Preparation method of glutathione

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of glutathione. The invention provides a preparation method of glutathione, a specific mutant of bifunctional glutathione synthetase and a mutant of polyphosphate kinase are used for participating in enzymatic synthesis of glutathione, and the conversion rate of glutathione synthesis can be further improved based on high activity and stability and high catalytic efficiency of the enzyme mutant.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for preparing glutathione. Background Art

[0002] Glutathione (GSH) is a tripeptide containing a γ-amide bond and a sulfhydryl group, and is composed of glutamic acid, cysteine and glycine. Glutathione can help maintain the normal function of the immune system, and has antioxidant and integrative detoxification effects, and is widely used in drugs, foods, cosmetics and health products.

[0003] The production methods of glutathione include extraction method, chemical synthesis method, direct fermentation method and enzyme catalysis method. Among them, the extraction method and the chemical synthesis method have been eliminated due to cost or quality problems. At present, the direct fermentation method and the enzyme catalysis method are mainly used to produce GSH. At present, yeast is mainly used for direct fermentation to produce GSH. However, due to problems such as long fermentation cycle, complex products, difficult separation and extraction, and low fermentation concentration, its production cost is relatively high. In recent years, the production of GSH by enzyme catalysis method has developed rapidly and has a tendency to gradually replace the fermentation method.

[0004] The enzymatic synthesis of glutathione mainly uses three amino acids, L-glutamic acid, glycine and L-cysteine as precursors, and generates GSH under the catalytic action of an enzyme at appropriate temperature and pH conditions in the presence of ATP and Mg 2+ This process requires the dependence on glutathione synthetase and a continuous adenosine triphosphate (ATP) energy supply system.

[0005] Bifunctional glutathione synthetase is the key enzyme for synthesizing glutathione, which has both γ-glutamylcysteine (γ-GC) synthetase (γ-GCS) activity and glutathione synthetase (GS) activity. However, the enzyme activity and stability of the wild-type bifunctional glutathione synthetase GshF are both poor, and it is difficult to meet the requirements of industrial production. Finding an enzyme with higher activity and stability has always been a research hotspot in this field.

[0006] The direct use of ATP raw materials has a high cost price, which limits the application of glutathione bifunctional enzyme in industrial production. Therefore, the synthesis of ATP and the construction of an ATP recycling system are another urgent problems to be solved in this enzymatic process. Polyphosphate kinase (PPK) is a class of enzymes that use polyphosphate as a phosphate donor to catalyze the formation of ATP from ADP or AMP. PPKs are divided into two categories, PPK1 and PPK2. PPK1 prefers to use ATP as a substrate for ATP degradation; the PPK2 family can be further divided into three categories. PPK2-I prefers to use ADP as a substrate to generate ATP, PPK2-II prefers to use AMP as a substrate to generate ADP, and PPK2-III uses both ADP and AMP as substrates to generate ATP. The efficiency of PPK determines whether the ATP synthesis recycling system meets the requirements of industrial applications.

[0007] However, the enzyme activities and stabilities of the existing glutathione synthase and polyphosphate kinase mutants are still relatively low, the production cost is relatively high, and the industrial application is restricted, resulting in a relatively low conversion rate of glutathione synthesis. Summary of the Invention

[0008] The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, an object of the present invention is to provide a method for preparing glutathione, which uses specific mutants of bifunctional glutathione synthase and mutants of polyphosphate kinase to participate in the enzymatic synthesis of glutathione. Based on the high activity, stability and high catalytic efficiency of the enzyme mutants, the conversion rate of glutathione synthesis can be further improved.

[0009] To this end, the first aspect of the present invention provides a method for preparing glutathione. According to an embodiment of the present invention, the preparation method includes:

[0010] Using L-glutamic acid or its salt, glycine, and L-cysteine as precursors, and using mutants of bifunctional glutathione synthase and mutants of polyphosphate kinase to carry out an enzymatic reaction to synthesize glutathione,

[0011] wherein the amino acid sequence of the mutant of bifunctional glutathione synthase is obtained by site-directed mutagenesis of at least one of positions 597, 607, 609, 610, 728, and 733 of the amino acid sequence of wild-type bifunctional glutathione synthase,

[0012] The amino acid sequence of the mutant of polyphosphate kinase is obtained by site-directed mutagenesis of at least one of positions 77, 103, and 106 of the amino acid sequence of wild-type polyphosphate kinase.

[0013] The inventor used a new artificial intelligence software tool to perform protein modeling on the sequence of wild-type bifunctional glutathione synthetase, and speculated that the second domain (amino acids 520 - 754) was responsible for catalyzing the synthesis of GSH from γ-Glu-Cys and glycine. Through semi-flexible molecular docking of its partial protein region and small molecule ligands, amino acids within the ligand range were subjected to site-directed saturation virtual screening. By calculating the binding free energy of the ligand and the protein, mutation sites at positions 597, 607, 609, 610, 728, and 733 were screened out. Further experiments confirmed that mutations at these sites would enhance the enzyme activity of bifunctional glutathione synthetase, improve enzyme stability, be more suitable for industrial production, be applicable to the preparation of glutathione, and improve the efficiency of glutathione preparation.

[0014] The inventor performed protein homology modeling on the amino acid sequence of polyphosphate kinase through Swiss-model, and used analysis software to find multiple amino acid sites with high RMSF values. Through further site-directed mutagenesis, a large number of mutants of polyphosphate kinase were obtained. Among them, it was found that based on the amino acid sequence of wild-type polyphosphate kinase, site-directed mutagenesis at at least one of positions 77, 103, and 106 resulted in mutants of polyphosphate kinase with high enzyme activity, good stability, low production cost, being suitable for industrial production, and applicable to the preparation of glutathione.

[0015] Using the preparation method of the present invention to synthesize glutathione, based on the high activity, stability, and high catalytic efficiency of the enzyme mutants, the conversion rate of glutathione synthesis can be further improved.

[0016] According to the embodiments of the present invention, the amino acid sequence of the wild-type bifunctional glutathione synthetase is as shown in SEQ ID NO:1.

[0017] The present invention performed site-directed mutagenesis on the bifunctional glutathione synthetase GshFST (GenBank: WP_011226281.1) derived from Streptococcus thermophilus, and found that mutants obtained by site-directed mutagenesis at at least one of positions 597, 607, 609, 610, 728, and 733, compared with the wild type, could further improve enzyme activity and had good stability.

[0018] The amino acid sequence of the wild-type bifunctional glutathione synthetase shown in SEQ ID NO:1 is as follows:

[0019] MTLNQLLQKLEATSPILQANFGIERESLRVDRQGQLVHTPHPSCLGARSFHPYIQTDFCEFQMELIT

[0020] PVAKSTTEARRFLGAITDVAGRSIATDEVLWPLSMPPRLKAEEIQVAQLENDFERHYRNYLAEKYGTKL

[0021] QAISGIHYNMELGKDLVEALFQESDQTDMIAFKNALYLKLAQNYLRYRWVITYLFGASPIAEQGFFDQ

[0022] EVPEPMRSFRNSDHGYVNKEEIQVSFVSLEDYVSAIETYIEQGDLIAEKEFYSAVRFRGQKVNRSFLDK

[0023] GITYLEFRNFDLNPFERIGISQTTMDTVHLLILAFLWLDSPENVDQALAQGHALNEKIALSHPLEPLPSE

[0024] AKTQDIVTALDQLVQHFGLGDYHQDLVKQVKAAFADPNQTLSAQLLPYIKDKSLAEFALNKALAYHD

[0025] YDWTAHYALKGYEEMELSTQMLLFDAIQKGIHFEILDEQDQFLKLWHQDHVEYVKNGNMTSKDNYV

[0026] VPLAMANKTVTKKILADASFPVPSGDEFTSLEEGLAYYPLIKDKQIVVKPKSTNFGLGISIFQEPASLDN

[0027] YQKALEIAFAEDTSVLVEEFIPGTEYRFFILDGRCEAVLLRVAANVIGDGKHTIRELVAQKNANPLRGRD

[0028] HRSPLEIIELGDIEQLMLAQQGYTPDDILPEGKKVNLRRNSNISTGGDSIDVTETMDSSYQELAAAMATS

[0029] MGAWACGVDLIIPDETQIATKENPHCTCIELNFNPSMYMHTYCAEGPGQAITTKILDKLFPEIVAGQT。

[0030] According to an embodiment of the present invention, the amino acid sequence of the wild-type polyphosphate kinase is as shown in SEQ ID NO:35.

[0031] In the present invention, by performing site-directed mutagenesis on the polyphosphate kinase DpPPK2-III (GenBank: WP_013615652) derived from Deinococcus proteolyticus, it is found that mutants obtained by performing site-directed mutagenesis on at least one of the 77th, 103rd, and 106th positions can further improve the enzyme activity and have good stability compared to the wild type.

