast spores obtained in the two steps (3) to obtain a diploid sporulating yeast strain with sporulation performance;

[0041] (5) The spores produced by the diploid sporulating yeast strain obtained in step (4) are the haploid ascospores of the brewer's yeast.

[0042] Preferably, step (3) of the method for preparing haploid ascospores of Saccharomyces cerevisiae includes: constructing transformation plasmids of GSH1, GSH2 and PKY1 respectively, and inserting secretion signal peptide genes. Preferably, the secretion signal peptide gene is one or a combination of two or more of MFα1, SUC2, PHO5 and INU, more preferably MFα1. 1 Then, the expression was transformed in the same yeast haploid spores;

[0043] Alternatively, GSH1, GSH2, and PKY1 can be expressed in the same plasmid, secretion signal peptides can be inserted into each plasmid, and the recombinant plasmid can be transformed into yeast haploid spores to form transformant spores.

[0044] The present invention also provides a haploid ascospore of Saccharomyces cerevisiae, wherein the haploid ascospore wall of the Saccharomyces cerevisiae is fixed with γ-glutamylcysteine ​​synthase, glutathione synthase and pyruvate kinase.

[0045] Preferably, the haploid ascospores of *Saccharomyces cerevisiae* contain a recombinant vector expressing γ-glutamylcysteine ​​synthase, glutathione synthase, and pyruvate kinase;

[0046] Preferably, the recombinant vector contains the base sequences described in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24;

[0047] Preferably, the recombinant vector contains the base sequence described in SEQ ID NO.21.

[0048] Preferably, the haploid ascospores of *Saccharomyces cerevisiae* are obtained from wild *Saccharomyces cerevisiae* strains with one or more combinations of cell wall β-glucan synthesis genes knocked out or inactivated, and capable of co-expressing the GSH1 gene, GSH2 gene and PYK1 gene.

[0049] Preferably, the wild yeast strain is Saccharomyces cerevisiae FX-2, with the preservation number CCTCC NO:M2016418;

[0050] Preferably, the cell wall β-glucan synthesis gene is one or a combination of two or more of KRE1, KRE6 and KRE9; preferably KRE1.

[0051] The present invention also provides the application of the aforementioned haploid ascospores of Saccharomyces cerevisiae in the preparation of glutathione.

[0052] The present invention also provides a glutathione synthesis immobilization enzyme, wherein the glutathione synthesis immobilization enzyme comprises the following components:

[0053] Using haploid ascospores of Saccharomyces cerevisiae as a fixation carrier, γ-glutamylcysteine ​​synthase, glutathione synthase and pyruvate kinase were immobilized.

[0054] Preferably, the haploid ascospores of *Saccharomyces cerevisiae* are obtained from wild *Saccharomyces cerevisiae* strains with one or more combinations of cell wall β-glucan synthesis genes knocked out or inactivated, and capable of co-expressing the GSH1 gene, GSH2 gene and PYK1 gene.

[0055] Preferably, the wild yeast strain is Saccharomyces cerevisiae FX-2, with the preservation number CCTCC NO:M2016418;

[0056] Preferably, the cell wall β-glucan synthesis gene is one or a combination of two or more of KRE1, KRE6, and KRE9; preferably, it is KRE1.

[0057] Alternatively, GSH 1, GSH 2, and PKY1 can be expressed in the same plasmid, secretion signal peptides can be inserted into them respectively, and the recombinant plasmid can be transformed into yeast haploid spores to form transformant spores.

[0058] Preferably, the recombinant vector contains the base sequences described in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24;

[0059] Preferably, the recombinant vector contains the base sequence described in SEQ ID NO.21.

[0060] The present invention also provides the application of the glutathione synthesis immobilization enzyme in the preparation of glutathione.

[0061] The beneficial effects of this invention are:

[0062] This invention uses haploid ascospores of Saccharomyces cerevisiae that simultaneously produce GSH1, GSH2 and pyruvate kinase as immobilized enzyme catalysts to catalyze the synthesis of glutathione. This eliminates the need for the exogenous addition of the three enzymes, significantly reducing raw material costs. The resulting product is easy to purify, and the prepared glutathione has high purity.

[0063] Immobilizing enzymes using yeast spores as carriers significantly reduces the large number of unknown impurities present in the enzymatic synthesis of glutathione. The resulting glutathione can be applied clinically, while also improving the reusability of the enzyme. A low-cost in-situ ATP regeneration tandem enzyme-catalyzed glutathione production method was constructed, achieving a substrate conversion rate of over 90% and reducing production costs. Attached Figure Description

[0064] Figure 1 The image shown is an electrophoresis diagram verifying the KRE1 gene knockout results in Example 1;

[0065] Figure 2 The image shown is a plasmid map constructed in Example 2;

[0066] Figure 3 The image shown is an electrophoresis diagram of plasmids obtained in haploid yeast in Example 2. Detailed Implementation

[0067] This invention provides a method for synthesizing glutathione, which uses haploid ascospores of Saccharomyces cerevisiae as an immobilized enzyme catalyst to catalyze the reaction of substrates to synthesize glutathione.

