NL2038973A

NL2038973A - Bi-functional glutathione synthase mutant and application thereof

Info

Publication number: NL2038973A
Application number: NL2038973A
Authority: NL
Inventors: Ma Chengwei; Jin Xiang; Cui Mengfei
Original assignee: Shaanxi Lifewe Bioengineering Co Ltd; Beijing Lifewe Biotechnology Inst Co Ltd; Gansu Lifewe Bioengineering Co Ltd
Priority date: 2024-05-21
Filing date: 2024-10-31
Publication date: 2024-12-17
Also published as: CN118207172A; CN118207172B

Abstract

The present invention discloses a bi-functional glutathione synthase mutant and application thereof to the production of glutathione. Based on streptococcus sanguis- 5 derived bi-functional glutathione synthase GshF, a mutant with higher enzymatic activity is obtained by a site-directed mutation technology. The mutant can reduce product inhibition and increase the production output and transformation rate of the glutathione, thereby effectively solving the problems of low yield, low transformation rate and the like in the production process of the glutathione by an enzymatic synthesis method and 10 having great significance in the large-scale and high-yield production of the glutathione.

Description

BI-FUNCTIONAL GLUTATHIONE SYNTHASE MUTANT AND

APPLICATION THEREOF

TECHNICAL FIELD

[0001] The present invention relates to the technical fields of protein engineering and enzyme catalysis, and in particular, to a glutathione synthase mutant and application thereof to the production of glutathione.

BACKGROUND

[0002] Glutathione (y-glutamyl-L-cysteinyl-glycine, GSH) is a tripeptide containing y- amido bond and sulfydryl, including glutamic acid, cysteine and glycine, and is the richest non-protein thiol compound in eukaryocyte and prokaryote. GSH has numerous important physiological functions, can directly or indirectly participate in life activities such as synthesis of DNA and protein and transport of amino acid, can help maintain the normal immune system function, and has the effects of antioxidation and integrated detoxification. Clinically, GSH has been successfully applied to the treatment of various diseases such as cancer, Alzheimer's disease, diabetes, AIDS and hepatopathy.

Additionally, due to the important physiological functions such as antioxidation, GSH is also widely applied in the industries of food, cosmetics and the like.

[0003] Although the demand for GSH keeps on increasing, the industrial production efficiency of GSH is difficult to improve, besides, due to the high price, the application and development in the fields of medicine, food and the like are limited. Therefore, it is urgent to develop a production method with high production efficiency and low cost.

[0004] Currently, GSH is primarily produced by a solvent extraction method, a chemical synthesis method, a microbiological fermentation method and an enzymatic method. The solvent extraction is to extract GSH from the raw materials of animal and plant tissues or yeast, however, this method has the disadvantages of high cost, low product yield, organic solvent contamination and the like, thereby having gradually become obsolete. The synthesis of GSH by the chemical synthesis method is limited due to problems of difficult separation of active products, long production time, high raw material cost, high pollution and the like. The microbiological fermentation method is currently a main method for the industrial production of GSH. By this method, the cheap raw material is transformed into GSH via microbial cells by utilizing the autophage pathway of the glutathione production strains. Among various microbial fermentation methods, the yeast fermentation process is relatively mature; however, this process still has the problems of low production output, complex downstream process, product intracellular inhibition and the like, and is difficult to fully meet the market demand.

[0005] GSH production by the enzymatic method is currently the focus of research.

This method is to complete the synthesis of GSH in the presence of Mg?" and the ongoing energy supply by ATP under the in vitro condition, by taking the glutathione synthetase system as the catalyst, adding three amino acids such as glutamic acid, cysteine and glycine or salt corresponding thereto into a proper buffer system as a substrate. The GSH production by the traditional enzymatic method includes two steps of reaction respectively catalyzed by y-glutamyl cysteine synthetase (y-GCS) and glutathione synthetase (GS). Due to the poor activity of y-GCS, the glutamyl cysteine synthesis reaction catalyzed by y-GCS becomes the rate-limiting step in GSH synthesis, resulting in that the GSH production output is always difficult to increase.

[0006] Recently, it has been discovered that some gram-positive bacteria-derived glutathione synthase GshFs have bi-catalytic functions of y-GCS and GS, and can produce GSH through in vitro one-step enzymatic catalysis. This catalysis process can be completed only by the substrate of the three amino acids, Mg** and ATP, and has advantages of high transformation efficiency, strong specifity, mild reaction condition, simple operation, convenient purification, low cost and the like, thereby being expectable to achieve high-yield and high-efficiency production of GSH. However, the existing method for synthesizing GSH through one-step catalysis enzymatic method of

GshF still has the problems of low enzymatic activity, low production output, low transformation rate and the like, and still cannot meet the requirements of the industrial production of GSH. It is of great significance to increase GSH production output by molecular modification of the key enzyme-glutathione synthase to improve the enzymatic activity thereof.