[0032] The amino acid sequence of the wild-type polyphosphate kinase shown in SEQ ID NO:35 is as follows:

[0033] MNSKASQQLQVPPGQKVRLADYSTDTFEGSAALDKEQVKQATEQLQQRLSELQEKLYAEGKQSL

[0034] LIILQARDGGGKDSTVSRVMGAFNPNGVHVANFKAPTDLELQHDFLWRIHQQVPSHGMIAVFNRSHYE

[0035] DVLVTRVHGLIDDARAQQNLEHIVNFEKLLSDAGTRIVKFYLHLSPEEQKARMEDRLNDPAKHWKFNP

[0036] SDLKDRALWHEYTAAYEDALATSRSYAPWYIIPADRKWLRDYLISEILVQTLEEMNPQFPTAHFEAAYY

[0037] LIEDIPSR.

[0038] According to an embodiment of the present invention, compared with the amino acid sequence of the wild-type bifunctional glutathione synthetase, the amino acid sequence of the mutant of the bifunctional glutathione synthetase has at least one of the following mutations (1)-(8):

[0039] (1) Lysine at position 597 is mutated to arginine;

[0040] (2) Lysine at position 607 is mutated to arginine;

[0041] (3) Alanine at position 609 is mutated to glutamate;

[0042] (4) Alanine at position 609 is mutated to arginine;

[0043] (5) Asparagine at position 610 is mutated to tyrosine;

[0044] (6) The tyrosine at position 728 is mutated to tryptophan;

[0045] (7) The proline at position 733 is mutated to histidine;

[0046] (8) The proline at position 733 is mutated to arginine.

[0047] The inventors found that mutants of the bifunctional glutathione synthetase having at least one of the mutations (1)-(8) above have high enzyme activity and good stability and are suitable for industrial production.

[0048] According to an embodiment of the present invention, compared with the amino acid sequence of the wild-type bifunctional glutathione synthetase, the amino acid sequence of the mutant of the bifunctional glutathione synthetase has the following mutations 1)-12):

[0049] 1) The lysine at position 597 is mutated to arginine; or

[0050] 2) The lysine at position 607 is mutated to arginine; or

[0051] 3) The alanine at position 609 is mutated to glutamate; or

[0052] 4) The alanine at position 609 is mutated to arginine; or

[0053] 5) The asparagine at position 610 is mutated to tyrosine; or

[0054] 6) The tyrosine at position 728 is mutated to tryptophan; or

[0055] 7) The proline at position 733 is mutated to histidine; or

[0056] 8) The proline at position 733 is mutated to arginine; or

[0057] 9) The tyrosine at position 728 is mutated to tryptophan, and the lysine at position 597 is mutated to arginine; or

[0058] 10) The tyrosine at position 728 is mutated to tryptophan, and the lysine at position 607 is mutated to arginine; or

[0059] 11) The tyrosine at position 728 is mutated to tryptophan, and the asparagine at position 610 is mutated to tyrosine; or

[0060] 12) The tyrosine at position 728 is mutated to tryptophan, and the proline at position 733 is mutated to histidine.

[0061] The inventors found that mutants of the bifunctional glutathione synthetase with the mutations of the above 1)-12) have high enzyme activity, further enhanced enzyme stability, are more suitable for industrial production, are applicable to the preparation of glutathione, and can improve the efficiency of glutathione preparation.

[0062] According to an embodiment of the present invention, compared with the amino acid sequence of the wild-type polyphosphate kinase, the amino acid sequence of the mutant of the polyphosphate kinase has a mutation of any one of the following (1)-(4):

[0063] (1) Aspartic acid at position 77 is mutated to asparagine;

[0064] (2) Leucine at position 103 is mutated to asparagine;

[0065] (3) Glutamine at position 106 is mutated to lysine;

[0066] (4) Leucine at position 103 is mutated to asparagine, and glutamine at position 106 is mutated to lysine.

[0067] The inventors found that mutants of the polyphosphate kinase with any one of the mutations of the above (1)-(4) have high enzyme activity, good stability, low production cost, and are suitable for industrial production.

[0068] According to an embodiment of the present invention, compared with the amino acid sequence of the wild-type bifunctional glutathione synthetase, the amino acid sequence of the mutant of the bifunctional glutathione synthetase has a mutation in which tyrosine at position 728 is mutated to tryptophan and lysine at position 607 is mutated to arginine;

[0069] Compared with the amino acid sequence of the wild-type polyphosphate kinase, the amino acid sequence of the mutant of the polyphosphate kinase has a mutation in which leucine at position 103 is mutated to asparagine and glutamine at position 106 is mutated to lysine.

[0070] According to an embodiment of the present invention, the preparation method further includes:

[0071] Mixing buffer, Mg 2+ , AMP, L-glutamic acid or its salt, glycine, L-cysteine, glycine, L-cysteine, sodium hexametaphosphate, sodium sulfite, a mutant of bifunctional glutathione synthetase, and a mutant of polyphosphate kinase, and reacting under the condition of 35-45 °C to synthesize glutathione.

[0072] According to an embodiment of the present invention, the working concentration of the mutant of the bifunctional glutathione synthetase is 30-40 g / L, and the working concentration of the mutant of the polyphosphate kinase is 20-25 g / L.

[0073] According to an embodiment of the present invention, the working concentration of the sodium hexametaphosphate is 28 - 32 mM.

[0074] According to a preferred embodiment of the present invention, the reaction temperature is 40 - 45 °C.

[0075] According to an embodiment of the present invention, the buffer solution is Tris buffer solution, with a working concentration of 140 - 160 mM and a pH of 8 - 9.

[0076] According to an embodiment of the present invention, the working concentration of the Mg 2+ is 40 - 60 mM.

[0077] According to an embodiment of the present invention, the working concentration of the AMP is 3 - 5 mM.

[0078] According to an embodiment of the present invention, the salt of L - glutamic acid is sodium glutamate, and the working concentration of the sodium glutamate is 150 - 250 mM.

[0079] According to an embodiment of the present invention, the working concentration of the glycine is 150 - 250 mM.

[0080] According to an embodiment of the present invention, the working concentration of the L - cysteine is 75 - 125 mM.

[0081] According to an embodiment of the present invention, the working concentration of the sodium sulfite is 8 - 12 g / L.

[0082] According to a preferred embodiment of the present invention, the preparation method further includes:

[0083] Mix 150 mM Tris (pH 8.5), 50 mM MgCl2, 4 mM AMP, 200 mM sodium glutamate, 200 mM glycine, 100 mM cysteine, 30 mM sodium hexametaphosphate, 10 g / L sodium sulfite, 35 g / L mutant of bifunctional glutathione synthetase, and 22.5 g / L mutant of polyphosphate kinase, and react at 42 °C to synthesize glutathione.

[0084] The additional aspects and advantages of the present invention will be partly given in the following description, partly become obvious from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The above - mentioned and / or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, wherein:

[0086] Figure 1 shows the three - dimensional spatial structure of bifunctional glutathione synthetase;

[0087] Figure 2 It shows the SDS-PAGE gel electrophoresis analysis results of the genetically engineered bacteria expressing the bifunctional glutathione synthase mutants H1-H14 with correct sequencing of the mutants H1-H14;

[0088] Figure 3 It shows the SDS-PAGE gel electrophoresis analysis results of the genetically engineered bacteria expressing the bifunctional glutathione synthase mutants H15-H19 of the double mutants H15-H19;

[0089] Figure 4 It shows the determination of single-site directed mutation sites of DpPPK2-III by a modeling tool, where A shows the RMSF values of sites D77, K98, T101, L103, Q106, and F264 in the whole protein; B shows the three-dimensional model structure of the DpPPK2-III protein;

[0090] Figure 5 It shows the SDS-PAGE gel electrophoresis analysis results of the genetically engineered bacteria expressing the polyphosphate kinase mutants H1-H10 with correct sequencing of the mutants H1-H10;

[0091] Figure 6 It shows the yields of glutathione at different reaction times in the enzymatic synthesis method of glutathione in Example 17. Detailed implementation mode

[0092] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as limiting the present invention.

[0093] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise stated, the meaning of "plurality" is two or more.

[0094] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the values between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0095] To facilitate a better understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein shall have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0096] As used herein, the term "comprising" or "including" is an open-ended expression, meaning it includes the content specified in the present invention but does not exclude other aspects.

[0097] As used herein, the terms "optionally", "optional" or "option" generally mean that the subsequent event or condition may or may not occur, and this description includes the cases where the event or condition occurs and the cases where it does not occur.

