Method for the biosynthesis of gamma-glu-xaa flavor dipeptides

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a biosynthesis method of gamma-Glu-Xaa flavor dipeptide. A GCL enzyme with high activity of synthesizing gamma-Glu-Val (gamma-EV) is obtained through screening and mutation, the amino acid sequence of the GCL enzyme is shown as SEQ ID NO: 33, the GCL enzyme has wide substrate selection range and high catalytic efficiency; a synthesis process of biosynthesizing gamma-EV and other gamma-glutamyl dipeptides is developed based on the GCL enzyme, the synthesis process has simple steps and low cost, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a biosynthesis method of γ-Glu-Xaa flavor dipeptide. BACKGROUND

[0002] In the 1990s, researchers of Japan Ajinomoto separated and identified γ-glutamyl cysteine (γ-Glu-Cys-Gly, GSH), γ-glutamyl-S-allyl-L-cysteine (γ-L-glutamyl-S-allyl-L-cysteine, GSAC), γ-glutamyl-S-allyl-L-cysteine sulfoxide (γ-L-glutamyl-S-allyl-L-cysteine sulfoxide, GSACSO), γ-glutamyl-S-propenyl-L-cysteine sulfoxide (γ-L-glutamyl-S-propenyl-L-cysteine sulfoxide, γ-Glu-PeCSO) and other kokumi components in garlic and onion. German scientists later separated and identified γ-glutamyl-leucine (γ-Glu-Leu), γ-glutamyl-valine (γ-Glu-Val, γ-EV), γ-glutamyl-cysteine-β-alanine (γ-Glu-Cys-β-Ala), γ-glutamyl-glutamic acid (γ-Glu-Glu), γ-glutamyl-methionine (γ-Glu-Met), γ-glutamyl-glutamine (γ-Glu-Gln) and other 13 kinds of kokumi components in lima bean and cheese. All of them are γ-glutamyl peptides. In 2010, researchers of Ajinomoto found that 46 kinds of γ-glutamyl peptides such as γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly, γ-EVG), glutathione and γ-glutamyl-serine (γ-Glu-Ser, γ-ES) play a kokumi role in enhancing sweet, salty, fresh and other basic tastes by activating calcium sensing receptors. Among them, γ-glutamyl-cysteine (γ-Glu-Cys) and γ-EV are reported to have anti-inflammatory activity by activating CaSR.

[0003] At present, various glutamyl cysteine ligases GCL are tried to be used for the synthesis of γ-Glu-Xaa flavor dipeptide, but overall, the substrate specificity of glutamyl cysteine ligase is strong, which leads to less substrate selectivity, and there are problems of low enzyme activity, high cost and inability to synthesize more valuable γ-EV. Therefore, it is of important practical significance to develop GCL enzymes with low cost, wide substrate selectivity and high catalytic efficiency and biosynthesis pathways of γ-Glu-Xaa flavor dipeptide. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to provide a biosynthesis method of γ-Glu-Xaa flavor dipeptide.

[0005] The present application provides a glutamyl cysteine ligase variant, wherein the glutamic acid at position 62 of the amino acid sequence as shown in SEQ ID: 25 is mutated to aspartic acid; in particular, the amino acid sequence of the glutamyl cysteine ligase variant is as shown in SEQ ID NO: 33.

[0006] The present application provides the use of the glutamyl cysteine ligase variant and / or glutamyl cysteine ligase in the synthesis of γ-Glu-Xaa dipeptide.

[0007] The γ-Glu-Xaa dipeptide comprises one or more of γ-Glu-Ser, γ-Glu-Ile, γ-Glu-Lys, γ-Glu-Glu, γ-Glu-His, γ-Glu-Gly, γ-Glu-Gln, γ-Glu-Ala, γ-Glu-Val, γ-Glu-Asn, γ-Glu-β-Ala, γ-Glu-Met and / or γ-Glu-Thr.

[0008] The amino acid sequence of the glutamyl cysteine ligase is as shown in SEQ ID NO: 1 (GCL01), SEQ ID NO: 3 (GCL02), SEQ ID NO: 5 (GCL03), SEQ ID NO: 7 (GCL04), SEQ ID NO: 9 (GCL05), SEQ ID: 13 (GCL07), SEQ ID: 15 (GCL08), SEQ ID: 17 (GCL09), SEQ ID: 19 (GCL010), SEQ ID: 21 (GCL11), SEQ ID: 23 (GCL012) and / or SEQ ID: 25 (GCL013).

[0009] The test results in the specific embodiments of the present application show that the glutamyl cysteine ligase with the amino acid sequence shown in SEQ ID NO: 1 (GCL01) can be used for the synthesis of γ-Glu-Ser, γ-Glu-Lys, γ-Glu-Glu, γ-Glu-His and γ-Glu-Ala; the glutamyl cysteine ligase with the amino acid sequence shown in SEQ ID NO: 3 (GCL02) can be used for the synthesis of γ-Glu-Ile, γ-Glu-Glu and γ-Glu-His; the glutamyl cysteine ligase with the amino acid sequence shown in SEQ ID NO: 5 (GCL03) can be used for the synthesis of γ-Glu-Ser and γ-Glu-Gly; the glutamyl cysteine ligase with the amino acid sequence shown in SEQ ID NO: 7 (GCL04) can be used for the synthesis of γ-Glu-Ser; the glutamyl cysteine ligase with the amino acid sequence shown in SEQ ID NO: 9 (GCL05) can be used for the synthesis of γ-Glu-Ser; the glutamyl cysteine ligase with the amino acid sequence shown in SEQ ID NO: 13 (GCL07) can be used for the synthesis of γ-Glu-Gln; the glutamyl cysteine ligase with the amino acid sequence shown in SEQ ID NO: 15 (GCL08) can be used for the synthesis of γ-Glu-Ser; the glutamyl cysteine ligase with the amino acid sequence shown in SEQ ID NO: 17 (GCL09) can be used for the synthesis of γ-Glu-Val, γ-Glu-Met; the glutamyl cysteine ligase with the amino acid sequence shown in SEQ ID NO: 19 (GCL010) can be used for the synthesis of γ-Glu-Val, γ-Glu-Lys; the glutamyl cysteine ligase with the amino acid sequence shown in SEQ ID NO: 21 (GCL11) can be used for the synthesis of γ-Glu-Val, γ-Glu-Lys, γ-Glu-Thr;

[0010] The present application provides a combination enzyme comprising at least one of glutamyl cysteine ligase (GCL enzyme) and / or the glutamyl cysteine ligase variant described in the present application and polyphosphate kinase.

[0011] Further, in the combination enzyme described in the present application,

[0012] The amino acid sequence of the glutamyl cysteine ligase is shown in SEQ ID: NO: 23 (GCL012) and / or SEQ ID: NO: 25 (GCL013);

[0013] The amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO: 29.