[0068] The glutathione synthesis method provided by this invention has a high substrate conversion rate, yields a product with low impurity content, and is easy to purify.

[0069] The method of this invention uses yeast spore microcapsules to immobilize enzymes and catalyze the reaction, generating a glutathione concentration of up to 27.7 g / L with a substrate conversion rate of over 90% and a 90% reduction in exogenous protein impurities in the reaction solution, which can meet the requirements of new process technology standards for pharmaceutical raw materials. The immobilized enzymes can be reused more than 7 times, and the generated glutathione concentration remains stable.

[0070] In one specific embodiment of the present invention, the method for preparing the Saccharomyces cerevisiae haploid ascospore immobilization enzyme used in the synthesis method provided by the present invention includes the following steps:

[0071] Screen a diploid yeast strain that has a fast production rate and strong sporulation capacity.

[0072] The preferred yeast strain is Saccharomyces cerevisiae FX-2. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on August 1, 2016, with accession number CCTCC NO:M2016418. This strain has been described in the patent publication text with publication number CN108220175A.

[0073] Diploid yeast was cultured in liquid medium for 20 hours, centrifuged to collect cells, washed three times with deionized sterile water, added to spore-forming liquid medium and cultured for 24-28 hours. Tetradical ascospores were selected under a microscope, and a sufficient number of haploids were obtained by dissecting the ascospores with a microneedle to obtain a sufficient number of α / α type haploid cells.

[0074] Using the principle of homologous recombination, primers for knocking out the yeast β-glucan synthesis gene were designed. Using a plasmid carrying the resistance gene as a template, PCR amplification was performed to obtain the PCR product carrying the resistance gene, i.e., the gene knockout fragment. The gene knockout fragment was concentrated by ethanol precipitation. The PCR product was transformed into multiple haploid strains using the lithium acetate conversion method. The bacterial culture was then cultured in a resistance solid medium, and positive mutant strains with good growth were selected.

[0075] Preferably, a homologous recombination knockout primer for the yeast glucan synthase gene KRE1 is designed. Using a plasmid with an resistance selection marker as a template, the amplified product carrying the resistance gene is obtained by PCR amplification. Then, the amplified PCR product is transformed into haploid yeast cells using the lithium acetate transformation method. After resistance selection, successfully transformed haploid spores are selected.

[0076] PCR amplification primers were designed separately, and yeast DNA was used as a template to amplify the promoters, secretory signal peptides, expression genes, and terminators of the GSH1, GSH2, and pyruvate kinase genes, respectively. Then, restriction enzymes and DNA ligase were used to construct gene plasmids with resistance selection markers, which were then transformed into the haploid spores mentioned above.

[0077] In one embodiment of the present invention, the promoter and terminator of the GSH1, GSH2 or pyruvate kinase gene in the strain that provides the GSH1, GSH2 or pyruvate kinase gene are used as the promoter and terminator in the present invention.

[0078] Preferably, the secretory signal peptide is selected from one or more combinations of MFα1, SUC2, PHO5, and INU.

[0079] Then, haploid cells were hybridized and fused to obtain diploids with high expression activity of GSH1, GSH2, and pyruvate kinase genes. Diploid mutant strains that could produce spores were selected as the production strains for immobilized enzymes.

[0080] The enzyme-producing strain was cultured in a seed culture medium. After the seed culture was completed, it was inoculated into a fermentation culture medium. Through fermentation, GSH1, GSH2, and pyruvate kinase were expressed and accumulated in the yeast cells. After the fermentation culture was completed, the cells were collected by centrifugation, washed with deionized sterile water to remove impurities from the fermentation broth, and the collected cells were added to a sporulation culture medium. The culture was terminated when the yeast sporulation rate reached more than 70%.

[0081] Preferably, the sporulating yeast is separated by centrifugation, washed with deionized sterile water to remove salt and impurities from the sporulation medium. The cell walls are disrupted using snail enzyme to release the yeast spores. After centrifugation, the cells are washed with deionized sterile water to remove yeast cell wall fragments and impurities. The resulting microcapsules contain GSH1, GSH2, and pyruvate kinase immobilized on the yeast spore walls and then freeze-dried.