[0098] According to a specific embodiment of the present invention, the present invention provides a method for preparing glutathione, comprising:

[0099] Using L-glutamic acid or its salt, glycine, and L-cysteine as precursors, and utilizing mutants of bifunctional glutathione synthetase and mutants of polyphosphate kinase to carry out an enzyme-catalyzed reaction to synthesize glutathione,

[0100] wherein the amino acid sequence of the mutant of bifunctional glutathione synthetase is obtained by site-directed mutagenesis of at least one of positions 597, 607, 609, 610, 728, and 733 in the amino acid sequence of wild-type bifunctional glutathione synthetase,

[0101] and the amino acid sequence of the mutant of polyphosphate kinase is obtained by site-directed mutagenesis of at least one of positions 77, 103, and 106 in the amino acid sequence of wild-type polyphosphate kinase.

[0102] The site-directed mutagenesis therein may be single-point mutagenesis or multi-point combined mutagenesis.

[0103] Regarding wild-type bifunctional glutathione synthase, including but not limited to bifunctional glutathione synthase GshFST from Streptococcus thermophilus (GenBank: WP_011226281.1), the amino acid sequence of which is shown in SEQ ID NO:1. Bifunctional glutathione synthase derivatives having at least 80% (such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%) or more, or at least 90% (such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%) or more homology with wild-type bifunctional glutathione synthase GshFST (GenBank: WP_011226281.1). These bifunctional glutathione synthase derivatives maintain the γ-glutamylcysteine (γ-GC) synthase (γ-GCS) activity and glutathione synthase (GS) activity of the bifunctional glutathione synthase itself, only the amino acid sequence is slightly different compared to the amino acid sequence of the bifunctional glutathione synthase shown in wild-type SEQ ID NO:1. The above-mentioned bifunctional glutathione synthase GshFST from Streptococcus thermophilus and its derivatives are all included within the scope of the wild-type bifunctional glutathione synthase described in the present invention.

[0104] Regarding wild-type polyphosphate kinases, including but not limited to polyphosphate kinase DpPPK2-III from Deinococcus proteolyticus (GenBank: WP_013615652), whose amino acid sequence is shown in SEQ ID NO: 35. Derivatives of polyphosphate kinases with at least 80% (such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%) or more, or at least 90% (such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%) homology with polyphosphate kinase DpPPK2-III of wild-type polyphosphate kinase from Deinococcus proteolyticus. These derivatives of polyphosphate kinases maintain the enzyme activity of catalyzing AMP or ADP of the polyphosphate kinase itself, only the amino acid sequence is slightly different compared with the amino acid sequence of the polyphosphate kinase shown in wild-type SEQ ID NO: 35. The above-mentioned polyphosphate kinase DpPPK2-III from Deinococcus proteolyticus and its derivatives are all included within the scope of the wild-type polyphosphate kinases described in the present invention.

[0105] According to a specific embodiment of the present invention, the above site-directed mutagenesis can be one of the following (single-point mutation) or a combination of two or more of them (multi-point mutation): (1) Lysine K at position 597 is mutated to arginine R; (2) Lysine K at position 607 is mutated to arginine R; (3) Alanine at position 609 is mutated to glutamic acid; (4) Alanine at position 609 is mutated to arginine; (5) Asparagine N at position 610 is mutated to tyrosine Y; (6) Tyrosine Y at position 728 is mutated to tryptophan W; (7) Proline P at position 733 is mutated to histidine H; (8) Proline P at position 733 is mutated to arginine R.

[0106] According to a preferred embodiment of the present invention, compared with the amino acid sequence of the wild-type bifunctional glutathione synthetase, the mutant of the bifunctional glutathione synthetase provided by the present invention has the following mutations 1)-12):

[0107] 1) Lysine at position 597 is mutated to arginine (mutant K597R); or

[0108] 2) Lysine at position 607 is mutated to arginine (mutant K607R); or

[0109] 3) Alanine at position 609 is mutated to glutamic acid; or

[0110] 4) The alanine at position 609 is mutated to arginine; or

[0111] 5) The asparagine at position 610 is mutated to tyrosine (mutant N610Y); or

[0112] 6) The tyrosine at position 728 is mutated to tryptophan (mutant Y728W); or

[0113] 7) The proline at position 733 is mutated to histidine (mutant P733H); or

[0114] 8) The proline at position 733 is mutated to arginine (mutant P733R); or

[0115] 9) The tyrosine at position 728 is mutated to tryptophan, and the lysine at position 597 is mutated to arginine (mutant Y728W-K597R); or

[0116] 10) The tyrosine at position 728 is mutated to tryptophan, and the lysine at position 607 is mutated to arginine (mutant Y728W-K607R); or

[0117] 11) The tyrosine at position 728 is mutated to tryptophan, and the asparagine at position 610 is mutated to tyrosine (mutant Y728W-N610Y); or

[0118] 12) The tyrosine at position 728 is mutated to tryptophan, and the proline at position 733 is mutated to histidine (mutant Y728W-P733R).

[0119] According to a specific embodiment of the present invention, the above site-directed mutation can be one of the following (single-point mutation) or a combination of two or more of them (multi-point mutation): (1) Aspartic acid D at position 77 is mutated to asparagine N; (2) Leucine L at position 103 is mutated to asparagine N; (3) Glutamine Q at position 106 is mutated to lysine K.

[0120] According to a preferred embodiment of the present invention, the amino acid sequence of the mutant of the polyphosphate kinase has a mutation of any one of the following (1)-(4):

[0121] (1) Aspartic acid D at position 77 is mutated to asparagine N (mutant D77N);

[0122] (2) Leucine L at position 103 is mutated to asparagine N (mutant L103N);

[0123] (3) Glutamine Q at position 106 is mutated to lysine K (mutant Q106K);

[0124] (4) The leucine L at position 103 was mutated to asparagine N (mutant L103N), and the glutamine Q at position 106 was mutated to lysine K (mutant L103N-Q106K).

[0125] In a preferred embodiment of the present invention, the inventors separately inoculated the bifunctional glutathione synthetase mutant H16 containing the double-point mutations K607R and Y728W into a fermentation medium for fermentation culture, and obtained the wet cells of the mutant from the fermentation product; the wet cells were disrupted to obtain a cell lysate, and the supernatant of the lysate was obtained by centrifugation to obtain a crude enzyme solution.

[0126] In a preferred embodiment of the present invention, the inventors separately inoculated the polyphosphate kinase mutant H13 containing the double-point mutations L103N and Q106K into a fermentation medium for fermentation culture, and obtained the wet cells of the mutant from the fermentation product; the wet cells were disrupted to obtain a cell lysate, and the supernatant of the lysate was obtained by centrifugation to obtain a crude enzyme solution.

[0127] In order to further optimize the optimal conditions for synthesizing glutathione, the inventors conducted an orthogonal experiment with 8 factors and 5 levels to explore the optimal pH, substrate concentration and other conditions for synthesizing glutathione.

[0128] According to a preferred embodiment of the present invention, the method for preparing glutathione comprises:

[0129] Mix 150 mM Tris (pH 8.5), 50 mM MgCl2, 4 mM AMP, 200 mM sodium glutamate, 200 mM glycine, 100 mM cysteine, 30 mM sodium hexametaphosphate, 10 g / L sodium sulfite, 35 g / L mutant of bifunctional glutathione synthetase and 22.5 g / L mutant of polyphosphate kinase at working concentration, and react at 42 °C to synthesize glutathione.

[0130] The present invention provides that the nucleic acid sequence encoding the bifunctional glutathione synthetase mutant H16 is as shown in SEQ ID NO: 61, and the amino acid sequence is as shown in SEQ ID NO: 62. The nucleic acid sequence of the polyphosphate kinase mutant H13 is as shown in SEQ ID NO: 63, and the amino acid sequence is as shown in SEQ ID NO: 64

[0131]

[0132] The amino acid sequence encoded by the bifunctional glutathione synthetase mutant H16 is shown in SEQ ID NO: 62: MTLNQLLQKLEATSPILQANFGIERESLRVDRQGQLVHTPHPSCLGARSFHPYIQTDFCEFQMELITPVAKSTTEARRFLGAITDVAGRSIATDEVLWPLSMPPRLKAEEIQVAQLENDFERHYRNYLAEKYGTKLQAISGIHYNMELGKDLVEALFQESDQTDMIAFKNALYLKLAQNYLRYRWVITYLFGASPIAEQGFFDQEVPEPMRSFRNSDHGYVNKEEIQVSFVSLEDYVSAIETYIEQGDLIAEKEFYSAVRFRGQKVNRSFLDKGITYLEFRNFDLNPFERIGISQTTMDTVHLLILAFLWLDSPENVDQALAQGHALNEKIALSHPLEPLPSEAKTQDIVTALDQLVQHFGLGDYHQDLVKQVKAAFADPNQTLSAQLLPYIKDKSLAEFALNKALAYHDYDWTAHYALKGYEEMELSTQMLLFDAIQKGIHFEILDEQDQFLKLWHQDHVEYVKNGNMTSKDNYVVPLAMANKTVTKKILADASFPVPSGDEFTSLEEGLAYYPLIKDKQIVVKPKSTNFGLGISIFQEPASLDNYQKALEIAFAEDTSVLVEEFIPGTEYRFFILDGRCEAVLLRVAANVIGDGKHTIRELVAQRNANPLRGRDHRSPLEIIELGDIEQLMLAQQGYTPDDILPEGKKVNLRRNSNISTGGDSIDVTETMDSSYQELAAAMATSMGAWACGVDLIIPDETQIATKENPHCTCIELNFNPSMYMHTWCAEGPGQAITTKILDKLFPEIVAGQT。