[0014] The GCL enzyme has strong substrate preference; in substrate selection, the GCL enzyme in nature has good activity on substrates Glu and Cys, and general activity on Glu and Ser, but generally poor activity on Glu and Val, for example, the GCL derived from Streptococcus agalactiae can generate γ-Glu-Cys and γ-Glu-Ser, and the GCL derived from Clostridium acetobutylicum can only generate γ-Glu-Cys. Through screening, the present application obtains GCL enzymes (GCL013 and GCL12, wherein GCL013 is more superior) with wide substrate range and high conversion activity, which have the function of synthesizing three kinds of flavor dipeptides γ-Glu-Val (γ-EV), γ-Glu-Ser (γ-ES) and γ-Glu-Asn (γ-EN), and then through modification, a mutant (the amino acid sequence is shown as SEQ ID NO: 33) with higher activity of synthesizing γ-Glu-Val (γ-EV) is obtained. The mutant is used together with other enzymes, such as polyphosphate kinase, for bidirectional synthesis of γ-Glu-Xaa flavor dipeptides, and has high conversion efficiency, low cost and wide industrial application prospect for bidirectional synthesis.

[0015] The present application provides a nucleic acid comprising a nucleic acid encoding the glutamyl cysteine ligase variant of the present application, and / or the combination enzyme of the present application, and / or a nucleic acid encoding a glutamyl cysteine ligase;

[0016] The amino acid sequence of the glutamyl cysteine ligase is shown as SEQ ID NO: 1 (GCL01), SEQ ID NO: 3 (GCL02), SEQ ID NO: 5 (GCL03), SEQ ID NO: 7 (GCL04), SEQ ID NO: 9 (GCL05), SEQ ID: NO: 13 (GCL07), SEQ ID: NO: 15 (GCL08), SEQ ID: NO: 17 (GCL09), SEQ ID: NO: 19 (GCL010) and / or SEQ ID: NO: 21 (GCL11).

[0017] The present application provides an expression unit comprising an expression element and the nucleic acid of the present application.

[0018] The present application provides a recombinant vector comprising the nucleic acid of the present application or the expression unit of the present application.

[0019] The present application provides a host cell transfected or transformed with the recombinant vector of the present application.

[0020] The nucleic acid of the present application can be DNA, RNA, cDNA or PNA. In the embodiments of the present application, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g. promoters or transcription terminators). The nucleic acid can be linear or circular in topology. The nucleic acid can be part of a vector (such as an expression or cloning vector) or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the aid of recombination, enzymatic or chemical techniques. The RNA form is mRNA and the like obtained by transcription of a gene.

[0021] In the present application, the nucleic acid is optimized, which includes but is not limited to: codon usage bias, elimination of secondary structures (such as hairpin structures) that are not conducive to expression, alteration of GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splice sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA instability regions, repeat sequences (direct repeats, inverted repeats, etc.) and restriction sites that can affect cloning;

[0022] In specific embodiments of the present application, after codon optimization,

[0023] The nucleotide sequence of the nucleic acid encoding the glutamyl cysteine ligase variant GCL13 (E62D) shown in the amino acid sequence of SEQ ID NO: 34 is shown in SEQ ID NO: 34;

[0024] The nucleotide sequence of the nucleic acid encoding the glutamyl cysteine ligase (GCL12) with the amino acid sequence of SEQ ID NO: 23 is shown in SEQ ID NO: 24;

[0025] The nucleotide sequence of the nucleic acid encoding the glutamyl cysteine ligase (GCL13) with the amino acid sequence of SEQ ID NO: 25 is shown in SEQ ID NO: 26;

[0026] The nucleotide sequence of the nucleic acid encoding the polyphosphate kinase (SlPPK2) is shown in SEQ ID NO: 30.

[0027] The present application also provides an expression unit, which refers to a DNA sequence from the start of a promoter to the end of a terminator. The promoter and the terminator can also include regulatory fragments between or on both sides, which can include promoters, enhancers, transcription termination signals, polyadenylation sequences, replication origins, nucleic acid restriction sites, and homologous recombination sites, such as enhancers of promoters, poly(A) signals, etc., which are operably linked to the nucleic acid sequence.

[0028] The present application provides a recombinant vector, which contains the nucleic acid of the present application or the expression unit of the present application.

[0029] The terms "comprising", "having", or "including", or any of their grammatical variations, when used in this specification, shall be taken to specify the presence of stated features but not to preclude the presence of additional features or steps. In some embodiments, the term "comprising" is used, which means that other steps, ingredients or components can be added.

[0030] The use of any and all examples, or exemplary language herein, for example, only the better illustrate the present application, and is not intended to be limiting of the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0031] Further, the source of the vector of the present application includes plant, animal, bacteria, fungi, bacteriophage, or virus, which are not limited by the present application. The recombinant vector of the present application refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule comprising a desired coding sequence and suitable nucleic acid sequences or elements necessary for the expression of the operably linked coding gene in a specific host organism. The nucleic acid sequences or elements necessary for the expression in a model animal or mammalian cell include promoters, ribosome binding sites, and possibly other sequences. It is known that prokaryotic cells use promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, can integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably, as the plasmid is the most commonly used form of vector currently. However, the present application is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or potentially genomic inserts. In specific embodiments, the nucleic acid encoding the fusion protein provided by the present application can be constructed in various prokaryotic (bacterial) expression vectors. For example, pET series vectors, more specifically pET28a.

[0032] Further, the method of transformation includes chemical transformation and electroporation; the method of transfection includes calcium phosphate coprecipitation, artificial liposome method, viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, and / or lentivirus transfection, etc.

[0033] The host cell of the present application can be derived from a plant, an animal, a bacterium, a fungus, a bacteriophage or a virus, and the present application does not limit the source of the host cell. The host cell of the present application is transformed or transfected with a vector constructed using recombinant DNA technology, and the transformed host cell has the ability to replicate the vector encoding the protein or to express the desired protein. In a specific embodiment of the present application, the host cell is derived from a bacterium, specifically from Escherichia coli, and more specifically from Escherichia coli BL21 (DE3).

[0034] The present application provides at least one of the following I) to V) for use in the synthesis of a γ-Glu-Xaa dipeptide:

[0035] I) a combination enzyme according to the present application;

[0036] II) a nucleic acid according to the present application;

[0037] III) a recombinant vector according to the present application;

[0038] IV) a host cell according to the present application;

[0039] V) a culture obtained by culturing the host cell according to the present application.

[0040] wherein Xaa represents an amino acid residue.

[0041] Further, the γ-Glu-Xaa dipeptide includes one or more of γ-Glu-Ser, γ-Glu-Ile, γ-Glu-Lys, γ-Glu-Glu, γ-Glu-His, γ-Glu-Gly, γ-Glu-Gln, γ-Glu-Ala, γ-Glu-Val, γ-Glu-Asn, γ-Glu-β-Ala, γ-Glu-Met and / or γ-Glu-Thr.