SUMMARY

[0007] To increase the output and substrate transformation rate in the process of producing GSH by enzymatic catalysis, according to the present invention, mutation and screening are performed to streptococcus sanguinis-derived bi-functional glutathione synthase GshF sourced to obtain a GshF mutant that has high enzymatic activity to catalyze glutamic acid, cysteine, glycine and ATP to synthesize glutathione (as referred to hereinafter, enzymatic activity, reactive enzymatic activity, enzymatic function, catalytic function or similar expressions with the same meaning, unless otherwise specified, all refer to the activity or function that catalyzes glutamic acid, cysteine, glycine and ATP to synthesize glutathione).

[0008] An objective of the present invention is to provide a bi-functional glutathione synthase mutant and application thereof to the production of GSH.

[0009] To achieve the objective of the present invention, according to a first aspect, the present invention provides a bi-functional glutathione synthase mutant. The mutant includes the following amino acid sequences:

[0010] 1) mutation from R to A or E or L of amino acid at the 184th site in the amino acid sequence shown in SEQ ID NO:1 of the streptococcus sanguinis-derived bi- functional glutathione synthase;

[0011] 2) mutation from N to F or Y of amino acid at the 215th site in the amino acid sequence shown in SEQ ID NO:1 of the streptococcus sanguinis-derived bi-functional glutathione synthase;

[0012] 3) mutation from S to T of amino acid at the 216th site in the amino acid sequence shown in SEQ ID NO:1 of the streptococcus sanguinis-derived bi-functional glutathione synthase;

[0013] 4) mutation from D to H of amino acid at the 217th site in the amino acid sequence shown in SEQ ID NO:1 of the streptococcus sanguinis-derived bi-functional glutathione synthase;

[0014] 5) mutation from H to F of amino acid at the 325th site in the amino acid sequence shown in SEQ ID NO:1 of the streptococcus sanguinis-derived bi-functional glutathione synthase;

[0015] 6) mutation from N to I or L or V of amino acid at the 328th site in the amino acid sequence shown in SEQ ID NO:1 of the streptococcus sanguinis-derived bi- functional glutathione synthase; and

[0016] 7) mutation from Q to D of amino acid at the 329th site in the amino acid sequence shown in SEQ ID NO:1 of the streptococcus sanguinis-derived bi-functional glutathione synthase.

[0017] According to a second aspect, the present invention provides a nucleic acid molecule encoding the glutathione synthase mutant.

[0018] According to a third aspect, the present invention provides a biomaterial containing the nucleic acid molecule. The biomaterial includes, but is not limited to recombinant DNA, an expression cassette, a transposon, a plasmid vector, a virus vector or an engineered bacterium.

[0019] According to a fourth aspect, the present invention provides a recombinant microorganism. The recombinant microorganism is constructed by introducing the nucleic acid molecule encoding the glutathione synthase mutant into the microorganism via plasmid or by integrating the same to the chromosome of the microorganism by means of genetic engineering.

[0020] According to a fifth aspect, the present invention provides application of the glutathione synthase mutant or the nucleic acid molecule or the biomaterial or the recombinant microorganism to the production of GSH.

[0021] According to a sixth aspect, the present invention provides a preparation method for GSH. The method uses the bi-functional glutathione synthase mutant.

[0022] The preparation method includes the following steps:

[0023] (1) culturing the recombinant microorganism expressing the glutathione synthase mutant;

[0024] (2) performing cell rupture treatment on the cultured bacterial cells, the ruptured cell solution obtained therefrom being a reactive enzyme solution containing the glutathione synthase mutant; and

[0025] (3) using the reactive enzyme solution to catalyze the reaction of glutamic acid, cysteine, glycine and ATP to synthesize the glutathione.

[0026] Further, the enzymatic catalytic reaction system in step (3) includes: the glutamic acid, the cysteine, the glycine, the ATP, magnesium chloride, and the reactive 5 enzyme solution containing the glutathione synthase mutant.

[0027] Furthermore, each 1 litre of the enzymatic catalytic reaction system includes: 100-200 mmol of glutamic acid, 100-200 mmol of cysteine, 100-200 mmol of glycine, 100-300 mmol of ATP, 50-150 mmol of magnesium chloride, and reactive enzyme solution containing the glutathione synthase mutant obtained by rupturing 5-20g of bacterial cells.

[0028] The reaction conditions are: 40-50°C and pH 7.0-8.0.

[0029] The GSH synthesis reaction is a high energy consumption reaction, with 2 molecules of ATP to be consumed to produce 1 molecule of GSH. However, on one hand,

ATP raw material has high price, and on the other hand, too high ATP concentration may result in reduced enzymatic activity. Therefore, during production, considering various factors such as cost reduction, an ATP regeneration system including a small amount of

ATP, polyphosphate kinase (PPK) and sodium hexametaphosphate can be used to replace high concentration ATP to continuously supply a large amount of ATP in the synthesis process of GSH.