[0133] The coding sequence of the polyphosphate kinase mutant H13 is shown in SEQ ID NO: 63:

[0134] ATGAATAGTAAAGCTTCACAGCAACTACAAGTCCCGCCTGGTCAGAAGGTGCGTTTGGCGGATTAC

[0135] AGCACCGATACCTTTGAGGGTAGCGCAGCGCTCGATAAAGAACAGGTTAAGCAAGCAACGGAACA

[0136] GCTGCAACAGCGTTTGTCTGAACTGCAAGAAAAGCTGTATGCGGAAGGTAAACAGAGCCTGTTGA

[0137] TCATTCTGCAAGCTAGAGACGGCGGTGGCAAGGACAGCACCGTTAGCCGCGTGATGGGTGCATTTA

[0138] ACCCAAATGGTGTTCACGTGGCCAACTTCAAAGCGCCAACGGATAATGAGCTGAAACACGATTTTC

[0139] TGTGGCGTATTCATCAGCAAGTTCCGTCGCACGGCATGATTGCCGTTTTTAACCGTAGTCATTATGA

[0140] GGACGTTTTGGTCACCCGCGTTCACGGCCTGATTGACGACGCGCGTGCGCAGCAAAACCTGGAGC

[0141] ACATTGTGAACTTCGAGAAGTTGCTCTCTGATGCTGGCACCCGCATCGTGAAGTTCTACCTGCACCT

[0142] GTCCCCGGAAGAACAAAAGGCACGTATGGAAGACCGCTTGAATGATCCGGCGAAACATTGGAAAT

[0143] TCAACCCGTCCGACCTAAAAGATCGTGCTTTATGGCATGAATATACCGCGGCGTACGAGGACGCCCT

[0144] TGCGACGAGCCGTTCCTATGCACCGTGGTATATCATCCCGGCTGATCGTAAATGGCTGCGTGACTAC

[0145] CTGATCAGCGAGATCCTGGTGCAGACCCTGGAGGAGATGAATCCGCAGTTTCCGACTGCGCATTTCGAGGCTGCGTACTACCTGATTGAAGACATCCCGTCACGCTAA。

[0146] The amino acid sequence encoded by the polyphosphate kinase mutant H13 is shown in SEQ ID NO: 64 as follows:

[0147] MNSKASQQLQVPPGQKVRLADYSTDTFEGSAALDKEQVKQATEQLQQRLSELQEKLYAEGKQSLLIIL

[0148] QARDGGGKDSTVSRVMGAFNPNGVHVANFKAPTDNELKHDFLWRIHQQVPSHGMIAVFNRSHYEDV

[0149] LVTRVHGLIDDARAQQNLEHIVNFEKLLSDAGTRIVKFYLHLSPEEQKARMEDRLNDPAKHWKFNPSD

[0150] LKDRALWHEYTAAYEDALATSRSYAPWYIIPADRKWLRDYLISEILVQTLEEMNPQFPTAHFEAAYYLIEDIPSR。

[0151] The solution of the present disclosure will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. For those without specific techniques or conditions noted in the embodiments, the techniques or conditions described in the literature in this field or according to the product specifications are followed. Those reagents or instruments without the manufacturer noted are all conventional products that can be obtained through commercial purchase.

[0152] Materials and Methods

[0153] 1) Materials: Plasmid pET28a was purchased from Changsha Abiway Biotech Co., Ltd.; Restriction endonucleases such as BamHI and HindIII, restriction endonucleases such as NdeI and XhoI, high-fidelity enzyme premix, and one-step rapid cloning kit were purchased from Shanghai Yisheng Biotech Co., Ltd.; Synthesized primers, E. coli BL21(DE3) competent cells, DNA marker, plasmid extraction kit, DNA gel recovery and purification kit, kanamycin sulfate, and isopropyl-β-D-thiogalactoside were all purchased from Shanghai Sangon Biotech Co., Ltd.; Chemical reagents were all of analytical grade from Sinopharm. The operation steps for plasmid extraction were referred to the plasmid extraction kit instruction manual; The operation steps for DNA gel recovery were referred to the DNA gel recovery and purification kit instruction manual; The operation steps for DNA fragment ligation were referred to the one-step rapid cloning kit instruction manual.

[0154] 2) LB medium (g / L): Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, sterilized at 121 °C for 20 min.

[0155] 3) Substrate reaction solution for bifunctional glutathione synthetase GshFST: 20 mM sodium L-glutamate, 20 mM glycine, 20 mM L-cysteine, 20 mM magnesium chloride, 40 mM adenosine triphosphate (ATP), pH 8.0;

[0156] 4) Substrate reaction solution for polyphosphate kinase DpPPK2-III: 6 mM adenosine diphosphate (ADP) or adenosine monophosphate (AMP), 20 mM sodium hexametaphosphate, 20 mM magnesium chloride, pH 8.0;

[0157] 5) Determination of the enzyme activity of bifunctional glutathione synthetase: Accurately measure the supernatant of the sonicated bacterial solution, add it to the pre-warmed substrate reaction solution at 37 °C to make the final concentration of the bacteria 5 mg / mL. Then, stir and react in a 37 °C water bath for 10 min, add an equal volume of 20% trichloroacetic acid solution to terminate the reaction, shake well and centrifuge, and take the supernatant for HPLC analysis, using GSH (reduced glutathione) and GSSG (oxidized glutathione) standards as controls;

[0158] 6) Determination of enzyme activity: Accurately measure the supernatant of the sonicated bacterial solution, add it to the pre-warmed substrate reaction solution at 37 °C to make the final concentration of the bacteria 5 mg / mL. Then, stir and react in a 37 °C water bath for 10 min, add an equal volume of 20% trichloroacetic acid solution to terminate the reaction, shake well and centrifuge, and take the supernatant for HPLC analysis, using the ATP standard as a control;

[0159] 7) Fermentation medium (g / L): peptone 10 g / L, yeast powder 5 g / L, glycerol 10 g / L, disodium hydrogen phosphate dodecahydrate 20 g / L, potassium dihydrogen phosphate 7 g / L, ammonium chloride 5 g / L, anhydrous sodium sulfate 1 g / L, and magnesium sulfate heptahydrate 1 g / L, antifoaming agent 0.5 ml / L, sterilized at 121 °C for 30 min;

[0160] 8) HPLC analysis method: Hypersil ODS-2 C18 chromatographic column (4.6×250 mm, 5 μm); flow rate 1.0 mL / min; detection wavelength 210 nm; mobile phase: methanol: 0.05 mol / L potassium dihydrogen phosphate (pH 2.30) = 10:90.

[0161] 9) Definition of enzyme activity unit: Under the conditions of pH 8.0 and temperature 37 °C, the amount of enzyme required to produce 1 micromole (μmol) of GSH / ATP per unit time is defined as 1 U.

[0162] Example 1: Construction of a genetically engineered bacterium of wild-type bifunctional glutathione synthetase GshFST

[0163] (1) Synthesis of wild-type gshF gene

[0164] According to the information publicly available in the NCBI database, the bifunctional glutathione synthetase GshFST (GenBank: WP_011226281.1) derived from Streptococcus thermophilus was selected, and its amino acid sequence is shown in SEQ ID NO:1. According to the codon preference rule of Escherichia coli, the gene coding sequence was codon-optimized. After designing and adding BamHI and HindIII restriction enzyme sites at both ends of the coding sequence, it was sent to Nanjing Genscript Biotech Co., Ltd. for artificial synthesis (SEQ ID NO:2).