[0042] The present application provides a method for preparing a γ-Glu-Xaa dipeptide, which includes synthesizing a γ-Glu-Xaa dipeptide using at least one of the following i) to v):

[0043] i) a combination enzyme according to the present application;

[0044] ii) a nucleic acid according to the present application;

[0045] iii) a recombinant vector according to the present application;

[0046] iv) a host cell according to the present application;

[0047] v) a culture obtained by culturing the host cell according to the present application.

[0048] The application screens and mutates to obtain a GCL enzyme with high activity of synthesizing gamma-EV, the amino acid sequence of which is shown in SEQ ID NO: 15, which has a wide range of substrates and high catalytic efficiency; and develops a synthesis process of biological synthesis of gamma-EV and other gamma-glutamyl dipeptides based on the enzyme, which has simple steps, low cost and wide application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 GCL01-08 candidate enzyme shake flask expression electrophoresis map is shown;

[0050] Figure 2 Structural clustering analysis of glutamyl cysteine ligase (GCL) is shown;

[0051] Figure 3 GCL09-13 candidate enzyme shake flask expression electrophoresis map is shown;

[0052] Figure 4 GCL enzyme activity screening is shown;

[0053] Figure 5 Mass spectrum results of GCL13 enzyme synthesis of gamma-ES product peak are shown;

[0054] Figure 6 Mass spectrum results of GCL13 enzyme synthesis of gamma-EV product peak are shown;

[0055] Figure 7 Expression electrophoresis map of GCL-E62D enzyme is shown;

[0056] Figure 8 Mass spectrum results of GCL13 enzyme synthesis of gamma-EN product peak are shown;

[0057] Figure 9 Reaction formula of GCL enzyme synthesis of gamma-ES is shown;

[0058] Figure 10 Reaction formula of GCL enzyme synthesis of gamma-EV is shown. DETAILED DESCRIPTION

[0059] The application provides a biological synthesis method of gamma-Glu-Xaa flavor dipeptide, and those skilled in the art can refer to the content herein and appropriately improve process parameters to realize. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the application. The method and application of the application have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the application, to realize and apply the technical content of the application.

[0060] Amino acid sequence of GCL01 : MKIQHIIHENQLGLLFQQGSFGLEKESQRVTADGAIVTTPHPAVFGNRRYHPYIQTDFAESQLELITPPTKKLEDTFRWLSVIHEVVQRSLPEEEYIFPLSMPAGLPAEEQIRVAQLDNPEDVAYREYLVKIYGKNKQMVSGIHYNFQLSPDLITRLFRLQNEYQSAVDFQNDLYLKMAKNFLRYQWILLYLLAATPTVESAYFKDGSPLAKGQFVRSLRSSQYGYVNDPEINVSFDSVEKYVESLEHWVSTGKLIAEKEFYSNVRLRGAKKAREFLTTGIQYLEFRLFDLNPFEIYGISLKDAKFIHVFALFMIWMDHTADQEEVELGKARLAEVAFEHPLEKTAYAVEGELVLLELLSMLEQIGAEPELFEIVKEKLTQFTDPSKTVAGRLVRAIEQAGSDQQLGAQLAQQYKAQAFERFYALSAFDNMELSTQALLFDVIQKGIHTEILDENDQFLCLKYGDHIEYVKNGN (SEQ ID NO: 1);

[0061]

[0062] Amino acid sequence of GCL02: MIIDRLLQRSHSHLPILQATFGLERESLRIHQPTQRVAQTPHPKTLGSRNYHPYIQTDYSEPQLELITPIAKDSQEAIRFLKAISDVAGRSINHDEYLWPLSMPPKVREEDIQIAQLEDAFEYDYRKYLEKTYGKLIQSISGIHYNLGLGQELLTSLFELSQADNAIDFQNQLYMKLSQNFLRYRWLLTYLYGASPVAEEDFLDQKLNNPVRSLRNSHLGYVNHKDIRISYTSLKDYVNDLENAVKSGQLIAEKEFYSPVRLRGSKACRNYLEKGITYLEFRTFDLNPFSPIGITQETVDTVHLFLLALLWIDSSSHIDQDIKEANRLNDLIALSHPLEKLPNQAPVSDLVDAMQSVIQHFNLSPYYQDLLESVKRQIQSPELTVAGQLLEMIEGLSLETFGQRQGQIYHDYAWEAPYALKGYETMELSTQLLLFDVIQKGVNFEVLDEQDQFLKLWHNSHIEYVKNGN (SEQ ID NO: 3);

[0063]

[0064] Amino acid sequence of GCL03: MNLRHIIKQNHLELLFQQGSFGLEKESQRVRHDGSVVTSAHPKAFGNRSFHPYIQTDFAESQLELITPPNKKLEDTFRWLQTIHEVVWRTLPEDEFIFPLSMPAGLPPEKDIKVAQLDNSVDVAYREHLVKSYGAYKQMVSGIHYNFQLDPALIEALFKAQSDCRSAVEFQNSLYLKMAKNFLRYQWILLYLLSATPTVDANYFREGTALKPNQYVRSLRSSQYGYVNAPEIVVSFDSIPQYVETLEHWVNSGKLIAEKEFYSNVRLRGAKKAREFLHTGIQYLEFRLFDLNPFEPYGMSLHDAQFIHYFILLMIWLDETADQAGVELGKARLLEVAFEDPRQETVYRDEGERILNALITMLKDLGTDENAVKSAQEKLSQFAYPERTLCARLVAAIEQAGGYQKLGAELAQRNKAQAFERFYALTAFDNMELSTQALMFDAIQKGLQMEILDENDQFLRLQFGEHFEYVKNGN (SEQ ID NO: 5);

[0065] Nucleotide sequence of GCL03: atgaacctgcgccatattattaaacagaaccatctggaactgctgttccagcagggctcgtttggcttggaaaaagaaagccagcgtgtgcgccatgatggcagc gtggtgaccagcgcgcacccg aaagcctttggcaatcgcagctttcatccgtacattcagaccgattttgccgaaagccagctggagctgattaccc cgccgaat

[0066] Amino acid sequence of GCL04: MKLNQLLQALPKDTHILEGHFGIEREGLRVTQEGHVSQREHPQSLGSRNRHPYIQTDFSEAQLELITPVSKSTKEIRRRLGAITDVAQRSLEADEVIWPLSMPPYLQEAEIHIAKLDNPSEVAYRSHLADKYGVLLQSISGIHYNFEIGSQPLDKLFQESGYSSRVTFKNDLYMKLARQFLTYRWLLTYLYGASPLAEREFYSHSPKRAVRSLRSSKAYGYNNKEEVKVSFQSIERYIEDIEKAVASGHLSMEKEFYSPIRLRGAKHNHDYLEQGVTYMEFRSFDLNPFDSLGMSQETLDTFHLIILALLWMDEGETSDKTIEMSKAINEAVALAHPLDALPQEADPKPLLLAMQEVIEHFDLDEIYQTTLTNITRVIQNPEETMAAKLLKAIENNSLMTFGTKMANNYHQLAWQAPYALKGYESMELSTQMVMFDAIQKGLHLEILDERDQFIKLWHQDHIEYVKNGN (SEQ ID NO: 7);