[0082] In one specific embodiment of the present invention, the method for synthesizing glutathione includes the following steps:

[0083] 1) Construct a diploid Saccharomyces cerevisiae strain with sporulation ability, characterized by knockout of the yeast cell wall β-glucan synthesis gene, high expression of γ-glutamylcysteine ​​synthase, glutathione synthase and pyruvate kinase on the spore surface.

[0084] 2) Haploid spores of Saccharomyces cerevisiae were obtained by fermentation culture and used as microencapsulated immobilization enzymes for glutathione synthesis.

[0085] 3) Using yeast spore microcapsule-immobilized enzymes as catalysts, an in-situ ATP regeneration reaction system with phosphoenolpyruvate (PEP) as the ATP energy donor was constructed.

[0086] 4) Catalytic synthesis of glutathione.

[0087] 5) Separate, purify and dry to obtain glutathione.

[0088] Preferably, glutamate, cysteine, and glycine are used as substrates, and GSH1 and GSH2 are used as glutathione synthases. Glutathione is generated through a two-step reaction. In the first step, glutamate and cysteine ​​consume one molecule of ATP under the action of GSH1 enzyme to generate gamma-glutamylcysteine ​​and ADP. In the second step, gamma-glutamylcysteine ​​and glycine consume one molecule of ATP under the action of GSH2 enzyme to obtain glutathione and ADP. An in-situ ATP regeneration system is composed of PEP, pyruvate kinase, potassium ions, and magnesium ions. That is, PEP is catalyzed by pyruvate kinase to transfer the phosphate group originally attached to the oxygen atom to ADP, thereby generating ATP and pyruvate. This step of the reaction requires metal ions such as magnesium and potassium as catalysts.

[0089] Preferably, after the catalytic synthesis of glutathione is completed, the immobilized enzyme is recovered by centrifugation. The recovered immobilized enzyme is washed multiple times with deionized sterile water, centrifuged, freeze-dried, and then reused.

[0090] Preferably, glutathione is purified using a cation exchange resin. The main mechanism is that glutathione carries a positive charge under acidic conditions and is relatively stable under acidic conditions. After glutathione is exchanged and adsorbed using the cation exchange resin, it is then eluted with sodium chloride using a gradient to obtain purified glutathione, removing unreacted amino acids, enzymes, inorganic salts, and other impurities. To further improve the purity of glutathione, it needs to be concentrated and then crystallized with ethanol. The concentration includes two steps: membrane concentration and vacuum evaporation concentration. Membrane concentration is used to remove a large amount of salt, while vacuum evaporation concentration is used to obtain a higher glutathione concentration. Glutathione is easily oxidized in an aerobic environment; therefore, an anaerobic environment must be maintained in the glutathione purification process.

[0091] The sources of the reagents and instruments used in the embodiments of this invention are shown in Table 1 below.

[0092] Table 1. Information on the Sources of Reagents and Instruments

[0093] Reagents / Instruments Manufacturer for sale DNA Gel Recovery Kit Sangon Biotech (Shanghai) Co., Ltd. Plasmid DNA Extraction Kit Sangon Biotech (Shanghai) Co., Ltd. PCR instrument Eppendorf, Germany PCR product recovery kit Sangon Biotech (Shanghai) Co., Ltd. 1*PBS buffer Sangon Biotech (Shanghai) Co., Ltd. T4 DNA ligase Takara Bio Engineering (Dalian) Co., Ltd. optical microscope General Electric Electric thermostatic incubator Shanghai Sanfa Scientific Instruments Co., Ltd. Lithium acetate Sangon Biotech (Shanghai) Co., Ltd. DNA ligase Takara Bio Engineering (Dalian) Co., Ltd. snail enzyme Sangon Biotech (Shanghai) Co., Ltd. freeze dryer EYELA Japan glutamic acid Sinopharm Chemical Reagent Co., Ltd. Cysteine Sinopharm Chemical Reagent Co., Ltd. glycine Sinopharm Chemical Reagent Co., Ltd. Sodium chloride Sinopharm Chemical Reagent Co., Ltd. High-speed refrigerated centrifuge Hitachi, Japan Cation exchange resin D001 Shanghai Yuanye Biotechnology Co., Ltd.

[0094] Example 1: Preparation of haploid spores of Saccharomyces cerevisiae FX-2 and knockout of the β-glucan synthesis gene KRE1

[0095] Saccharomyces cerevisiae FX-2 was cultured in liquid YPD medium at 30°C in shake flasks for 12 hours. The cells were collected by centrifugation, washed three times with deionized sterile water, and then diluted and transferred to basic sporulation medium. The cells were then cultured at 30°C for 18-24 hours, and sporulation was observed under a microscope. The basic sporulation medium consisted of 10g potassium acetate and 20g agar per 1000ml of distilled water.