[0030] Furthermore, to reduce the usage amount of ATP, an ATP regeneration system including a small amount of ATP, polyphosphate kinase (PPK) and sodium hexametaphosphate can be used to replace a large amount of ATP in the enzymatic catalytic reaction system.

[0031] The enzymatic catalytic reaction system includes: the glutamic acid, the cysteine, the glycine, the ATP, sodium hexametaphosphate, polyphosphate kinase, magnesium chloride, and the reactive enzyme solution containing the glutathione synthase mutant.

[0032] Further, each 1 litre of the enzymatic catalytic reaction system includes: 100- 200 mmol of glutamic acid, 100-200 mmol of cysteine, 100-200 mmol of glycine, 1-10 mmol of ATP, 50-200 of mmol sodium hexametaphosphate, 50-200 mg of PPK, 50-150 mmol of magnesium chloride, and reactive enzyme solution containing the glutathione synthase mutant obtained by rupturing 5-20g of bacterial cells.

[0033] The reaction conditions are: 40-50°C and pH 7.0-8.0.

[0034] Furthermore, the cysteine may be pure cysteine or crude cysteine.

[0035] The crude cysteine may be a serine-to-cysteine transformation reaction solution or a cysteine fermentation solution obtained through the fermentation of a cysteine production strain.

[0036] The serine-to-cysteine transformation reaction solution refers to the transformation solution from the serine-to-cysteine transformation via enzymatic catalysis, and the enzyme refers to an enzyme that can catalyze the serine to synthesize cysteine, such as cysteine synthase and tryptophan synthase.

[0037] The serine-to-cysteine transformation reaction solution can be prepared by a catalytic system as follows: L-serine, sodium hydrosulfide, phosphopyridoxal, sodium hydrogen phosphate, and cysteine synthase.

[0038] The serine-to-cysteine transformation reaction solution can be prepared by a catalytic system as follows: L-serine, sodium hydrosulfide, phosphopyridoxal, sodium hydrogen phosphate, and tryptophan synthase.

[0039] By the above technical solutions, the present invention at least has the following advantages and beneficial effects:

[0040] according to the present invention, by performing mutation and screening on the wild type bi-functional glutathione synthase to obtain high-activity mutants

GshFR¥E GshFH32F GshFN28 GshFN228L and GshF®°29P in particular, the enzymatic activity of mutants GshFR!*E GshFN2! and GshF™ 2 respectively reach 1.44 times, 1.43 times and 1.53 times of that of the wild type, all of such mutants have increased the generation amount of GSH. The present invention has the advantages of greatly increasing the GSH production output and reducing the cost, and has great significance in large-scale and high-yield production of GSH.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 is the comparison of the relative enzymatic activity of glutathione synthase mutants;

[0042] FIG. 2 is the GSH production output results utilizing the reactive enzyme solution containing glutathione synthase mutants and with pure cysteine as a substrate, and

[0043] FIG. 3 is the GSH production output results utilizing the reactive enzyme solution containing glutathione synthase mutants and with the serine-to-cysteine transformation reactive solution as the substrate.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] To increase GSH production output, the present invention provides a bi- functional glutathione synthase mutant, which is obtained by performing mutation at the following selected sites based on the amino acid sequence of the bi-functional glutathione synthase shown in SEQ ID NO: 1:

[0045] arginine at the 184th site mutated to alanine or leucine or glutamic acid;

[0046] asparaginate at the 215th site mutated to phenylalanine or tyrosine;

[0047] serine at the 216th site mutated to threonine;

[0048] asparaginate at the 217th site mutated to histidine;

[0049] histidine at the 325th site mutated to phenylalanine;

[0050] asparaginate at the 328th site mutated to leucine or isoleucine or valine; and

[0051] glutamine at the 329th site mutated to asparaginate.

[0052] The present invention further provides a gene encoding the mutant.

[0053] The present invention further provides a vector containing the gene containing the mutant.

[0054] The present invention further provides a recombinant microorganism expressing the bi-functional glutathione synthase mutant.

[0055] The present invention further provides a method of producing GSH by utilizing the bi-functional glutathione synthase mutant.

[0056] In one embodiment of the present invention, the recombinant microorganism cell takes escherichia coli as a host cell, and the bi-functional glutathione synthase mutant is expressed in the recombinant cell.

[0057] These examples are only intended to illustrate the present invention rather than to limit the scope thereof. Unless otherwise specified, all examples are based on normal experimental conditions (conditions provided by Sambrook J & Russell DW, Molecular

Cloning: a Laboratory Manual, 2001) or conditions recommended by manufacturer’s instructions.