[0067]

[0068] Amino acid sequence of GCL05: MTIDRLLEKLDPASPILQATFGLERESLRMTEAGTLASTPHPSSLGSRTFHPYIQTDFSEQQLELITPIASSTKEAHRLLGAITDVTGRSISREELMWPLSMPPQLREDEIEIAHLENTFELRYRQGLAEKYGKRLQTISGIHYNIELGEDLMTALFEASDFTSLKDFKNALYLKLARNFLRFRWFLTYLYGAAPLAEAGFYDKQVEAPVRSLRNSQHGYVNEEHVHVSFSSLESYVSDIEAYVESGDLSAEKEFYSPVRFRGQKRNRDYLEQGITYLELRCFDLNPFNVLGISQETLDTVHLFLLALLWLDDLTDSDAQLQAAYALNDAIALAHPLTPLPPEADSSAILQAMENVIQHFNLPDSYHQLLAQVKATLTEPKLTLAGQLLPHIEKNSLLDFGLAKAREYRDYAWTAPYALKGYEKMELSTQMLMFDALQKGLHLEILDENDQFLKLWHGQHVEYVKNGN (SEQ ID NO: 9);

[0069]

[0070] Amino acid sequence of GCL06: MKMNQLLQQLDAASPVLQANFGIERESLRVDLEGRVAQTPHPEVCGARSYHPYIQTDFSEFQLELITPVAKSTQEAKRLLGAITDVAGRSINRNEHMWPLSMPPHITEADIQIAQLEDDFERHYRSYLADKYGTKLQAISGIHYNIELGADLVQALYQVSDYRDKKSFKNDLYLKLAQNFLRYSWLLTYLYGAAPLAEKGFYKEAPQGPVRSYRNSDYGYVNKPNIQVSYASLEAYITDIERYVKEGELLAEKEFYSAVRFRGQKPNRQFLDQGITYLELRCFDLNPFEVTGIDQATMDTVHLLLLALVWMDQADKEEVDGLLQEAHELNNQIALAHPLDPLPNKADAAPVLAAMEAVIAHFRLGRSYQDLLDDIRLTLTDPHRTLSGQLLPHIENHSLANFGREKATEYQNYAWTAPYALKGYENMELSTQMLLFDVIQQGVHFEILDEEDQFLKLWHDQHVEYVKNGN (SEQ ID NO: 11);

[0071]

[0072] Amino acid sequence of GCL07: MTLNQLLQKLEATSPILQANFGIERESLRVDRQGQLVHTPHPSCLGARSFHPYIQTDFCEFQMELITPVAKSTTEARRFLGAITDVAGRSIATDEVLWPLSMPPRLKAEEIQVAQLENDFERHYRNYLAEKYGTKLQAISGIHYNMELGKDLVEALFQESDQTDMIAFKNALYLKLAQNYLRYRWVITYLFGASPIAEQGFFDQEVPEPMRSFRNSDHGYVNKEEIQVSFVSLEDYVSAIETYIEQGDLIAEKEFYSAVRFRGQKVNRSFLDKGITYLEFRNFDLNPFERIGISQTTMDTVHLLILAFLWLDSPENVDQALAQGHALNEKIALSHPLEPLPSEAKTQDIVTALDQLVQHFGLGDYHQDLVKQVKAAFADPNQTLSAQLLPYIKDKSLAEFALNKALAYHDYDWTAHYALKGYEEMELSTQMLLFDAIQKGIHFEILDEQDQFLKLWHQDHVEYVKNGN (SEQ ID NO: 13);

[0073]

[0074] MTVDQILQNSSTSLPILQGTFGLEHESLRIDRQSNHLSQRSHPDRLGSRNFHPYIQTDYSEPQIELITPISQSTKEARRFLRAITDVAGRSIAKEDYLWPLSMPPQVSEDEIVIAHLEDAFEREYREHLAKVYGKRLQTISGIHYNFGLGTDLLNQLFQQSNYQDLVAFKNDLYLKLAQNFIKYRWFLTYLYGASPIAEKGFFDHDWDSPVRSIRNSSLGYVNHESVKISYSSLKQYVADIEACVADGRLIAEKEFYSPVRLRGSKRSRDFLERGITYL

[0075] EFRCFDIDPFDQQGIAQETLDTVHLLVLALLWLDGPEAVDKELAAAEKLNEHIAKANPLEPLPNQAPIE

[0076] DLLQAMRAVIEHFSLPPYYSQLVDQLETQLADPRLTIGGRLAKEIKGASLEEFGRKQGQTFSNLAWQAPYALKGFETMELSTQLIMFDVIQKGIQLEILDESDQFLKLTLGDHTEYVKNGN (SEQ ID NO: 15);

[0077]

[0078] Amino acid sequence of GCL09 (Leptospira interrogans): MKTKELTQSKIEEVSLEILLRHAVKAKHGLEKESMRVNPDGTLSGTTHPIHLGSSLTNHYIKTDFAEPQLEYATHPRPKVEANIRELQDLHIFTIRKLENELIWPFSMPPVLPEEENEIPLGQYGTSHSGRWKTIYRHGLGLRYGRRMQTISGVHYNFSFSKVFLRQFLGKEISNFTKEEISSLYLHVIRNFLRRVHFLTYLTGSSPVFDFTFLPNPGSLKFEKHKNFTLYSTYATSLRMSEIGYTSKVQDTLGIHYNSLEEYVDRMCYAVHTPYPKYVSFSENKDAQLNPNYLQIENEFYSPIRPKQIPKGDERPLDALLQRGIEYIEIRSLDIDPYSPVGVCRSNLAFTQLILLDSLLKVSPSISEEENFSLKENLNSVIWEGRNPELKINVNGSKRNFQEAGAEYSESLRHYAKILDLHTGRRTYQEAIDFQIKKWKNPDKTPSGKLLSEILKRNIEFREKGIELAQENKRMFSYLEYSPGTLMKMEKETIRSFQEKEELEKQEIQTQYPTVKLCNH (SEQ ID NO: 17);

[0079]