[0165] The sequence of the gshF gene coding sequence after codon optimization is shown in SEQ ID NO:2:

[0166]

[0167] (2) Construction of wild-type GshFST pET28a vector

[0168] Primers for constructing the design vector. Using 1-F (5'-atcgtggcgggtcaaaccaagcttgcggccgcactc-3', as shown in SEQ ID NO: 3) / 1-R (5'-tagctgattcaatgtcatggatccgcgacccatttg-3', as shown in SEQ ID NO: 4) as primers, and the purchased pET28a vector as the template, 2×Hieff Plus PCR Master Mix(With Dye) high-fidelity enzyme premix was used to amplify the linearized vector by PCR; using 2-F (5'-caaatgggtcgcggatccatgacattgaatcagcta-3', as shown in SEQ ID NO: 5) / 2-R (5'-gagtgcggccgcaagcttggtttgacccgccacgat-3', as shown in SEQ ID NO: 6) as primers, and the gshF gene DNA fragment (SEQ ID NO: 2) synthesized by GenScript as the template, the gshF gene DNA fragment with the sequences at both ends exactly the same as those at both ends of the linearized vector at the 5' and 3' ends was amplified by high-fidelity enzyme premix PCR; detected by 1% agarose gel electrophoresis, and the amplified linearized vector (5.4 kb) and gshF gene (2.3 kb) DNA fragments were recovered using a DNA gel recovery and purification kit. Using Hieff Plus One Step Cloning Kit one-step rapid cloning kit to perform homologous recombination on the gel-recovered linearized vector and gshF gene fragment. The recombination system was: 2.5 μL of pET28a linearized vector, 2.5 μL of gshF gene fragment, 2×Hieff Enzyme Premix 5 μL. React at 50 °C for 30 min.

[0169] (3) Construction of wild-type GshFST genetic engineering bacteria

[0170] The homologous recombination product obtained in (2) was transformed into Escherichia coli BL21(DE3) competent cells by heat shock method. The transformed product was spread on an LB plate containing 50 μg / mL kanamycin sulfate and cultured overnight at 37 °C. Then, positive transformants were selected and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The transformants with correct sequencing contained the plasmid vector GshFST-pET28a with the wild-type gshF gene, and this transformant was also the wild-type GshFST genetic engineering bacteria.

[0171] Example 2: Determination of a single site-directed mutation site of GshFST

[0172] The protein modeling of the sequence of bifunctional glutathione synthase was performed using the online software of the new artificial intelligence software tool RoseTTA Fold2 (http: / / robetta.bakerlab.org / ), and its three-dimensional protein spatial structure was obtained as Figure 1 shown. The PyMol was used to perform visual analysis and processing on its model. The PROCHECK and Verify3D programs were used to evaluate the model. The Ramachandran plot is used to elaborate the ratio of two dihedral angles ( and ψ) in the peptide plane to indicate the allowed and disallowed conformations of amino acid residues. The reliability of the protein model was evaluated by the Psi / Phi Ramachandran plot calculated by PROCHECK. The compatibility of the side-chain amino acid residues of the protein was evaluated by the Verify3D software. The verification was passed if at least 80% of the amino acid residues scored ≥ 0.2. The molecular docking was performed using the glide-dock molecular docking program in the Schrodinger Suites software. Based on the reported binding sites in the literature, the simulated structure was analyzed, and it was observed that the structure of GshFST is a homodimer, and each monomer contains 2 domains and a connecting region. It is speculated that the first domain (1-440) is responsible for catalyzing the reaction of glutamate and cysteine to generate the γ-glutamyl-cysteine (γ-Glu-Cys) intermediate, and the second domain (520-754) is responsible for catalyzing the synthesis of GSH from γ-Glu-Cys and glycine. The presence of the γ-Glu-Cys intermediate was significantly observed during the reaction of GshFST catalyzing the synthesis of GSH from glutamate, cysteine and glycine. It is speculated that the main reason for restricting the enzyme activity of GshFST is in the second domain. Therefore, the rationally designed points were screened, mainly focusing on the second domain. The semi-flexible molecular docking of its protein partial region and small molecule ligand was performed, and the amino acids within the range of its ligand were subjected to site-directed saturation virtual screening, and the binding free energy of the ligand and the protein was calculated by Rosetta Flex ddG. The program was used to calculate the binding free energy of various docking complexes respectively. The lower the value, the following Table 1 is the screened table. The binding free energy in the table indicates the higher the affinity between the receptor and the ligand, so as to improve the enzyme activity and obtain the mutation sites.

[0173] Table 1:

[0174] Mutation site <![CDATA[ΔG (Binding) (Kcal / mol) <!-- 11 -->]]> H598R -32.8 K607R -31.3 A609E -35.1 A609R -30.3 N610Y -30.7 H617Y -42.6 H711F -31.2 T713W -33.2 Y728W -38.1 A730T -39.6 A730L -31.5 P733H -32.6 P733R -34.9 K597R -35.9 WT -29.1

[0175] Example 3: Preparation and expression of GshFST single mutants

[0176] Site-directed mutagenesis was performed using the original plasmid as a template to obtain the recombinant plasmid GshFST-X-pET28a carrying the mutant bifunctional glutathione synthetase-encoding gene. The site-directed mutagenesis method was as follows:

[0177] The primer sequences used to design the mutants are shown in Table 2 (the bold and underlined parts in the primer sequences are the bases corresponding to the mutation sites):

[0178] Table 2:

[0179]

[0180]

[0181] Using K597R-F / 2-R as primers and the GshFST-pET28a plasmid as a template, a small fragment gene with the K597R mutation site was amplified by PCR using the 2×Hieff Plus PCRMaster Mix (With Dye) high-fidelity enzyme premix. Using 1-F / K597R-R as primers and the GshFST-pET28a plasmid as a template, large fragment DNAs with sequences identical to the two ends of the small fragment gene at the 5' and 3' ends were amplified by PCR using the high-fidelity enzyme premix. The amplified DNA fragments of different sizes were detected by 1% agarose gel electrophoresis, and the DNA of the amplified fragments of different sizes was recovered using a DNA gel recovery and purification kit. The DNA fragments of different sizes recovered by gel were subjected to homologous recombination using the Hieff Plus One Step Cloning Kit one-step rapid cloning kit. The recombination system was: 2.5 μL of large fragment DNA, 2.5 μL of small fragment DNA, and 5 μL of 2×Hieff Enzyme Premix. React at 50 °C for 30 min.

[0182] The obtained homologous recombination product was transformed into Escherichia coli BL21(DE3) competent cells by heat shock method. The transformed product was spread on an LB plate containing 50 μg / mL kanamycin sulfate. After overnight culture at 37°C, positive transformants were selected and sent to Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. for sequencing. The transformants with correct sequencing contained the plasmid vector GshFST-K597R-pET28a with the single mutant gshF gene, and this transformant was also the genetically engineered bacterium of the single mutant GshFST-K597R, numbered H1. Engineered bacteria H2 with H598R mutation, H3 with K607R mutation, H4 with A609E mutation, H5 with A609R mutation, H6 with N610Y mutation, H7 with H617Y mutation, H8 with H711F mutation, H9 with T713W mutation, H10 with Y728W mutation, H11 with A730T mutation, H12 with A730L mutation, H13 with P733H mutation, and H14 with P733R mutation were constructed in the same manner.

[0183] The genetically engineered bacteria of mutants H1 - H14 with correct sequencing were respectively inoculated into test tubes containing 4 mL of LB medium with 50 μg / mL kanamycin. After overnight culture at 37°C and 200 rpm, they were transferred to 50 mL of LB medium and cultured at 37°C and 200 rpm until the OD 600 reached 0.6 - 0.8. IPTG was added to a final concentration of 0.2 mM, and the culture continued at 20°C and 200 rpm for 18 h. After the fermentation broth was centrifuged at 9000 rpm for 5 min, the supernatant was removed, and the cell pellet was retained. The cell pellet was resuspended in water to a cell concentration of 100 mg / mL, sonicated for 10 min, and the precipitate was removed by centrifugation. The supernatant was retained for SDS-PAGE gel electrophoresis analysis. The results were as Figure 2 shown, indicating that these mutant proteins were all normally expressed.

[0184] Example 4: Enzyme activity assay of GshFST single mutants

[0185] 50 μL of enzyme solution was added to a centrifuge tube containing 950 μL of substrate solution (containing 20 mM L-sodium glutamate, 20 mM glycine, 20 mM L-cysteine, 20 mM magnesium chloride, and 40 mM ATP in water), and the mixture was placed at 37°C for reaction for 10 min. After the reaction ended, an equal volume of 20% trichloroacetic acid solution was added to terminate the reaction. After shaking well, the mixture was centrifuged, and the supernatant was taken for HPLC analysis, with GSH (reduced glutathione) and GSSG (oxidized glutathione) standards as controls respectively.

[0186] HPLC analysis method: ODS-2 HypersilTM (4.6×250 mm, 5 μm); flow rate 1.0 mL / min; detection wavelength 210 nm; mobile phase for GshFST enzyme reaction: methanol: 0.05 mol / L potassium dihydrogen phosphate (pH 2.30) = 10:90.