[0096] For the method of preparing haploid spores, refer to the second edition of "Guide to the Use of Yeast Genetic Methods".

[0097] The haploid yeast strains were inoculated into YPD liquid medium and cultured overnight at 30°C with shaking.

[0098] Using plasmid pFA6a-kanMX6 as a template, KRE1 knockout primers were designed as follows:

[0099] The upstream primer sequence for KRE1 is shown in SEQ ID NO.1:

[0100]

[0101] The downstream primer sequence for KRE1 is shown in SEQ ID NO.2:

[0102]

[0103] The PCR product of the knockout gene fragment was amplified using plasmid pFA6a-kanMX6 as a template. The reaction conditions were as follows: 98℃ for 2 min, step 1 at 98℃ for 20 s, step 2 at 56℃ for 30 s, step 3 at 68℃ for 105 s, and step 4 at 68℃ for 2 min. The first three steps were repeated for 35 cycles. After the PCR reaction, the PCR product was detected by electrophoresis, and the target band, consistent with the amplified fragment size, was recovered and stored at low temperature for later use.

[0104] Take 500 μL of haploid yeast culture, centrifuge for 1 min, collect the cells, wash once with sterile water, centrifuge to remove the supernatant, resuspend the cells in 1 mL of lithium acetate (LiAC) (0.1 mol·L⁻¹), centrifuge for 30 s, and then completely remove the supernatant with a pipette; add 50 μL of lithium acetate (LiAC) (0.1 mol·L⁻¹) to resuspend the cells; pipette 50 μL of the resuspended solution into a new 1.5 mL centrifuge tube, and then add the following reagent solutions in sequence: 240 μL 50% polyethylene glycol, 16 μL 2 mol·L⁻¹ lithium acetate (LiAC), 5 μL pre-denatured salmon sperm DNA, 50 μL… The PCR amplification product was recovered and mixed by vortexing. The mixture was placed in a metal bath heater and reacted at 30°C for 30 min and then at 42°C for 20 min. After centrifugation for 1 min, the supernatant was completely removed by pipette. The bacterial cells were then resuspended in 100 μL of sterile water and spread onto kanamycin culture plates. The plates were inverted and incubated in a 30°C incubator until single colonies were visible to the naked eye. Once the single colonies had grown to a suitable size, several single colonies were streaked onto kanamycin culture plates and incubated in a 30°C incubator for 2–3 days. After incubation, the plates were stored at 4°C.

[0105] Kanamycin culture medium components: Add 10 parts Angel yeast extract FM888, 20 parts tryptone, 5 parts sodium chloride, 2 parts agar powder, and 0.1 parts kanamycin to every 1000 parts of sterile water.

[0106] Using the upstream and downstream primers of KRE1 as primers, PCR verification was performed using the obtained target strain and Saccharomyces cerevisiae FX-2 as templates, respectively. The results are as follows: Figure 1 As shown. Figure 1 Lane FX-2 represents the amplification results using *Saccharomyces cerevisiae* FX-2 as a template, and lane △KRE1 represents the amplification results using the target strain prepared above as a template. Figure 1It can be seen that the Saccharomyces cerevisiae FX-2 strain contains the KRE1 gene, while the KRE1 gene of the target strain prepared above has been successfully knocked out.

[0107] Example 2: Construction of haploid mutant strains

[0108] Saccharomyces cerevisiae FX-2 strain was cultured in YPD liquid medium and shake-cultured at 30°C for 18 hours. The cells were then separated by centrifugation, washed, and yeast DNA was extracted. For specific methods, please refer to the second edition of "Experimental Guide to Yeast Genetics Methods".

[0109] Using Saccharomyces cerevisiae FX-2 strain DNA as a template, secreting signal peptide MFα1, PCR amplification primers for GSH1, GSH2 and PYK2, as well as upstream and downstream primers for the secretion signal peptide were designed. The promoter of each gene is the 2000bp upstream region of each gene, and the terminator of each gene is the 500bp downstream region of each gene, as detailed below.