[0058] Example 1 Construction of glutathione synthase GshF mutant

[0059] 1. Acquisition of genes

[0060] The coding sequence of the streptococcus sanguinis-derived bi-functional glutathione synthase GshF (SsGshF) was subjected to codon optimization, and then was synthesized to pET28A by Shanghai Logen Biotech LLC. to obtain an expression vector pET28A-SsGshF, and the nucleotide coding sequence of the SsGshF after codon optimization was shown in SEQ ID NO:2.

[0061] 2. Construction of mutant recombinant strain

[0062] pET28A-SsGshF was taken as a template, a mutant primer pair (as shown in

Table 1) was used to conduct mutation of the coding genes of GshF at corresponding sites, a PCR amplification product was subjected to digestion by Dpn I for 1 hour, and then was transferred into escherichia coli DH5a competent cell, and monoclone was picked for sequencing identification. A plasmid containing mutant genes was transferred into escherichia coli BL21(DE3) to respectively obtain mutant recombinant strains

BL21-GshFR!$#4 BL21-GshFR!!E BL21-GshFR34 BL21-GshFN2F BL21-

GshFN25Y BL21-GshFS219T BL21-GshEP2VH BL21-GshF82F, BL21-GshFN328L

BL21-GshFN°28L BL21-GshFN328V BL21-GshF®?2D and the unmutated vector pET28A-SsGshF was transferred into escherichia coli BL21(DE3) to obtain a recombinant strain BL21-GshF expressing wild type GshF as control.

Table 1 GshF gene site-directed mutation primer

Mutation site Primer sequence (5'—3')

R184A gtaatttcctgcstttc GC Gtggattctgacctacctg ggtcagaatccaC(GCgaaacgcaggaaattacge gtaatttcctgegtttcGA Atggattctgacctacctg

R184E ggtcagaatccaTTC gaaacgcaggaaattacgc gtaatttcctgcgttteCTGtggattctgacctacctg

R184L ggtcagaatccaC AGgaaacgcaggaaattacgc ccgtagctttegtTTTagegattacggctacgtc

N215F gccgtaatcgctAAAacgaaagctacggatcggc ccgtagctttegtTATagcgattacggctacgtc

N215Y gccgtaatcgctATAacgaaagctacggatcggc gtagctttcgtaacA CC gattacggctacgtcaacg

S216T cgtagccgtaatcGGTgttacgaaagctacggatc ctttcgtaacagcCATtacggctacgtcaacgacg

D217H gacgtagccgtaATGgctgttacgaaagctac ctgaaagcagcaTTTgacctgaaccagaaaattg

H325F ctggttcaggtcAAAtgctgctttcagtgcgg gcacacgacctgATTcagaaaattgcttgtag

N328I gcaattttctg A ATcaggtcgtgtgetgcttte gcacacgacctgCTGcagaaaattgcttgtag

N328L gcaattttetgCAGcaggtcgtgtgctgctttc gcacacgacctgGTGcagaaaattgcttgtag

N328V gcaattttctgCACcaggtcgtgtgetgcttte cacgacctgaacGATaaaattgcttgtagtcatccg

Q329D caagcaattttATCgttcaggtcgtgtgetgc

[0063] Example 2 Enzymatic activity test of glutathione synthase mutant

[0064] Recombinant protein induced expression

[0065] The mutant recombinant strain and the control recombinant strain constructed in Example 1 were respectively subjected to shaking culture in LB culture media at 37°C, when the bacteria solution concentration reached ODo0=1.0, IPTG with the final concentration of 0.4 mM was added for induced culture at 30°C for 20 h, and then centrifugal collection of bacterial cells was conducted.

[0066] 2. Enzymatic activity test of glutathione synthase mutant

[0067] 1g of the bacterial cells each of the glutathione synthase mutant recombinant strain and the control recombinant strain collected above was respectively taken, the bacterial cells were re-suspended using sterile water, cell rupture was conducted by a high pressure homogenizer at 800-1000bar to obtain a ruptured cell solution, and the ruptured cell solution was as added into | mL of enzymatic activity reaction system for enzymatic activity determination.

[0068] Other components and the content thereof in the enzymatic activity reaction system are as follows: glutamic acid of 20 mM, cysteine of 20 mM, glycine of 20 mM,

ATP of 20 mM and magnesium chloride of 20 mM. The reaction conditions of enzymatic activity determination are: 45°C and pH 8.0. After the reaction, 1M of isovolumetric hydrochloric acid was added to end the reaction and obtain a test sample, and the GSH content of the sample was tested using HPLC to calculate the relative enzymatic activity.

[0069] The enzymatic activity results are as shown in FIG.1, mutants GshFR!®*E,

GshF™25F GshFN 2 GshFN28L and GshF©2°P obtain glutathione synthase activity higher than the wild type, where the enzymatic activities of mutants GshFRI34E

GshFN3281 and GshFN°?*L respectively reach 1.44 times, 1.43 times and 1.53 times of the wild type.