[0080] Amino acid sequence of GCL10 (Desulfofustis): MNQLLPERLEQIEAGGVGHHLAAALHGIEKEGLRVTGSGRIAPTPHPRSLGAALTNKFITTDFSESLLEFITPALPEPATALEILRALHHYLYTSLDDELIWAASMPCWIEDVAEIPIARYGSSNLGRMKHIYRLGLQHRYGKMMQSIAGIHYNYSLPEPFLRGYRDLLEGSETMQSFTSAAYFAMMRNFRRHSWLLLFLFGASPVVSRSFLKGSRRGLDTLTEDSLYLPWATSLRMSDLGYSNAAQSAIAVCFNTLPTYIATLAGAINTPHPEYERIGVVVDGEYRQLSNTILQIENEYYSDIRPKRAPEPGESALQALNRAGVEYVEVRSTDVNPLLPVGIDLEQARFMEAFLLTCLLMGDTPVSEEECRVIAENHHRVTTRGREPRLMLSTPWGETVLEELARRLLHQCGLTAQLLDTEHDTTDYAQSVAAQHAKIDDPNLTPSARIIESIKNSALSYNEWVLALSKQHKKTITSRPVAADIIEQLANEAKDSLLRQSRIEEDDTVDFDTFMRRYRTHQTDIAASIGDK (SEQ ID NO: 19);

[0081] Nucleotide sequence of GCL10 (codon optimized): atgaaccagctgctgccggaacgcctggaacagattgaagcgggcggcgtgggccatcatctggccgccgcgctgcatggcattgaaaaagaaggcctgcg cgtgaccggtagc ggccgcattgcgccgaccccgcatccgcgctcactgggtgcggcgctgaccaacaaattcattaccaccgatttta gcgaaagcctgctg

[0082] Amino acid sequence of GCL11 (Beta proteobacteria bacterium): MAERVVPFQERLHALPRAVLQGLLRGVEKESLRVRPDGPLATTPHPERLGAALTHPYVTTDFSESQLELITGVHSGAEACLSELTEIHQVVYRAIGDEMLWCGSMPCGLPADDAIPIGRFGNSNIGRAKSVYRMGLAHRYGRRMQMISGIHYNFSLPDPLSNDAYFALLRNFRRHSWLLLYLFGASPAVCSSFVAGRAHELEHLSQGTLFLPHATSLRMGRLGYQSDAQASLAVSYNSLESYAASLQEALTKPYPAYAAIGIRKGDDYRQLATSLLQIENEFYGTIRPKRVIRTGERPLHALRERGVEYVEVRLMDLDPFCPIGITASTIRFLDVFLLHCLLCDSPPDTPQELAAILRNKQRVAARGREPGLLLTRGSQEVALSEWGGQVLAECEPIAAALDAVNAITAHREALATAVTALNDSAATPSARVLQAMDRDHGGSYVRFVLAQSLAHRDTVLKRPLAAEVGERFARLAETSLLEQRQIEAADTVPFEIYRQQYLSPLRLNE (SEQ ID NO: 21);

[0083]

[0084] Amino acid sequence of GCL12 (Methanobrevibacter ruminantium): MDKIFNLSQIKNNLSSEQLKEGSFGIEWECLRVKENGELSLSPHPEIFGDKLTNPYITTDFSESQIEIITPAFDTIDEAFSFFSFMSDLVNSSLSDDEYLWFQSLPCILPESDKIPIAKYKGRGLGEESMEYRKGLAKKYGLKKQLISGIHFNFSFKEELIESFYDGVIGDKEKISYKEFKDSLYLKISRNYIRYVWLIIYLTGCSVAVHNSFTLDCQKLMNHKDNQGSVYSDRGPSFRNSSCGYKNLEHLYPSYASVEEFTRDIQSFIDNGHLSEAKELYTQIRLKPRNPSDLLGSLNNNGIQYLEIRTLDINPFYKCGLIKNDMNFLHMFMIYLLIKEESDYESWQEDALYNEEKTAECGYEDGVKLIKDGKEVVLKDWALSILDEMEMMVESLNLENKSVIELMKLKIINSDLTYGRRLARLVEKDGYIESQLKLSRNNKLRSKYLVEETNLLNDERFKEYVPIALQGLSK (SEQ ID NO: 23);

[0085]

[0086] Amino acid sequence of GCL13 (Hoyosella subflava): MLKVDPLENPDESAPKIAFRRCPSVGVEEEFLLHDTARGMLAPRAPELLTRLADAVSGACSEMQNTQIELVTEPETTLEAMATGLLRNREQASAAASEMGLRLLPAGMPPQMSLQAPPARASIDDSRYRKIFARYGAAAERYQGCGTHVHVEVPDRETAVAVLGHLTPWLPLLVALGGNSAYAGGADTRFSSWRIATQRRFPVSGLPPEFRSATHYDETVDALVDAGLLEDGHTCFWLARLSDRYPTIEIRASDVGLTADDSVLQAALGRALVMTALSDLEKGHPPPTINPMWGDAAVWTAARHGLEGPALDPFTGRKITVSEYTTMLLDKTTDALCEAGDYYAVRTLLAALKKRGSGAQLQRCAARGGHAAVTSMLHGATCQAAGLGRNAGLILNAAQRDWSDAQMENEQSKSQPETTPPAEDQTNLVHEEHDPDTGSQPKVAGGAIIFLTAAIAIAFLIVAVYVGGQAFNAW (SEQ ID NO: 25);

[0087]

[0088] Amino acid sequence of EcGCL (Escherichia coli): MIPDVSQALAWLEKHPQALKGIQRGLERETLRVNADGTLATTGHPEALGSALTHKWITTDFAEALLEFITPVDGDIEHMLTFMRDLHRYTARNMGDERMWPLSMPCYIAEGQDIELAQYGTSNTGRFKTLYREGLKNRYGALMQTISGVHYNFSLPMAFWQAKCGDISGADAKEKISAGYFRVIRNYYRFGWVIPYLFGASPAICSSFLQGKPTSLPFEKTECGMYYLPYATSLRLSDLGYTNKSQSNLGITFNDLYEYVAGLKQAIKTPSEEYAKIGIEKDGKRLQINSNVLQIENELYAPIRPKRVTRSGESPSDALLRGGIEYIEVRSLDINPFSPIGVDEQQVRFLDLFMVWCALADAPEMSSSELACTRVNWNRVILEGRKPGLTLGIGCETAQFPLPQVGKDLFRDLKRVAQTLDSINGGEAYQKVCDELVACFDNPDLTFSARILRSMIDTGIGGTGKAFAEAYRNLLREEPLEILREEDFVAEREASERRQQEMEAADTEPFAVWLEKHA (SEQ ID NO: 27);

[0089]

[0090] Amino acid sequence of SlPPK2 (Sulfurovum lithotrophicum): MKKNIYKKELYKLQVELVKFQKYVIEENVAVCLVLEGRDTAGKDGTIKRFTEHLSPREARTVALGVPSDKEKKSWYFQRYVPHLPSAGEIVFFNRSWYNRAGVEKVMGFCTKKQYKAFMEEVGSFEQMLTHSNIRFFKYYLDITKKEQKKRLEARKTDPLKQWKLSPIDAKAQKMWDAYSKARDDMFNKTSFIYAPWYVVHTDDKKEARINIMKHFLSLNDYPDKDKALLVYDHDVICKFDPVCYEKEMIAP (SEQ ID NO: 29);