[0110] The upstream primer sequence HY0 for the GSH1 promoter region is shown in SEQ ID NO.3:

[0111]

[0112] The downstream primer sequence HY1 for the GSH1 promoter region is shown in SEQ ID NO.4:

[0113]

[0114] The upstream primer sequence HY2 for the GSH1 secretion signal peptide MFα1 is shown in SEQ ID NO. 5:

[0115]

[0116] The downstream primer sequence HY3 for the GSH1 secretion signal peptide MFα1 is shown in SEQ ID NO. 6:

[0117]

[0118] The upstream primer sequence HY4 for GSH1 is shown in SEQ ID NO.7:

[0119]

[0120] The downstream primer sequence HY5 for GSH1 is shown in SEQ ID NO. 8:

[0121]

[0122] The upstream primer sequence HY6 for the GSH2 promoter region is shown in SEQ ID NO. 9:

[0123]

[0124] The downstream primer sequence HY7 for the GSH2 promoter region is shown in SEQ ID NO.10:

[0125]

[0126] The upstream primer sequence HY8 for the GSH2 secretion signal peptide MFα1 is shown in SEQ ID NO.11:

[0127]

[0128] The downstream primer sequence HY9 for the GSH2 secretion signal peptide MFα1 is shown in SEQ ID NO.12:

[0129]

[0130] The upstream primer sequence HY10 for GSH2 is shown in SEQ ID NO.13:

[0131]

[0132] The downstream primer sequence HY11 for GSH2 is shown in SEQ ID NO.14:

[0133]

[0134] The upstream primer sequence HY12 for the PKY2 promoter region is shown in SEQ ID NO.15:

[0135]

[0136] The downstream primer sequence HY13 for the PKY2 promoter region is shown in SEQ ID NO.16:

[0137]

[0138] The upstream primer sequence HY14 for the PYK2 signal peptide MFα1 is shown in SEQ ID NO.17:

[0139]

[0140] The downstream primer sequence HY15 for the PYK2 signal peptide MFα1 is shown in SEQ ID NO.18:

[0141]

[0142] The upstream primer sequence HY16 for PYK2 is shown in SEQ ID NO.19:

[0143]

[0144] The downstream primer sequence HY17 for PYK2 is shown in SEQ ID NO.20:

[0145]

[0146] Using *Saccharomyces cerevisiae* strain FX-2 DNA as a template, primers HY0-HY17 were added to the PCR reaction system. The reaction conditions were as follows: 98℃ for 2 min, first step 98℃ for 20 s, second step 56℃ for 30 s, third step 68℃ for 6 min, and fourth step 68℃ for 6 min. The first to third steps were repeated for 35 cycles. After the PCR reaction, the PCR product was detected by electrophoresis. If the amplified fragment size matched the target band, the PCR product was purified and recovered, and stored at low temperature for later use. The target PCR product sequence is shown in SEQ ID NO. 21.

[0147]

[0148]

[0149]

[0150] The GSH1 gene sequence is shown in SEQ ID NO.22:

[0151]

[0152]

[0153] The GSH2 gene sequence is shown in SEQ ID NO.23:

[0154]

[0155]

[0156] The PYK2 gene sequence is shown in SEQ ID NO.24:

[0157]

[0158]

[0159] Constructing transformation plasmids:

[0160] The restriction enzyme digestion reaction system for the PCR product was as follows: 8 μL of PCR amplified, purified, and recovered product, 2 μL of digestion buffer, 0.5 μL of restriction enzyme SCAI, 0.5 μL of restriction enzyme AseI, and 20 μL of sterile water. The digestion reaction was carried out at 37°C for 3 hours, followed by gel electrophoresis for recovery.

[0161] Using plasmid PBR322 as the expression vector, enzyme digestion was performed. The reaction system was as follows: 2 μL vector, 2 μL enzyme digestion buffer, 0.5 μL restriction enzyme SCAI, 0.5 μL restriction enzyme AseI, and 16 μL sterile water. The enzyme digestion reaction was carried out at 37°C for 3 h, and then electrophoresis was performed for gel recovery.

[0162] The target gene was ligated to the enzyme-digested vector to obtain the expression plasmid vector. The reaction system was as follows: 2.5 μL of the enzyme-digested target gene fragment, 1 μL of the enzyme-digested vector, and 3.5 μL of ligase (Ligation Mighty Mix).

[0163] The obtained plasmid map is as follows Figure 2 As shown.

[0164] The transforming plasmid was transferred into the KRE1 gene knockout haploid yeast competent cells prepared in Example 1. The cells were first cultured on tetracycline plates for 28 hours. After colonies grew, they were selected and selectively inoculated at the same location using ampicillin agar plates and tetracycline agar plates, respectively. Strains that could grow on tetracycline agar but not on ampicillin agar were identified as target strains. Further selection and purification were performed. Using HY0 and HY17 as primers, the plasmid extracted from the target strain was used as a template for electrophoresis to verify whether the plasmid containing the target gene had been transferred into the *Saccharomyces cerevisiae* cells. The results are as follows: Figure 3 As shown, from Figure 3 It can be seen that the plasmid containing the target gene has been successfully transferred into the Saccharomyces cerevisiae cells.