[0187] The results of relative enzyme activities measured for each single mutant are shown in Table 3:

[0188] Table 3:

[0189] Name Mutation site Relative enzyme activity WT - 100.0％ H1 K597R 130.0％ H2 H598R 104.9％ H3 K607R 142.4％ H4 A609E 111.5％ H5 A609R 119.9％ H6 N610Y 120.8％ H7 H617Y 45.0％ H8 H7I1F 74.8％ H9 T713W 84.0％ H10 Y728W 166.3％ H11 A730T 108.1％ H12 A730L 94.2％ H13 P733H 125.6％ H14 P733R 122.7％

[0190] From the results in Table 3, it can be seen that the enzyme activities of the mutants containing single point mutations of K597R (H1), K607R (H3), A609E (H4), A609R (H5), N610Y (H6), Y728W (H10), P733H (H13), and P733R (H14) are significantly increased compared to the wild type. Among them, the enzyme activity of the mutant H10 containing Y728W increases the most, which is 1.66 times that of the wild type enzyme activity.

[0191] Example 5: Preparation and expression of GshFST double mutants

[0192] Select 6 variants from the mutant sites with relatively large increases (more than 20% increase) compared to the wild type enzyme activity, namely H1, H3, H6, H10, H13, and H14 in Table 3. Analyze the base sequences of the corresponding genes and analyze the amino acid mutation information, perform combined mutations and expressions. The method for constructing the expression vector and the preparation method are the same as in Example 3 to obtain double mutants, and the protein expression and relative enzyme activities of the obtained double mutants are measured using the same method as in Example 4. The results are as Figure 3 and shown in Table 4:

[0193] Table 4:

[0194] Name Mutation site Relative enzyme activity WT - 100.0％ H15 Y728W-K597R 189.9％ H16 Y728W-K607R 210.3％ H17 Y728W-N610Y 175.2％ H18 Y728W-P733H 123.8％ H19 Y728W-P733R 101.5％

[0195] From the results in Table 4, it can be seen that the enzyme activity of the mutant H16 containing the double point mutations of K607R and Y728W is the highest compared to the wild type enzyme activity, which is 2.10 times that of the wild type enzyme activity.

[0196] Example 6: Determination of the thermal stability of GshFST mutants with increased enzyme activity and the wild type

[0197] The protein thermal stability was determined using the supernatant of the lysates of the 12 high-enzymatic-activity mutants (H1, H3 - H6, H10, H13 - H18) and WT obtained in Example 4 and Example 5 above. The obtained supernatant of the lysates was treated at 30 °C, 35 °C, 40 °C, 45 °C, 55 °C, and 60 °C for 30 min respectively, then cooled in an ice-water bath, and subsequently taken out to 37 °C to measure its residual activity. The specific data are shown in Table 5 below:

[0198] Table 5:

[0199]

[0200]

[0201] The above results show that among the 12 high-enzymatic-activity mutants (H1, H3 - H6, H10, H13 - H18), the thermal stability of the 5 mutants (H10, H15 - H18) containing Y728W is slightly improved compared to the wild type WT. That is, at 40 °C, their activity begins to decline, and when the temperature rises to 45 °C, their activity only loses 20 - 26%, while the wild type and the mutants without the Y728W mutation lose 30 - 40% of their activity at 45 °C. The above examples illustrate that Y728W has a certain effect on improving enzymatic activity and thermal stability.

[0202] Example 7: Construction of a genetic engineering bacterium of wild-type polyphosphate kinase DpPPK2-III

[0203] (1) Synthesis of wild-type ppk2-III gene

[0204] According to the information publicly available in the NCBI database, polyphosphate kinase DpPPK2-III (GenBank: WP_013615652) derived from Deinococcus proteolyticus was selected, and its amino acid sequence is shown as SEQ ID NO:35. The gene coding sequence was codon-optimized according to the codon preference rule of Escherichia coli, and NdeI and XhoI restriction sites were designed and added at both ends of the coding sequence, and then sent to Nanjing Genscript Biotech Co., Ltd. for artificial synthesis (SEQ ID NO:36).

[0205] The sequence of the ppk2-III gene coding sequence after codon optimization is shown as SEQ ID NO:36:

[0206]

[0207] (2) Construction of wild-type DpPPK2-III pET28a vector

[0208] Primers for constructing the design vector. Using 1-F (5'-ctcgagcaccaccaccaccac-3', as shown in SEQ ID NO:37) / 1-R (5'-atggctgccgcgcggcaccag-3', as shown in SEQ ID NO:38) as primers, and the purchased pET28a vector as the template, 2×Hieff Plus PCR Master Mix(With Dye) high-fidelity enzyme premix was used for PCR amplification to obtain a linearized vector; using 2-F (5'-gtgccgcgcggcagccatatgaatagtaaagcttca-3', as shown in SEQ ID NO:39) / 2-R (5'-gtggtggtgctcgagttagcgtgacgggatgtc-3', as shown in SEQ ID NO:40) as primers, and the ppk2-III gene DNA fragment (SEQ ID NO:36) synthesized by GenScript as the template, high-fidelity enzyme premix was used for PCR amplification to obtain a ppk2-III gene DNA fragment with sequences at both the 5' and 3' ends that are exactly the same as the sequences at both ends of the linearized vector; detected by 1% agarose gel electrophoresis, and the amplified linearized vector (5.3 kb) and ppk2-III gene (0.9 kb) DNA fragments were recovered using a DNA gel recovery and purification kit. Using Hieff Plus One Step Cloning Kit for one-step rapid cloning, the linearized vector recovered from the gel and the ppk2-III gene fragment were subjected to homologous recombination. The recombination system was: 2.5 μL of pET28a linearized vector, 2.5 μL of ppk2-III gene fragment, 2×Hieff Enzyme Premix 5 μL. React at 50 °C for 30 min.

[0209] (3) Construction of wild-type DpPPK2-III gene engineering bacteria

[0210] The homologous recombination product obtained in (2) was transformed into Escherichia coli BL21(DE3) competent cells by heat shock method. The transformed product was spread on an LB plate containing 50 μg / mL kanamycin sulfate and cultured overnight at 37 °C. Then, positive transformants were selected and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The transformants with correct sequencing were plasmid vectors DpPPK2-III-pET28a containing the wild-type ppk2-III gene, and these transformants were also wild-type DpPPK2-III gene engineering bacteria.

[0211] Example 8: Determination of a single site-directed mutation site of DpPPK2-III

[0212] The sequence of polyphosphate kinase DpPPK2-III was subjected to protein homology modeling using the Siwss-model. The modeling template was selected as PDB ID: 7bmm, with a sequence similarity of 55%. The three-dimensional spatial structure of its protein was obtained, and PyMol was used to perform visual analysis and processing on the model. The model was evaluated using the PROCHECK and Verify-3D programs. The Ramachandran plot is used to illustrate the ratio of two dihedral angles ( and ψ) in the peptide plane to indicate the allowed and disallowed conformations of amino acid residues. The reliability of the protein model was evaluated through the Psi / Phi Ramachandran plot calculated by PROCHECK. The compatibility of the side-chain amino acid residues of the protein was evaluated using the Verify-3D software. The verification was passed if at least 80% of the amino acid residues had a score ≥ 0.2. Molecular dynamics simulations were performed using the Desmond molecular dynamics simulation program in the Schrodinger Suites software. Molecular dynamics simulations with a running duration of 20 ns were carried out, and it was found that the RMSF (Root Mean Square Fluctuation) values of the protein at sites D77, K98, T101, L103, Q106, and F264 were relatively high throughout the protein (as Figure 4 shown). Then, virtual screening of site-directed saturation mutations at the above sites was performed through Rosetta Flex ddG, and the binding free energies of the ligand and the protein (one mutant type and the wild type) were calculated respectively. The program was used to calculate the binding free energies of various docking complexes. The lower the value, the higher the calculated affinity between the receptor and the ligand, thereby improving the enzyme activity and obtaining its mutation sites.

[0213] Example 9: Preparation and expression of DpPPK2-III single mutants

[0214] Using the original plasmid (DpPPK2-III-pET28a) as a template for site-directed mutagenesis, a recombinant plasmid DpPPK2-III-X-pET28a carrying the coding sequence of the polyphosphate kinase mutant was obtained. The method of site-directed mutagenesis is as follows:

[0215] The primer sequences used to design and obtain the mutants are shown in Table 6 (the bold and underlined parts in the primer sequences are the bases corresponding to the mutation sites):

[0216] Table 6:

[0217]

[0218]

[0219] Using 2-F / D77N-R as the primer and DpPPK2-III-pET28a plasmid as the template, a small fragment gene with the D77N mutation site was amplified by PCR using 2×Hieff PlusPCR Master Mix (With Dye) high-fidelity enzyme premix; using D77N-F / 1-R as the primer and DpPPK2-III-pET28a plasmid as the template, large fragment DNAs with sequences exactly the same as the two ends of the small fragment gene at the 5' and 3' ends respectively were amplified by PCR with the high-fidelity enzyme premix; detected by 1% agarose gel electrophoresis, and the amplified large and small fragment DNAs were recovered using a DNA gel recovery and purification kit. Using Hieff Plus One Step Cloning Kit one-step rapid cloning kit to perform homologous recombination on the gel-recovered large and small fragment DNAs. The recombination system was: 2.5 μL of large fragment DNA, 2.5 μL of small fragment DNA, 5 μL of 2×Hieff Enzyme Premix. React at 50 °C for 30 min.