[0091] Nucleotide sequence of SlPPK2 (codon-optimized): atgaaaaaaaatatttataaaaaagagctgtacaagctgcaggttgaactggtgaaattccagaaatacgtgattgaagaaaatgttgcggtgtgcctggtactggaaggccgtgataccgccggcaaagatggcaccattaaacgctttaccgaacatctgagcccgcgcgaagcgcgcaccgtggcgctgggcgtgccgagcgataaagaaaaaaaaagctggtactttcagcgctatgtgccgcacctgccgagcgccggcgaaattgtgttttttaatcgcagctggtacaatcgcgcgggcgtggaaaaagtgatgggcttttgcaccaaaaaacaatacaaagcgtttatggaagaagtgggtagctttgaacagatgctgacccatagcaacattcgcttttttaaatactacctggatattaccaaaaaagaacagaaaaaacgcctggaagcccgcaaaaccgatccgctgaaacagtggaaactgagcccgattgatgcgaaagcgcagaaaatgtgggatgcgtacagcaaagcgcgcgatgacatgtttaacaaaaccagctttatttacgccccgtggtacgtggtgcataccgatgataaaaaagaagcgcgtattaatattatgaaacatttcctgtcactgaacgactacccggataaagataaagccctgctggtgtatgatcatgatgtgatttgcaaatttgatccggtgtgttatgaaaaagaaatgatcgcgccgtaa (SEQ ID NO:30);

[0092] GCL13-E62D-up: ggtgcgtgttcagatatgcagaataccc (SEQ ID NO: 31);

[0093] GCL13-E62D-dn: tgggtattctgcatatctgaacacgcac (SEQ ID NO: 32);

[0094] Amino acid sequence of GCL13-E62D (artificial): MLKVDPLENPDESAPKIAFRRCPSVGVEEEFLLHDTARGMLAPRAPELLTRLADAVSGACSDMQNTQIELVTEPETTLEAMATGLLRNREQASAAASEMGLRLLPAGMPPQMSLQAPPARASIDDSRYRKIFARYGAAAERYQGCGTHVHVEVPDRETAVAVLGHLTPWLPLLVALGGNSAYAGGADTRFSSWRIATQRRFPVSGLPPEFRSATHYDETVDALVDAGLLEDGHTCFWLARLSDRYPTIEIRASDVGLTADDSVLQAALGRALVMTALSDLEKGHPPPTINPMWGDAAVWTAARHGLEGPALDPFTGRKITVSEYTTMLLDKTTDALCEAGDYYAVRTLLAALKKRGSGAQLQRCAARGGHAAVTSMLHGATCQAAGLGRNAGLILNAAQRDWSDAQMENEQSKSQPETTPPAEDQTNLVHEEHDPDTGSQPKVAGGAIIFLTAAIAIAFLIVAVYVGGQAFNAW (SEQ ID NO:33);

[0095]

[0096] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market. The present application is further described below in combination with examples:

[0097] Example 1 Screening of GCL enzyme

[0098] 1. Screening of existing enzyme library

[0099] Eight candidate enzymes in Table 1 were selected, and reverse primers were designed for each of them. The corresponding GCL enzyme fragments were amplified from the original template using the universal primer T7, and were then ligated to the pET28a vector using NdeI and XhoI restriction enzymes, and were transformed into the BL21(DE3) expression host.

[0100] Table 1

[0101]

[0102]

[0103] 2. Expression and activity screening of fusion-evolved GCL enzyme

[0104] The saved glycerol bacteria were streaked on LB plates (Kan) and incubated overnight in a 37°C incubator. Then, three single colonies of appropriate size were picked from each plate and inoculated in 10 mL of LB liquid medium (Kan) and incubated at 37°C and 200 rpm for 6-8 h until turbidity. 1 mL was taken to make a glycerol stock, and the rest was transferred to 400 mL of LB liquid medium (Kan) and incubated at 37°C and 200 rpm for 2-3 h until the OD 600 = 0.6-1.0. After adding 0.1 mM IPTG, it was transferred to 20°C and induced at 200 rpm for 14-18 h. The bacterial cells were collected by low-temperature centrifugation, weighed, resuspended in 9 mL of lysis buffer (20 mM K2HPO4 / KH2PO4, 100 mM KCl, pH 7.6) per gram of wet bacterial cells, and then sonicated and centrifuged at high speed. The supernatant and precipitate were sampled and subjected to protein electrophoresis to detect the expression.

[0105] As shown in Figure 1 , GCL02 and GCL03 were expressed in both the supernatant and the precipitate, GCL01, GCL04, GCL05, GCL07 and GCL08 were mainly expressed in the supernatant, and GCL06 was expressed in the precipitate during the verification in the molecular construction stage, and did not undergo shake flask culture expression.

[0106] Take 0.1 mL of broken liquid to add 0.9 mL of reaction liquid (10 mM Glu, 10 mM Xaa (representing different amino acids), 15 mM ATP, 20 mM MgCl2, pH 8.0), 38°C water bath. Sample high performance liquid chromatography (HPLC) detection, the amount of ATP reduction of reaction 1h shown in Table 2, "-" indicates the reduction is less than 1 mM not to be counted. The preliminary activity of fusion evolution GCL enzyme to synthesize the following γ-Glu-Xaa dipeptide is not high, γ-Glu-Val, γ-Glu-Asn, γ-Glu-β-Ala, γ-Glu-Met and γ-Glu-Thr were not found to be synthesized, wherein GCL04, GCL05, GCL08 can only synthesize γ-Glu-Ser, GCL07 can only synthesize γ-Glu-Gln.

[0107] Table 2

[0108]

[0109] 3、Enzyme structure clustering analysis

[0110] Based on Table 1, GCL01-GCL08 cannot synthesize γ-Glu-Val (γ-EV); in order to explore the activity of independent evolution GCL enzyme to synthesize γ-Glu-Xaa dipeptide, the UniProt code of EcGCL enzyme is input into the structure clustering analysis website https: / / cluster.foldseek.com / , as shown in Table 3, EcGCL has been classified into a structural cluster consisting of 224 protein members, from which 6 candidate enzymes in Table 3 are selected for whole gene synthesis and construction in pET28a vector, and transformed into BL21 (DE3) expression host. Figure 2

[0111] Table 3

[0112] UniProt code Species origin Enzyme name Q72RD4 Leptospira interrogans GCL09 V4J9A7 Desulfofustis GCL10 A0A7W1AL24 Betaproteobacteria bacterium GCL11 D3E133 Methanobrevibacter ruminantium GCL12 F6EGD9 Hoyosella subflava GCL13 P0A6W9 Escherichia coli EcGCL

[0113] 4、Expression and activity screening of independent evolution GCL enzyme

[0114] The saved glycerol bacteria were streaked on LB plates (Kan) and incubated in a 37°C incubator overnight, then 3 single colonies of appropriate size were picked from each plate and inoculated in 10 mL of LB liquid medium (Kan) at 37°C and 200 rpm for 6-8 h until turbidity. 1 mL was taken to make glycerol bacteria preservation, and the rest was transferred to 400 mL of LB liquid medium (Kan) and incubated at 37°C and 200 rpm for 2-3 h until OD 600 ​= 0.6~1.0, after adding 0.1 mM IPTG, transfer to 20℃ 200 rpm to induce expression for 14~18 h. Collect the bacterial cells by low temperature centrifugation, weigh, resuspend according to wet bacterial cells 1 g:9 mL, add the crushing buffer (20 mM K2HPO4 / KH2PO4, 100 mM KCl, pH 7.6), ultrasonic crushing, take sample high speed centrifugation, and detect the expression of the supernatant and the precipitate respectively by protein electrophoresis.