[0165] The obtained strain was a haploid yeast with the KRE1 gene, which synthesizes cell wall glucan, knocked out, and containing a recombinant vector expressing γ-glutamylcysteine ​​synthase, glutathione synthase, and pyruvate kinase.

[0166] Example 3: Preparation of enzymes immobilized in yeast spore microcapsules

[0167] The haploid yeast spores obtained in Example 2 were hybridized and fused. The experimental method was based on the second edition of "Experimental Guide to Yeast Genetics Methods". A diploid mutant strain with good sporulation ability was selected as the target sporulation strain.

[0168] The obtained strain was cultured in a seed culture medium at pH 6.0 and 30°C for 15-30 hours. The seed culture medium consisted of 3g sucrose, 2g yeast extract, 0.1g magnesium sulfate, and 0.1g potassium dihydrogen phosphate per 100mL of water.

[0169] After seed culture, the yeast was inoculated into the fermentation medium at an inoculation rate of 5 wt%. The fermentation pH was 5.0-6.0, the temperature was 30℃, the aeration rate was 0.5-1.5 vvm, and the culture time was 20-25 hours. The yeast fermentation medium composition was as follows: molasses and ammonia were added during fermentation. For every 100 kg of water, 0.02 kg of ammonium dihydrogen phosphate, 2 kg of yeast extract, 0.02 kg of magnesium sulfate, and 0.01 kg of zinc sulfate were added. The flow rate of 30 wt% molasses was 1-3 kg / hour, and the flow rate of ammonia was 0.1-0.3 kg / hour.

[0170] After fermentation, the cells were collected by centrifugation, washed three times with deionized sterile water, and added to a sporulation medium. The pH was controlled at 5.0-6.0, the temperature at 30℃, and the culture was carried out for 28 hours to achieve a yeast sporulation rate of over 70%. The yeast sporulation medium consisted of: 1 kg of yeast extract, 2 kg of yeast peptone, 0.02 kg of glucose, and 1 kg of potassium acetate per 100 kg of water.

[0171] After the sporulation culture was completed, the cells were centrifuged, washed three times with deionized sterile water, and the dry matter content was controlled at about 15%. Snail enzyme (5‰) was added to break the cell wall. The pH was controlled at 6-7, the temperature was 37℃, and the cells were kept at this temperature for 1 hour. After centrifugation, the cells were washed several times with deionized sterile water to obtain free spores. The spores were then freeze-dried to obtain yeast spore microcapsules with enzyme immobilization.

[0172] The catalytic activity of enzymes immobilized in yeast spore microcapsules was detected using the method shown below.

[0173] Validation of yeast spore microcapsule-immobilized enzyme: Under laboratory conditions, 0.1 g of glutamic acid, 0.05 g of L-cysteine, 0.05 g of glycine, 0.05 g of magnesium chloride, 0.05 g of potassium chloride, 0.1 g of ATP, 0.2 g of PEP, and 5 g of yeast spore microcapsule-immobilized enzyme were dissolved in water and brought to a final volume of 100 ml. The reaction was carried out at 25 °C and pH 6.5 for 3 hours. The glutathione concentration was measured every 20 minutes during the reaction. The results are shown in Table 2 below.

[0174] Table 2

[0175] Enzymatic reaction process reaction time min Glutathione concentration (g / L) Sample 1 20 0.44 Sample 2 40 0.78 Sample 3 60 0.98 Sample 4 80 1.13 Sample 5 100 1.22 Sample 6 120 1.27 Sample 7 140 1.27 Sample 8 160 1.27 Sample 9 180 1.27

[0176] The detection of the substrate conversion rate (calculated based on cysteine) reaches 96.3%, indicating that the immobilized enzyme in the yeast spore microcapsules prepared has a high substrate conversion rate. The normal production requirement is that the substrate conversion rate (calculated based on cysteine) of the immobilized enzyme in the yeast spore microcapsules prepared should reach more than 90% to be qualified.

[0177] Example 4: Synthesis of glutathione by immobilized enzyme in yeast spore microcapsules

[0178] 1) Reaction with low-concentration substrates: In the reaction system, control the concentration of glutamic acid to be 0.1%, the concentration of L-cysteine to be 0.05%, the concentration of glycine to be 0.05%, the concentration of magnesium chloride to be 0.05%, the concentration of potassium chloride to be 0.05%, the concentration of ATP to be 0.1%, the concentration of PEP to be 0.2%, and the addition amount of the immobilized enzyme in yeast spore microcapsules to be 5%. Mix well, control the temperature at 25°C, pH 6.5, catalyze the synthesis of glutathione, and the reaction time is 2 hours. Finally, the concentration of glutathione obtained is 1.2 g / L, and the substrate conversion rate (calculated based on cysteine) reaches 95%.