[0220] The obtained homologous recombination product was transformed into Escherichia coli BL21(DE3) competent cells by heat shock method. The transformation product was spread on an LB plate containing 50 μg / mL kanamycin sulfate. After overnight culture at 37 °C, positive transformants were selected and sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing. The transformant with correct sequencing was the plasmid vector DpPPK2-III-D77N-pET28a containing the single mutant ppk2-III gene, and this transformant was also the genetically engineered bacterium of the single mutant DpPPK2-III-D77N, numbered H1. Genetically engineered bacteria H2 with the K98E mutation, H3 with the T101C mutation, H4 with the L103F mutation, H5 with the L103N mutation, H6 with the L103Y mutation, H7 with the Q106E mutation, H8 with the Q106K mutation, H9 with the Q106A mutation, and H10 with the F264P mutation were constructed in the same way.

[0221] The genetically engineered bacteria of the mutants H1-H10 with correct sequencing were respectively inoculated into test tubes containing 4 mL of LB medium, where the LB medium contained 50 μg / mL kanamycin. After overnight culture at 37 °C and 200 rpm, they were transferred to 50 mL of LB medium and cultured at 37 °C and 200 rpm until OD 600It was 0.6 - 0.8. IPTG was added to a final concentration of 0.2 mM, and the culture was continued at 20 °C and 200 rpm for 18 h. After centrifuging the fermentation broth at 9000 rpm for 5 min, the supernatant was removed, and the cell precipitate was retained. The cell precipitate was resuspended in water to a cell concentration of 100 mg / mL, sonicated for 10 min, the precipitate was removed by centrifugation, and the supernatant was retained for SDS-PAGE gel electrophoresis analysis. The results are as Figure 5 shown, and the results indicate that these mutant proteins were all normally expressed.

[0222] Example 10: Enzyme activity assay of DpPPK2-III single mutants

[0223] Take 50 μL of enzyme solution and add it to a centrifuge tube containing 950 μL of substrate solution (containing 6 mM ADP or AMP, 20 mM sodium hexametaphosphate, and 20 mM magnesium chloride in water), and place it at 37 °C for reaction for 10 min. After the reaction is completed, add an equal volume of 20% trichloroacetic acid solution to terminate the reaction, shake well and centrifuge, and take the supernatant for HPLC analysis with an ATP standard as a control.

[0224] HPLC analysis method: ODS-2 HypersilTM (4.6×250 mm, 5 μm); flow rate 1.0 mL / min; detection wavelength 210 nm; mobile phase for DpPPK2-III enzyme reaction: methanol: 0.05 mol / L potassium dihydrogen phosphate (pH 2.30) = 5:95.

[0225] The relative enzyme activity results measured for each single mutant are shown in Table 7:

[0226] Table 7:

[0227]

[0228]

[0229] From the results in Table 7, it can be seen that when generating ATP with AMP or ADP as substrates, the enzyme activities of the mutants containing single point mutations D77N (H1), L103N (H5), and Q106K (H8) were significantly increased compared to the wild type. Among them, the mutant H8 containing Q106K had the most increased enzyme activity, which was 1.5 times (using AMP as the substrate) and 1.2 times (using ADP as the substrate) that of the wild type enzyme activity.

[0230] Example 11: Preparation and expression of DpPPK2-III double mutants

[0231] Select the sites (D77N, L103N, Q106K) that can improve enzyme activity in single-point mutations for combined mutations and expression. The construction method of the expression vector and the preparation method are the same as those in Example 9 to obtain a combined mutant. The relative enzyme activity of the obtained combined mutant is measured by the same method as in Example 10, and the results are shown in Table 8 as follows:

[0232] Table 8:

[0233] Name Mutation site Relative enzyme activity WT - 100.0％ H11 D77N-L103N 89.9％ H12 D77N-Q106K 97.5％ H13 L103N-Q106K 172.3％ H14 D77N-L103N-Q106K 72.0％

[0234] From the results in Table 8, it can be seen that the enzyme activity of mutant H13 with double-point mutations of L103N and Q106K is the highest compared to that of the wild type, which is 1.7 times that of the wild type enzyme activity.

[0235] Example 12: Determination of the thermal stability of the DpPPK2-III mutant with improved enzyme activity and the wild type

[0236] The protein thermal stability was measured using the supernatant of the lysates of the 4 highly enzyme-active mutants (H1, H5, H8, H13) and WT obtained in the above Examples 10 and 11. The obtained supernatant of the lysates was treated at 30 °C, 35 °C, 40 °C, 45 °C, 55 °C, and 60 °C for 30 min and then cooled in an ice-water bath. Subsequently, it was taken out and the residual activity was measured at 3 ℃. The specific data are shown in Table 9 below:

[0237] Table 9:

[0238]

[0239] The results in Table 9 show that the thermal stability of the 4 highly enzyme-active mutants (H1, H5, H8, H13) is basically unchanged compared to that of the wild type WT. That is, at 40 °C, their activity begins to decline. When the temperature rises to 45 °C, their activity loses about 50%. When the temperature rises to 60 °C, the activity is basically completely lost.

[0240] Example 13: Fermentation and culture of strains

[0241] 1 mL of the genetically engineered bacteria strains of glutathione synthetase mutant H16 and polyphosphate kinase mutant H13 were respectively taken and activated on an LB slant medium, and cultured at 37 °C for 16 h; one inoculation loop of bacterial growth was picked from the slant medium and transferred to an LB seed medium containing 100 mL, and cultured with shaking at 37 °C and 160 rpm for 13 h. The seeds were inoculated into a 5 L fermenter containing 3 L of fermentation medium at a volume ratio of 8%, the temperature was controlled at 37 °C, the pH was maintained at 7.0 during the fermentation process, the aeration rate was controlled at 2 vvm, the rotation speed was controlled at 550 rpm, and the dissolved oxygen was maintained at 30% by adjusting the rotation speed and the aeration rate. After 8 h of fermentation, the temperature was lowered and stabilized at 25 °C, and IPTG with a final concentration of 0.2 mM was added to induce for about 18 h. When the OD of the fermentation no longer increased, the fermentation ended.

[0242] The fermentation broth was centrifuged at 6000 rpm for 20 min, and the supernatant was discarded to obtain wet bacterial cells. The bacterial cells were resuspended in water to a final concentration of 100 g / L, and the bacterial solution was ultrasonically disrupted to obtain a cell lysate. After centrifugation, the supernatant of the lysate was obtained to obtain a crude enzyme solution.

[0243] Example 14: Enzymatic conversion using AMP / ADP as a substrate to produce GSH

[0244] In a 5 mL reaction system, 100 mM Tris (tris(hydroxymethyl)aminomethane), 4 mM sodium sulfite, 20 mM magnesium chloride, 12 mM sodium hexametaphosphate, 20 mM glycine, 20 mM sodium glutamate, 20 mM cysteine, 2 mM AMP or ADP, 35 g / L H16 and 17.5 g / L H13 were respectively added at a final concentration. The pH of the reaction system was adjusted to 8.0, and then the reaction was carried out in a 37 °C water bath for 30 min. After the reaction ended, an equal volume of 20% trichloroacetic acid solution was added to terminate the reaction. After shaking, it was centrifuged, and the supernatant was taken for HPLC analysis, using GSH (reduced glutathione) and GSSG (oxidized glutathione) standard products as controls respectively;

[0245] The results showed that: after 30 min of reaction, when using AMP as a substrate, 1.60 g / L of glutathione could be produced (the theoretical yield was 6.14 g / L), and the conversion rate was 26.1%; when using ADP as a substrate, 1.65 g / L of glutathione could be produced, and the conversion rate was 26.9%. It was shown that when using H13 and H16 to synthesize glutathione, the synthesis effects using AMP and ADP as substrates were not very different. Considering that AMP was cheaper than ADP in production, the present invention selected AMP as a substrate to synthesize glutathione.