[0115] As shown in Figure 3 GCL12 and GCL13 are mainly expressed in the supernatant, GCL10 and GCL11 are expressed in both the supernatant and the precipitate, GCL09 has low expression, and EcGCL is found to be expressed in the precipitate during the transformation stage, and the addition of a solubility-promoting tag still does not improve it, and no shake flask culture expression is performed.

[0116] Take 0.1 mL of the crushing solution and add 0.9 mL of the reaction solution (10 mM Glu, 10 mM Xaa, 15 mM ATP, 20 mM MgCl2, pH 8.0), and place in a 38℃ water bath. Take sample high performance liquid chromatography (HPLC) detection, and the ATP reduction amount of reaction 1 h is shown in Table 4, and "-" indicates that the reduction amount is less than 1 mM and is not counted. As shown in Figure 4 GCL13 enzyme may have higher conversion rate when synthesizing γ-Glu-Val (γ-EV), γ-Glu-Ser (γ-ES), γ-Glu-Asn (γ-EN) and other γ-glutamyl dipeptides compared with GCL12 enzyme.

[0117] Table 4

[0118]

[0119] Example 2 One-way reaction of GCL13 enzyme synthesizing γ-ES

[0120] Take glutamic acid (Glu) 0.736 g (50 mM), serine (Ser) 0.525 g (50 mM), adenosine triphosphate disodium (ATP-Na2) 2.756 g (50 mM), and magnesium chloride hexahydrate (MgCl2) 0.407 g (20 mM) and dissolve in 80 mL of pure water, adjust the pH to 8.0 and make up to 90 mL, preheat at 38℃, then add 10 mL of GCL13 crude enzyme solution obtained by resuspending according to wet bacterial cells:crushing buffer = 1:4 after ultrasonic crushing.

[0121] Take 100 μl of the reaction solution, quench with 60% acetonitrile 700 μl, and detect by high performance liquid chromatography (HPLC). After 2 h of reaction, Glu and Ser are consumed by 99% and 89% respectively, and the product peak is collected for mass spectrometry peptide sequence analysis, Figure 5The product was confirmed to be γ-ES.

[0122] Example 3 Bi-directional reaction of GCL13 enzyme for synthesis of γ-ES

[0123] An ATP regeneration system composed of polyphosphate kinase SlPPK2 derived from Sulfurovum lithotrophicum and sodium hexametaphosphate was introduced.

[0124] Sodium hexametaphosphate (6P) 0.917 g (15 mM), glutamic acid (Glu) 0.736 g (50 mM), serine (Ser) 0.525 g (50 mM), adenosine triphosphate disodium (ATP-Na2) 0.110 g (2 mM), magnesium chloride hexahydrate (MgCl2) 0.407 g (20 mM) were weighed into 70 mL of pure water, adjusted to pH 8.0 and made up to 80 mL, and 10 mL of GCL13 crude enzyme solution and 10 mL of SlPPK2 crude enzyme solution obtained by resuspending the wet cell according to the ratio of cell to breaking buffer = 1:4 and then ultrasonic breaking were added after preheating at 38°C.

[0125] 100 μl of the reaction solution was taken, 700 μl of 60% acetonitrile was added for quenching, and high performance liquid chromatography (HPLC) was used for detection. After 2 h of reaction, 99% and 100% of Glu and Ser were consumed, respectively.

[0126] Example 4 Laboratory scale-up of GCL13 enzyme for synthesis of γ-ES

[0127] Sodium hexametaphosphate (6P) 18.34 g (30 mM), glutamic acid (Glu) 14.71 g (100 mM), serine (Ser) 10.51 g (100 mM), adenosine triphosphate disodium (ATP-Na2) 1.10 g (2 mM), magnesium chloride hexahydrate (MgCl2) 4.07 g (20 mM) were weighed into 700 mL of pure water, adjusted to pH 8.0 and made up to 80 mL, and 100 mL of GCL13 crude enzyme solution and 100 mL of SlPPK2 crude enzyme solution obtained by resuspending the wet cell according to the ratio of cell to breaking buffer = 1:4 and then ultrasonic breaking were added after preheating at 38°C.

[0128] 100 μl of the reaction solution was taken, 700 μl of 60% acetonitrile was added for quenching, and high performance liquid chromatography (HPLC) was used for detection. After 4 h of reaction, 97% and 97% of Glu and Ser were consumed, respectively. After 5 h of reaction, the pH of the reaction solution was adjusted to 3.0, the supernatant was taken by centrifugation, and the sample of γ-ES was prepared by freeze-drying after filtration, with a purity of 97.14% and a content of 70.05% (nuclear magnetic quantification result).

[0129] Example 5 Unidirectional reaction of GCL13 enzyme for synthesis of γ-EV

[0130] Glu 0.736 g (50 mM), Val 0.586 g (50 mM), ATP-Na2 2.756 g (50 mM), MgCl2 0.407 g (20 mM) were dissolved in 80 mL pure water, adjusted to pH 8.0 and constant volume to 90 mL, 10 mL of GCL13 crude enzyme solution obtained by resuspension according to wet bacteria:crushing buffer = 1:4 and ultrasonic crushing was added after preheating at 38°C.

[0131] Take 100 μl of reaction solution, add 700 μl of 60% acetonitrile to quench, and detect by high performance liquid chromatography (HPLC). After 6 h of reaction, Glu, Val and ATP were consumed by 99%, 82% and 84%, respectively, and the product peak was collected for mass spectrometry peptide sequence analysis, Figure 6 The product was confirmed to be γ-EV.

[0132] Example 6 Bidirectional reaction of GCL13 enzyme for synthesis of γ-EV

[0133] Similarly, SlPPK2 derived from Sulfurovum lithotrophicum and an ATP regeneration system composed of sodium hexametaphosphate were introduced.

[0134] Glu 0.736 g (50 mM), Val 0.586 g (50 mM), ATP-Na2 2.756 g (50 mM), MgCl2 0.407 g (20 mM) were dissolved in 80 mL pure water, adjusted to pH 8.0 and constant volume to 90 mL, 10 mL of GCL13 crude enzyme solution obtained by resuspension according to wet bacteria:crushing buffer = 1:4 and ultrasonic crushing was added after preheating at 38°C.