[0179] 2) Reaction with high-concentration substrates: In the reaction system, control the concentration of glutamic acid to be 2%, the concentration of L-cysteine to be 1.2%, the concentration of glycine to be 1.2%, the concentration of magnesium chloride to be 0.2%, the concentration of potassium chloride to be 0.2%, the concentration of ATP to be 0.3%, the concentration of PEP to be 0.6%, and the addition amount of the immobilized enzyme in yeast spore microcapsules to be 10%. Mix well, control the temperature at 30°C, pH 7.5, catalyze the synthesis of glutathione, and the reaction time is 5 hours. Finally, the concentration of glutathione obtained is 27.7 g / L, and the substrate conversion rate (calculated based on cysteine) reaches 91%.

[0180] 3) Recovery and reuse of the immobilized enzyme in yeast spore microcapsules: After the above high-concentration substrate reaction, centrifuge and separate to collect the heavy phase, wash it 2 - 3 times with deionized sterile water, and freeze-dry after centrifugation to obtain the recovered immobilized enzyme in yeast spore microcapsules. Add it again to the above high-concentration substrate reaction system for reaction and then recover. After recycling 7 batches, the concentration of glutathione generated still reaches more than 25 g / L, indicating that the enzyme activity remains stable.

[0181] Example 5: Refinement of glutathione

[0182] After the high-concentration substrate reaction in Example 4 was completed, the mixture was centrifuged. The enzyme immobilized in yeast spore microcapsules was recovered in the heavy phase, while the light phase contained glutathione. The pH was adjusted to 3.0 with hydrochloric acid, and glutathione was adsorbed onto cation exchange resin D001. A gradient elution was performed using 0%, 1.5%, and 3% sodium chloride solutions, and the purified glutathione was collected in fractions. The glutathione was concentrated using a 150D nanofiltration membrane to a concentration of 84 g / L, and washed seven times with purified water. The glutathione was then concentrated by vacuum evaporation at 50°C and a vacuum of 0.08-0.09 MPa to a concentration of 415 g / L. An equal volume of 75% ethanol was added, and crystallization was carried out in a low-temperature anaerobic environment at 4°C. The crystals were collected by centrifugation and dried under vacuum at 50°C to constant weight to obtain high-purity reduced glutathione. The purity of glutathione was determined according to the content determination method in the national drug standard for glutathione, with approval number XGB2020-021. The purity was found to be 99.8%.

Claims

1. A glutathione synthesis immobilization enzyme, characterized in that, The glutathione synthesis immobilization enzyme comprises the following components: using haploid ascospores of *Saccharomyces cerevisiae* as the immobilization carrier, γ-glutamylcysteine ​​synthase, glutathione synthase, and pyruvate kinase are immobilized. The haploid ascospores of *Saccharomyces cerevisiae* are obtained from wild-type *Saccharomyces cerevisiae* strains with one or more combinations of cell wall β-glucan synthesis genes knocked out or inactivated, and capable of co-expressing the GSH1, GSH2, and PYK1 genes. The cell wall β-glucan synthesis genes are one or more combinations of KRE1, KRE6, and KRE9.

2. The glutathione synthesis immobilization enzyme according to claim 1, wherein, The wild yeast strain is Saccharomyces cerevisiae FX-2 ( Saccharomyces cerevisiae d FX-2), with accession number CCTCC NO: M2016418.

3. The glutathione synthesis immobilization enzyme according to claim 1, wherein, The cell wall β-glucan synthesis gene is KRE1.

4. The glutathione synthesis immobilization enzyme according to any one of claims 1-3, wherein, The co-expression of GSH1, GSH2 and PYK1 genes in the haploid ascospores of *Saccharomyces cerevisiae* is achieved by the following steps: GSH1, GSH2 and PYK1 are respectively constructed into transformation plasmids, secretion signal peptide genes are inserted, and then transformed and expressed in the same haploid yeast spores. Alternatively, GSH1, GSH2, and PKY1 can be expressed in the same plasmid, and secretion signal peptides can be inserted into them to obtain recombinant plasmids. The recombinant plasmids can then be transformed into yeast haploid spores to form transformant spores.

5. The glutathione synthase according to claim 4, wherein, The secretory signal peptide gene is one or a combination of two or more of MFα1, SUC2, PHO5 and INU.

6. The glutathione synthase according to claim 4, wherein, The secretory signal peptide gene is MFα1.

7. The glutathione synthesis immobilization enzyme according to claim 4, wherein, The recombinant plasmid contains the base sequences described in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24, or contains the base sequence described in SEQ ID NO.

21.

8. The glutathione synthesis immobilization enzyme according to claim 5, wherein, The recombinant plasmid contains the base sequences described in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24, or contains the base sequence described in SEQ ID NO.