[0070] Example 3 Product inhibition test of glutathione synthase mutant

[0071] Mutant proteins GshFR!3%E, GshFN323! and GshFN2%L with high enzymatic activity were selected to prepare a crude enzyme reactive solution to catalyze GSH synthesis in vitro, and the effect of product inhibition on the enzymatic activity of mutant proteins was tested.

[0072] 1. The mutant recombinant strains BL21-GshFR!**E BL21-GshFN23L BL21-

GshFN22sL and the control recombinant strain BL21-GshF were respectively subjected to shaking culture in LB culture media at 37°C, when the bacteria solution concentration reached ODgoo=1.0, IPTG with the final concentration of 0.4 mM was added for induced culture at 30°C for 20 h, and then centrifugal collection of bacterial cells was conducted.

[0073] 2. The bacterial cells of the mutant strains and the control strain were respectively re-suspended using sterile water, cell rupture was conducted by a high pressure homogenizer at 800-1000bar, the obtained ruptured cell solution, as a glutathione synthase reactive enzyme solution, was added into the enzymatic catalytic reaction system that synthesizes GSH for reaction.

[0074] The rest components and the content thereof in each IL of the enzymatic catalytic reaction system are as follows: 150 mmol of glutamic, 0-150 mmol of cysteine, 150 mmol of glycine, 2 mmol of ATP, 100 mmol of sodium hexametaphosphate, 100 mg of PPK, 100 mmol of magnesium chloride, and glutathione synthase reactive enzyme solution obtained by rupturing of 10 g of bacterial cells. The cysteine concentration is 0, 10, 50, 100 and 150 mM, respectively. The catalytic reaction system reacted at 45°C and 1000 rpm for 4 h, after the reaction, 100 pL of reaction solution was taken to end the reaction with isovolumetric IM hydrochloric acid, and GSH production output was tested using HPLC.

[0075] The GSH synthesis reaction is a high energy consumption reaction, with 2 molecules of ATP to be consumed to produce 1 molecule of GSH. However, on one hand,

[0076] The results are shown in Table 2, product inhibition occurs when the GSH product produced by wild type GshF reaches about 60 mM, and the GSH amount does not increase along with the increase of the substrate concentration anymore. Compared with the wild type GshF, mutants GshFR!+E, GshFN°28! and GshF™?* can reduce the effect of product inhibition, and increase the GSH production output to above 70 mM.

For the mutant GshF™2% only when the produced GSH content approximates 100 mM,

product inhibition occurs. The above results fully demonstrate that mutated glutathione synthase effectively reduces the effect of product inhibition, and increases GSH production output.

Table 2 GSH transformation at different adding amounts of cysteine (mM)

Adding Amount of Cysteine 0 10 50 100 150

Wild type GshF (WT) 0 9.8 40.5 60.1 61.1

GshFR!s#E 0 9.9 50.2 72.2 74.6

GshFN3 2 0 9.9 49.7 75.1 74.3

GshFN25L 0 10.0 49.8 93.5 98.7

[0077] Example 4 Production of GSH through the catalysis of glutathione synthase mutant

[0078] 1. The mutant strain BL21-GshFN2® and the control strain BL21-GshF were subjected to shaking culture in LB culture media at 37°C, and when the bacteria solution concentration reached ODgsoo=1.0, IPTG with the final concentration of 0.4mM was added for induced culture at 30°C for 20 h.

[0079] 2. The bacterial cells obtained from the culture were subjected to high pressure rupture, and the obtained ruptured cell solution, as a glutathione synthase reactive enzyme solution, was added into the enzymatic catalytic reaction solution that synthesizes GSH for catalytic reaction.

[0080] Each IL of the enzymatic catalytic reaction solution includes: 150 mmol of glutamic acid, 150 mmol of cysteine, 150 mmol of glycine, 2 mmol of ATP, 100 mmol of sodium hexametaphosphate, 100 mg of PPK, 100 mmol of magnesium chloride, and glutathione synthase reactive enzyme solution obtained by rupturing 10 g of bacterial cells. The reaction conditions are: 45°C, and pH maintained at 8.0 as adjusted by 2 M

H>SO4. Reaction solutions of 0.5 h, 1 h, 2 h, 3 h and 4 h were respectively taken, the reaction was ended by isovolumetric IM hydrochloric acid, and the GSH production within 4 hours was tested by HPLC.

[0081] The results are as shown in FIG.2, the wild type GshF reacted for 4 h, where the GSH production reaches 65.9 mM, and the transformation rate is 44%. While for reaction using mutant GshFN*?*®, after 3 h reaction, the production has reached 98.9 mM, and the transformation rate is increased to 67.0%. Compared with the wild type, the yield, transformation rate and production efficiency are greatly increased.