[0246] Example 15: Enzymatic conversion using sodium metaphosphate / sodium hexametaphosphate / sodium polyphosphate as a substrate to produce GSH

[0247] In a 5 mL reaction system, add Tris (100 mM final concentration), sodium sulfite (4 mM), magnesium chloride (20 mM), AMP (2 mM), glycine (20 mM), sodium glutamate (20 mM), cysteine (20 mM), sodium metaphosphate or sodium hexametaphosphate or sodium polyphosphate (12 mM), H16 (35 g / L) and H13 (17.5 g / L) respectively. Adjust the pH of the reaction system to 8.0, and then react in a 37 °C water bath for 30 min. After the reaction is completed, add an equal volume of 20% trichloroacetic acid solution to terminate the reaction. Shake well and centrifuge, and take the supernatant for HPLC analysis, using GSH (reduced glutathione) and GSSG (oxidized glutathione) standards as controls respectively;

[0248] The results showed that: after 30 min of reaction, when using sodium metaphosphate as the substrate, 0.40 g / L of glutathione could be produced (the theoretical yield was 6.14 g / L), and the conversion rate was 6.5%; when using sodium hexametaphosphate as the substrate, 1.63 g / L of glutathione could be produced, and the conversion rate was 26.5%; when using sodium polyphosphate as the substrate, 0.74 g / L of glutathione could be produced, and the conversion rate was 12.1%. It indicated that when using H13 and H16 to synthesize glutathione, the synthesis effect of GSH with sodium hexametaphosphate as the substrate was better than that of sodium metaphosphate and sodium polyphosphate.

[0249] Example 16: Orthogonal experiment to determine the optimal level combination for synthesizing glutathione

[0250] To study the influence of key parameters on GSH synthesis, an orthogonal experiment with eight factors (buffer concentration, pH value, magnesium chloride concentration, AMP concentration, substrate ratio, sodium hexametaphosphate concentration, enzyme dosage, reaction temperature) and five levels was carried out as shown in Table 10:

[0251] Table 10:

[0252]

[0253]

[0254] After preparing 25 reaction systems according to the above table, place them in a 37 °C water bath and react for 2 h. After the reaction is completed, add an equal volume of 20% trichloroacetic acid solution to terminate the reaction. Shake well and centrifuge, and take the supernatant for HPLC analysis, using GSH (reduced glutathione) and GSSG (oxidized glutathione) standards as controls respectively. The specific results are shown in Table 11:

[0255] Table 11:

[0256]

[0257] According to the analysis of experimental results, the R values of various factors were calculated, as shown in Table 12. The larger the R value, the greater the influence. From the R ranges of various factors, the concentration of sodium hexametaphosphate, temperature, substrate concentration ratio, and buffer concentration are the key factors affecting the glutathione yield. According to the magnitudes of the k values of different factors, the optimal level combination for synthesizing glutathione was determined as: 150 mM Tris, pH 8.5, 50 mM MgCl2, 4 mM AMP, 200 mM sodium glutamate, 200 mM glycine, 100 mM cysteine, 30 mM sodium hexametaphosphate, 35 g / L H16, 22.5 g / L H13, 42 °C.

[0258] Table 12:

[0259]

[0260] Example 17: Enzymatic Synthesis of Glutathione

[0261] In a 1-L reaction system, 150 mM Tris (pH 8.5), 50 mM MgCl2, 4 mM AMP, 200 mM sodium glutamate, 200 mM glycine, 100 mM cysteine, 30 mM sodium hexametaphosphate, 10 g / L sodium sulfite, 35 g / L H16, and 22.5 g / L H13 were successively added. After mixing, the reaction was stirred at 42 °C, and samples were taken every 2 h to detect the amount of glutathione generated. The specific results are as Figure 6 shown. Under these reaction conditions, H16 and H13 can synthesize 25.2 g / L of glutathione in 12 h, and the conversion rate can reach 82.1%.

[0262] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0263] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing glutathione, characterized in that, Comprising: Using L-glutamic acid or its salt, glycine, and L-cysteine as precursors, and carrying out an enzymatic reaction to synthesize glutathione by using a mutant of bifunctional glutathione synthetase and a mutant of polyphosphate kinase. Wherein, the amino acid sequence of the mutant of bifunctional glutathione synthetase is obtained by performing site-directed mutagenesis on at least one of positions 597, 607, 609, 610, 728, and 733 of the amino acid sequence of wild-type bifunctional glutathione synthetase. The amino acid sequence of the mutant of polyphosphate kinase is obtained by performing site-directed mutagenesis on at least one of positions 77, 103, and 106 of the amino acid sequence of wild-type polyphosphate kinase.

2. The preparation method according to claim 1, wherein The amino acid sequence of the wild-type bifunctional glutathione synthetase is as shown in SEQ ID NO:1; The amino acid sequence of the wild-type polyphosphate kinase is as shown in SEQ ID NO:

35.

3. The preparation method according to claim 2, wherein Compared with the amino acid sequence of wild-type bifunctional glutathione synthetase, the amino acid sequence of the mutant of bifunctional glutathione synthetase has at least one of the following mutations (1)-(8): (1) Lysine at position 597 is mutated to arginine; (2) Lysine at position 607 is mutated to arginine; (3) Alanine at position 609 is mutated to glutamic acid; (4) Alanine at position 609 is mutated to arginine; (5) Asparagine at position 610 is mutated to tyrosine; (6) Tyrosine at position 728 is mutated to tryptophan; (7) Proline at position 733 is mutated to histidine; (8) Proline at position 733 is mutated to arginine.

4. The preparation method according to claim 2, characterized in that, Compared with the amino acid sequence of wild-type bifunctional glutathione synthetase, the amino acid sequence of the mutant of bifunctional glutathione synthetase has the following mutations 1)-12): 1) Lysine at position 597 is mutated to arginine; or 2) Lysine at position 607 is mutated to arginine; or 3) Alanine at position 609 is mutated to glutamic acid; or 4) Alanine at position 609 is mutated to arginine; or 5) Asparagine at position 610 is mutated to tyrosine; or 6) Tyrosine at position 728 is mutated to tryptophan; or 7) Proline at position 733 is mutated to histidine; or 8) Proline at position 733 is mutated to arginine; or 9) Tyrosine at position 728 is mutated to tryptophan, and lysine at position 597 is mutated to arginine; or 10) Tyrosine at position 728 is mutated to tryptophan, and lysine at position 607 is mutated to arginine; or 11) Tyrosine at position 728 is mutated to tryptophan, and asparagine at position 610 is mutated to tyrosine; or 12) Tyrosine at position 728 is mutated to tryptophan, and proline at position 733 is mutated to histidine.

5. The preparation method according to claim 4, characterized in that, Compared with the amino acid sequence of wild-type polyphosphate kinase, the amino acid sequence of the mutant of polyphosphate kinase has any one of the following mutations (1)-(4): (1) Aspartic acid at position 77 is mutated to asparagine; (2) Leucine at position 103 is mutated to asparagine; (3) Glutamine at position 106 is mutated to lysine; (4) The leucine at position 103 is mutated to asparagine, and the glutamine at position 106 is mutated to lysine.

6. The preparation method according to claim 2, characterized in that, Compared with the amino acid sequence of the wild-type bifunctional glutathione synthetase, the amino acid sequence of the mutant of the bifunctional glutathione synthetase has mutations where tyrosine at position 728 is mutated to tryptophan and lysine at position 607 is mutated to arginine; Compared with the amino acid sequence of the wild-type polyphosphate kinase, the amino acid sequence of the mutant of the polyphosphate kinase has mutations where leucine at position 103 is mutated to asparagine and glutamine at position 106 is mutated to lysine.

7. The preparation method according to any one of claims 1-6, characterized in that, The preparation method further includes: Mix a buffer solution, Mg 2+ , AMP, L-glutamic acid or its salt, glycine, L-cysteine, glycine, L-cysteine, sodium hexametaphosphate, sodium sulfite, a mutant of bifunctional glutathione synthetase, and a mutant of polyphosphate kinase, and react under the condition of 35-45 °C to synthesize glutathione.

8. The preparation method according to claim 7, characterized in that, The working concentration of the mutant of the bifunctional glutathione synthetase is 30 - 40 g / L, and the working concentration of the mutant of the polyphosphate kinase is 20 - 25 g / L.

9. The preparation method according to claim 7, wherein The working concentration of the sodium hexametaphosphate is 28 - 32 mM; Optionally, the reaction temperature is 40 - 45 °C; Optionally, the buffer is Tris buffer with a working concentration of 140 - 160 mM and a pH of 8 - 9.

10. The preparation method according to claim 7, characterized in that, The Mg 2+ has a working concentration of 40 - 60 mM; Optionally, the working concentration of the AMP is 3 - 5 mM; Optionally, the salt of L-glutamic acid is sodium glutamate, and the working concentration of the sodium glutamate is 150 - 250 mM; Optionally, the working concentration of the glycine is 150 - 250 mM; Optionally, the working concentration of the L-cysteine is 75 - 125 mM; Optionally, the working concentration of the sodium sulfite is 8 - 12 g / L.

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