21.

9. The glutathione synthesis immobilization enzyme according to claim 6, wherein, The recombinant plasmid contains the base sequences described in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24, or contains the base sequence described in SEQ ID NO.

21.

10. A method for preparing glutathione, characterized in that, The preparation method comprises synthesizing glutathione using the glutathione synthesis immobilization enzyme described in any one of claims 1-9 to catalyze a substrate reaction, with the phosphoenolpyruvate ATP in situ regeneration reaction system as the energy supply system.

11. The method according to claim 10, characterized in that, Includes the following steps: (1) Prepare a reaction solution by mixing glutamic acid, L-cysteine, glycine, magnesium chloride, potassium chloride, ATP and PEP, add glutathione to synthesize immobilized enzyme, and stir to react; (2) After the reaction is completed, the reaction solution is separated, the fixed enzyme is recovered, and the supernatant is extracted and purified to obtain glutathione.

12. The method according to claim 11, characterized in that, In step (1), the concentrations of glutamate, L-cysteine, glycine, magnesium chloride, potassium chloride, ATP, PEP, and glutathione synthesis immobilization enzyme are 0.1-0.3 wt%, 0.2-0.6 wt%, and 5-10 wt%.

13. The method according to claim 11, characterized in that, In step (1), the catalytic reaction temperature is 20-30℃.

14. The method according to claim 12, wherein, In step (1), the catalytic reaction temperature is 20-30℃.

15. The method according to claim 11, wherein, In step (1), the pH value of the catalytic reaction is 6.5-7.

5.

16. The method according to claim 12, wherein, In step (1), the pH value of the catalytic reaction is 6.5-7.

5.

17. The method according to claim 13, wherein, In step (1), the pH value of the catalytic reaction is 6.5-7.

5.

18. The method according to claim 14, wherein, In step (1), the pH value of the catalytic reaction is 6.5-7.

5.

19. The method according to claim 11, wherein, In step (1), the catalytic reaction time is 2-5 hours.

20. The method according to any one of claims 11-19, characterized in that, In step (2), the refining process includes: 1) Adjust the pH to 2.5-3.0; 2) Cation exchange resins adsorb glutathione through exchange; 3) Gradient elution with 0-3% sodium chloride solution to collect glutathione in fractions; 4) Concentrate glutathione using nanofiltration membrane to achieve a glutathione concentration of 80-100 g / L, and wash with purified water after concentration. 5) Reduced pressure evaporation and concentration to achieve a glutathione concentration of 300-450 g / L; 6) Crystallize and dry to obtain reduced glutathione.

21. The method according to claim 20, characterized in that, In step 4) of the refining process, the nanofiltration membrane has a pore size of 100-200D.

22. The method according to claim 21, wherein, The nanofiltration membrane has a pore size of 150D.

23. The method of claim 20, wherein, In step 5) of the refining process, the temperature of the reduced pressure evaporation concentration is 45-55℃ and the vacuum degree is 0.08-0.09MPa.

24. The method according to claim 21, wherein, In step 5) of the refining process, the temperature of the reduced pressure evaporation concentration is 45-55℃ and the vacuum degree is 0.08-0.09MPa.

25. The method according to claim 20, wherein, In step 6) of the refining process, an equal volume of 75-80% ethanol is added, and crystallization is carried out in a low-temperature, oxygen-free environment at 4-8°C.

26. The method according to claim 21, wherein, In step 6) of the refining process, an equal volume of 75-80% ethanol is added, and crystallization is carried out in a low-temperature, oxygen-free environment at 4-8°C.

27. The method according to claim 23, wherein, In step 6) of the refining process, an equal volume of 75-80% ethanol is added, and crystallization is carried out in a low-temperature, oxygen-free environment at 4-8°C.

28. The method according to claim 20, wherein, In step 6) of the refining process, the obtained crystals are vacuum dried at 50-60°C.

29. The method according to claim 21, wherein, In step 6) of the refining process, the obtained crystals are vacuum dried at 50-60°C.

30. The method according to claim 23, wherein, In step 6) of the refining process, the obtained crystals are vacuum dried at 50-60°C.

31. The method according to claim 25, wherein, In step 6) of the refining process, the obtained crystals are vacuum dried at 50-60°C.

32. The method according to claim 26, wherein, In step 6) of the refining process, the obtained crystals are vacuum dried at 50-60°C.

33. The method according to claim 27, wherein, In step 6) of the refining process, the obtained crystals are vacuum dried at 50-60°C.

34. The use of the glutathione synthesis immobilization enzyme according to any one of claims 1-9 in the preparation of glutathione.

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