[0082] The above results indicate that, compared with the wild type GshF, when using the mutated glutathione synthase GshF*®2*L to catalyze the GSH synthesis, the reaction time is shortened by 25%, and the transformation rate is increased by 20% or more, thereby greatly improving GSH production output and efficiency, reducing the cost, and having great significance to the large-scale and high-yield production of GSH.

[0083] Example 5 Preparation of GSH under the catalysis of glutathione synthase mutant by utilizing serine-to-cysteine transformation reaction solution as substrate in replacement of pure cysteine

[0084] Due to that pure cysteine has high price, is easy to be oxidized into cystine and is not convenient for storage, in order to further reduce the cost, crude cysteine can be used as a substrate in replacement of pure cysteine for enzymatic synthesis of GSH. The unpurified crude cysteine can be, for example, a serine-to-cysteine transformation reaction solution or a fermentation solution from the production of cysteine by fermentation of the cysteine production strain. The serine-to-cysteine transformation reaction solution is taken as an example for specific implementation.

[0085] (I) Preparation of serine-to-cysteine transformation reaction solution

[0086] The purpose of preparing serine-to-cysteine transformation reaction solution is to obtain cysteine with higher concentration for subsequent GSH preparation by catalyzing the reaction of the substrate serine and the -SH donor, so as to reduce the high production cost of using pure cysteine. Therefore, any production strain and method of the enzyme in this field that express the catalysis of the synthesis of cysteine from serine can be used in this step.

[0087] By way of one specific example, the serine-to-cysteine transformation reaction solution can be prepared in accordance with the method and operation of utilizing the tryptophan synthase mutant that can catalyze the synthesis of cysteine from serine as described in Example 3 in the Specification of Patent ZL202410154368.7. Specifically:

[0088] (1) a strain W3110-TrpsQRDPPEIC-0AMAPA that expresses tryptophan synthase mutant was selected to inoculate in LB culture medium containing ampicillin for shaking culture at 37°C. When the concentration of the bacterial solution reached ODgpo=1.0,

IPTG with the final concentration of 0.4mM was added for induced expression, and culture was performed at 30°C for 20 h to obtain a recombinant cell culture containing tryptophan synthase.

[0089] (2) The bacterial cells in the recombinant cell culture was subjected to high pressure rupture, the obtained ruptured cell solution, as a tryptophan synthase reactive enzyme solution, was used in the enzymatic catalytic reaction solution that transforms serine to produce cysteine. The reaction solution includes: L-serine of 0.95 M, sodium hydrosulfide of 1.1 M, phosphopyridoxal of 0.4 mM, sodium hydrogen phosphate of 50 mM, and tryptophan synthase reactive enzyme solution (20 g of ruptured bacterial cell solution added in each 1 litre reaction solution). The reaction conditions are: reacting at 40°C, and pH maintained at 8.0 as adjusted by 2 M H:S0..

[0090] (3) After the catalytic reaction, the pH of the reaction solution was adjusted to 7, and heating was performed to 65-75°C to deactivate and denature the protein.

[0091] (4) Extraction filtration was performed on the reaction solution subjected to thermal treatment by using two layers of filter paper, bacterial debris and denatured protein were removed to obtain a supernatant clear solution from extraction filtration, and obtain supernate containing cysteine, the concentration of the cysteine in the supernate was controlled to 90g/L, then tris(2-carboxyethyl)phosphine hydrochloride was added until a final concentration of 50 mM to obtain the treated serine-to-cysteine transformation reaction solution.

[0092] (II) Production of GSH

[0093] (1) The bacterial cells in the cultures of recombinant mutant strain BL21-

GshFN°28L and control strain BL21-GshF that express glutathione synthase were subjected to high pressure rupture, and the obtained ruptured cell solution, as a glutathione synthase reactive enzyme solution, was added into the enzymatic catalytic reaction system that synthesizes GSH for catalytic reaction.

[0094] (2) Preparation of GSH by in vitro enzymatic catalytic reaction

[0095] Each IL enzymatic catalytic reaction system includes: 201.8 mL of serine-to- cysteine transformation reaction solution (L-cysteine molecular weight 121, it can be calculated that the cysteine in 1L reaction system is about: 90+121#201.8=150 mmol), 150 mmol of glutamic acid, 150 mmol of glycine, 2 mmol of ATP, 100 mmol of sodium hexametaphosphate, 100 mmol of magnesium chloride, 100 mg of PPK, and glutathione synthase reactive enzyme solution obtained by rupturing 10 g of bacterial cells.

[0096] The reaction conditions are: reacting at 40°C, and pH maintained at 8.0 as adjusted by 2 M H;SOx.

[0097] (3) Reaction solutions of 0.5 h, 1 h, 2 h, 3 h and 4 h were respectively taken, the reaction was terminated by 1M hydrochloric acid, and the GSH production within 4 hours was tested by HPLC.