[0135] Take 100 μl of reaction solution, add 700 μl of 60% acetonitrile to quench, and detect by high performance liquid chromatography (HPLC). After 6 h of reaction, Glu, Val and ATP were consumed by 99%, 82% and 84%, respectively, and the product peak was collected for mass spectrometry peptide sequence analysis,

[0136] Example 7 Rational design of GCL13 enzyme

[0137] To expand the substrate pocket of GCL13 enzyme to accommodate Val which is larger than Cys, Ser, GCL13-E62D mutant was designed, i.e. glutamic acid at position 62 was mutated to aspartic acid. The corresponding plasmid pET28a-GCL3-E62D was constructed using Quick-Change site-directed mutagenesis kit (Agilent) with pET28a-GCL3 as template, GCL13-E62D-up and GCL13-E62D-dn as primers, and transformed into BL21(DE3) expression host after sequencing success. Figure 7 GCL-E62D enzyme expression electrophoretogram is shown.

[0138] Example 8 Bidirectional reaction of GCL13-E62D enzyme to synthesize γ-EV

[0139] Sodium hexametaphosphate (6P) 0.917 g (15 mM), glutamic acid (Glu) 0.736 g (50 mM), valine (Val) 0.586 g (50 mM), adenosine triphosphate disodium (ATP-Na2) 0.110 g (2 mM), magnesium chloride hexahydrate (MgCl2) 0.407 g (20 mM) were weighed into 70 mL of pure water, adjusted to pH 8.0 and made up to 80 mL, and then 10 mL of GCL13-E62D crude enzyme solution obtained by resuspending according to wet bacteria:crushing buffer = 1:4 and ultrasonic crushing, and 10 mL of SlPPK2 crude enzyme solution were added after preheating at 38°C.

[0140] 100 μl of the reaction solution was taken, quenched with 700 μl of 60% acetonitrile, and detected by high performance liquid chromatography (HPLC). After 4 h of reaction, Glu and Val were consumed by 93% and 85%, respectively, and the substrate consumption rate was >80%, which had potential for industrial production.

[0141] Example 9 Unidirectional reaction of GCL13 enzyme to synthesize γ-EN

[0142] Glutamic acid (Glu) 0.736 g (50 mM), asparagine (Asn) 0.661 g (50 mM), adenosine triphosphate disodium (ATP-Na2) 2.756 g (50 mM), magnesium chloride hexahydrate (MgCl2) 0.407 g (20 mM) were weighed into 80 mL of pure water, adjusted to pH 8.0 and made up to 90 mL, and then 10 mL of GCL13 crude enzyme solution obtained by resuspending according to wet bacteria:crushing buffer = 1:4 and ultrasonic crushing was added after preheating at 38°C.

[0143] 100 μl of the reaction solution was taken, quenched with 700 μl of 60% acetonitrile, and detected by high performance liquid chromatography (HPLC). After 4 h of reaction, Glu was consumed by 62%; Asn could not be quantified because the peak position overlapped with that of adenosine monophosphate AMP.

[0144] Example 10 Bi-directional reaction of GCL13 enzyme to synthesize γ-EN

[0145] Also introduced is a ATP regeneration system composed of polyphosphate kinase SlPPK2 from Sulfurovum lithotrophicum and sodium hexametaphosphate.

[0146] Sodium hexametaphosphate (6P) 0.917 g (15 mM), glutamic acid (Glu) 0.736 g (50 mM), asparagine (Asn) (50 mM), adenosine triphosphate disodium (ATP-Na2) 0.110 g (2 mM), magnesium chloride hexahydrate (MgCl2) 0.407 g (20 mM) were weighed into 70 mL of pure water, dissolved, adjusted to pH 8.0 and made up to 80 mL, and after preheating at 38°C, 10 mL of GCL13 crude enzyme solution obtained by resuspending according to wet cell:crushing buffer = 1:4 and ultrasonic crushing was added, and 10 mL of SlPPK2 crude enzyme solution was added.

[0147] 100 μl of the reaction solution was taken, 700 μl of 60% acetonitrile was added for quenching, and high performance liquid chromatography (HPLC) detection was performed. After 2 h of reaction, 72% of Glu was consumed, the product peak was collected for mass spectrometry peptide sequence analysis, Figure 8 The product was confirmed to be γ-EN.

[0148] Example 11 Unidirectional reaction of GCL12 enzyme to synthesize γ-ES

[0149] Glutamic acid (Glu) 0.736 g (50 mM), serine (Ser) 0.525 g (50 mM), adenosine triphosphate disodium (ATP-Na2) 2.756 g (50 mM), magnesium chloride hexahydrate (MgCl2) 0.407 g (20 mM) were weighed into 80 mL of pure water, dissolved, adjusted to pH 8.0 and made up to 90 mL, and after preheating at 38°C, 10 mL of GCL12 crude enzyme solution obtained by resuspending according to wet cell:crushing buffer = 1:4 and ultrasonic crushing was added.

[0150] 100 μl of the reaction solution was taken, 700 μl of 60% acetonitrile was added for quenching, and high performance liquid chromatography (HPLC) detection was performed. After 4 h of reaction, 99% and 88% of Glu and Ser were consumed, respectively.

[0151] The above is only a preferred embodiment of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A variant of glutathione synthetase characterized in that, the amino acid sequence of which is based on SEQ ID: NO: 25 with glutamic acid at position 62 mutated into aspartic acid.

2. Use of the glutathione synthetase variant of claim 1 in the synthesis of a γ-Glu-Val dipeptide.

3. Use of a glutathione synthetase in the synthesis of a γ-Glu-Xaa dipeptide, characterized in that the γ-Glu-Xaa dipeptide is γ-Glu-Ser, γ-Glu-Ile, γ-Glu-Lys, γ-Glu-Glu, γ-Glu-His, γ-Glu-Gly, γ-Glu-Gln, γ-Glu-Ala, γ-Glu-Val, γ-Glu-Asn, γ-Glu-β-Ala, γ-Glu-Met and γ-Glu-Thr; the amino acid sequence of the glutathione synthetase is given in SEQ ID: NO: 23 or SEQ ID: NO:

25.

4. A combination enzyme characterized in that, comprising the glutathione synthetase variant of claim 1 and a polyphosphate kinase; the amino acid sequence of the polyphosphate kinase is given in SEQ ID NO:

29.

5. A nucleic acid, characterized in that, a nucleic acid encoding the combined enzyme of claim 4.

6. A recombinant vector, characterized in that, containing the nucleic acid of claim 5.

7. A host cell characterized in that, the recombinant vector of claim 6.

8. Use of at least one of the following I) to V) in the synthesis of a γ-Glu-Val dipeptide: I) the combined enzyme of claim 4; II) the nucleic acid of claim 5; III) the recombinant vector of claim 6; IV) the host cell of claim 7; V) a culture obtained by cultivating the host cell of claim 7.

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