[0098] The results are shown in FIG.3, after the wild type GshF reacts for 4h, the GSH production is 53.67 mM, and the cysteine transformation rate is 35.7% only. Whereas after the reaction using mutant GshF™28 for 4h, the production output is up to 95.36 mM, increased by 27.7% over the wild type GshF, the cysteine transformation rate reaches 63.6%, much higher than the wild type.

[0099] In addition, compared with the reaction using pure L-cysteine as substrate in

Example 3, when using concentrated serine-to-cysteine transformation reaction solution as a substrate, the transformation rate of the wild type GshF after 4h reaction is decreased from 44% to 35.7%. This indicates that the impurities in the serine-to-cysteine transformation reaction solution have great interference and effect on the enzymatic activity of the wild type GshF. Whereas in reaction using mutant GshF™2L the transformation rate remains basically unchanged, the production output after 4h reaction can still reach that of using pure cysteine as a substrate, indicating that the enzymatic activity of the mutated GshF is less affected by the impurities in the serine-to-cysteine transformation reaction solution, therefore it is more suitable for industrial production.

[00100] Based on the above description, in the present invention, by mutating the glutathione synthase GshF of streptococcus sanguinis, the glutathione synthase mutant with high enzymatic activity is obtained, thereby not only significantly increasing GSH production output and transformation rate, but also effectively improving production efficiency and reducing production cost with the activity not susceptible to impurity effect, and having great significance to the large-scale and high-yield production of GSH.

[00101] Although the present invention has been described in detail above with a general description and specific examples, some modifications or improvements can be made on the basis of the present invention, which 1s apparent to those skilled in the art.

Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the protection scope of the present invention

NL Shaanxi Lifewe Bioengineering Co., Ltd. CN 2024106272573 2024-05-21

Shaanxi Lifewe Bioengineering Co., Ltd. Xiang Jin BI-FUNCTIONAL GLUTATHIONE

SYNTHASE MUTANT AND APPLICATION THEREOF 2 750 AA PAT source 1..750 mol_type protein organism Streptococcus sanguinis

MTINQLLQKLDTASPILQATFGLERENLRVTTDGHLAQTAHPSQLGSRNFHPTIQTDFSEQQLE

LITPIAHSTKEARRLLGAISDVAGRSIDQSERLWPLSMPPQLTEEEIVIARLENDYERHYREGLA

KKYGKKLQAISGIHYNMELGKDLVTSLFQVSSYHSLKDFKNDLYLKLARNFLRFRWILTYLYGA

APWAEAGFYSQEISQPIRSFRNSDYGYVNDENIQVSYASLEQYITDIENYVQSGELSAEKEFYS

AVRFRGQKHNHAYLEQGITYLEFRCFDLNPFDHLGISQETLDTVHLFLLSLLWLDDVENVDTAL

KAAHDLNQKIACSHPLTALPDEADSSALLQAMEELIQHFELPTYYQTLLQQLKEALLNPQLTLS

GQLLPHIQQDSLMAFGLEKAEEYHRYAWTAPYALKGYENMELSTQMLLFDAIQKGLNVDILDE

NDQFLKLWHGHHVEYVKNGNMTSKDNYVIPLAMANKTVTKKILAEADFPVPAGAEFSSLEEG

LAYYPLIRDRQIVVKPKSTNFGLGISIFQEPASLESYRKALEIAFSEDAAVLVEEFIAGTEYRFFVL

DGQCEAVLLRVAANVVGDGQHTVRELVAIKNDNPLRGRDHRSPLEIIELGDIELLMLDQQGYG

PDDILPAGVKVDLRRNSNISTGGDSIDVTDSMHSSYKELAADMAKAMGAWACGVDLIIPDSS

AISTKENPNCTCIELNFNPSMYMHTYCAEGPGQSITPKILAKLFPEMD 2253 DNA PAT source 1..2253 mol_type genomic DNA organism Streptococcus sanguinis atgaccatcaaccagttactgcagaaactggataccgctagtcctattctgcaagcaacctttggtctggaacgtgaaa atctgagagttaccactgacggtcatctggcacaaaccgctcatcctagtcaactgggaagccgtaactttcatccgta catacagaccgactttagcgaacagcaactggaactgattacccctattgcacatagcaccaaagaagcacgtcgtct gctgggtgcaattagcgacgttgcaggtcgtagtattcctcaatctgaacgtctgtggccgctgagtatgccacctcaa ctgactgaagaggaaattgtcatcgcacgactggaaaacgattacgaacgtcattaccgcgaaggtctggcgaaaa agtacggcaagaagaaacaggcgatcagcggtatccactacaacatggagctgggcaaagatctggtcaccagtct gtttcaggttagcagctaccacagcctgaaggacttcaagaacgacctgtacctgaaactggcgcgtaatttcctgcgt ttccgctggattctgacctacctgtatggcgctgcaccgtgggcagaagcaggtttttacagtcaggaaatcagccagc cgatccgtagctttcgtaacagcgattacggctacgtcaacgacgagaacatccaggtaagctacgcgagcctggaa cagtatatcaccgatatcgaaaactacgttcagagcggcgaactgtctgcggaaaaagaattctacagcgcggttcgt tttcgcggtcagaaacataaccacgcgtacctggaacagggtattacctacctggagttccgttgcttcgatctgaaccc gttcgatcatctgggtattagccaggaaaccctggataccgtccacctgtttctgctgtctctgctgtggctggacgatgt tgaaaacgttgataccgcactgaaagcagcacacgacctgaaccagaaaattgcttgtagtcatccgctgaccgctct gccggatgaagcagatagttctgctctgctgcaggcaatggaagaactgatccagcacttcgaactgccgacctatta tcagaccctgctgcaacagctgaaagaagcactgctgaatccgcaactgaccctgtctggtcaactgctgccgcatatt cagcaagacagcctgatggcgttcggtctggaaaaagcggaagaataccaccgttatgcttggaccgcaccgtacgc actgaaaggctacgaaaacatggaactgtccacccaaatgctgctgtttgacgcgatccagaaaggcttaaacgtgg atattctggacgaaaatgatcagttcctgaaactgtggcacggccatcatgtggaatacgtgaaaaacggcaatatga ccagcaaagataattatgtgattccgctggcgatggcgaataagaccgtgaccaaaaaaattctggcggaagcgga ctttccggtgccggcgggtgcggaatttagcagtctggaagaaggcctggcctactacccgcttattcgtgatcgtcag atcgtcgtcaaaccgaaaagcaccaacttcggcctgggtattagcatcttccaggaaccggcatcactggaaagttat cgtaaagcactggaaattgccttttctgaagacgcagcggttctggttgaagaattcattgcaggtaccgaatatcgctt ctttgttctggacggtcagtgtgaagcagttctgttacgcgttgctgcaaacgtagttggcgacggtcaacataccgttc gcgaactggtcgcgatcaaaaacgataacccactgcgtggtcgagatcatcgtagtccactggaaatcatcgaactg ggcgatatcgagctgctgatgctggatcaacaaggttacggtccggacgatattctgcctgcaggcgttaaagtcgat ctgcgtcgtaacagcaacattagcaccggtggagatagtattgacgttaccgatagcatgcatagtagctacaaagaa ctggccgcagatatggctaaagcaatgggcgcttgggcttgcggcgttgatctgattattccagatagttctgctattag caccaaagaaaatccgaactgcacctgcatcgagctgaacttcaacccgtccatgtacatgcatacctattgcgcaga aggtccgggtcaaagtattacgcctaaaatcctggcgaagctgtttcctgaaatggattaa

Claims

Conclusions

1. Glutathione synthase mutant characterized in that it is mutated at the following positions based on an amino acid sequence shown in SEQ ID NO:1; mutated from R to E at the 184th position, mutated from H to F at the 325th position, mutated from N to I or L at the 329th position, and mutated from Q to D at the 329th position.

2. A nucleic acid molecule encoding a mutant according to claim 1.

3. Biological material containing the nucleic acid molecule according to claim 2, characterised in that it is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.

4. Recombinant micro-organism, characterised in that it is obtained by introducing a nucleic acid molecule according to claim 2 into the micro-organism via a plasmid or integrating it into the chromosome of the micro-organism via genetic engineering.

5. Use of the glutathione synthase mutant according to claim 1, the nucleic acid molecule according to claim 2, the biological material according to claim 3 or the recombinant microorganism according to claim 4 in the production of glutathione.

6. A method for synthesizing glutathione by in vitro catalysis synthesis of glutathione, characterized by the use of the glutathione synthase mutant according to claim 1.

7. The method according to claim 6, characterized in that it comprises the steps of: (1) culturing a microorganism according to claim 4 to express a glutathione synthase mutant; (2) performing cell disruption on the culture, the cell disruption solution thereby obtained being a reactive enzyme solution containing the glutathione synthase mutant; and (3) using the reactive enzyme solution to catalyze the reaction of glutamic acid, cysteine, glycine and ATP to synthesize glutathione.

8. A method according to claim 7, characterised in that the composition of the catalytic system of step (3) is selected from one of the following groups: (1) glutamic acid, cysteine, glycine, ATP, magnesium chloride and a reactive enzyme solution containing the glutathione synthase mutant; and (it) glutamic acid, cysteine, glycine, ATP, sodium hexametaphosphate, polyphosphokinase (PPK), magnesium chloride and a reactive enzyme solution containing the glutathione synthase mutant.

9. A method according to claim 7 or 8, characterised in that the cysteine is pure cysteine or unpurified cysteine.

10. A method according to claim 9, characterized in that the unpurified cysteine is a serine in cysteine conversion reaction solution or a cysteine fermentation solution obtained by fermentation of a cysteine producing strain.