Biosynthesis method of gamma-Glu-Xaa flavor dipeptide

Abstract

The invention relates to the technical field of biology, in particular to a biosynthesis method of gamma-Glu-Xaa flavor dipeptide. The GCL enzyme with high activity for 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 substrate selection range of the GCL enzyme is wide, and the catalytic efficiency of the GCL enzyme is high; a synthetic process for biologically synthesizing gamma-EV and other gamma-glutamyl dipeptides based on the enzyme is developed, and the synthetic process is simple in step, low in cost and wide in application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a biosynthetic method of gamma-Glu-Xaa flavor dipeptide. Background Art

[0002] In the 1990s, researchers at Ajinomoto Co., Ltd. of Japan separated and identified kokumi ingredients such as glutathione (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), and γ-glutamyl-S-1-propenyl-cysteine ​​sulfoxide (γ-L-glutamyl-S-propenyl-L-cysteine ​​sulfoxide, γ-Glu-PeCSO) from garlic and onions. German scientists later separated and identified 13 kinds of kokumi ingredients in oil beans and cheese, including γ-glutamyl-leucine (γ-Glu-Leu), γ-glutamyl-valine (γ-Glu-Val, γ-EV), γ-glutamyl-cysteinyl-β-alanine (γ-Glu-Cys-β-Ala), γ-glutamyl-glutamic acid (γ-Glu-Glu), γ-glutamyl-methionine (γ-Glu-Met), and γ-glutamyl-glutamine (γ-Glu-Gln), all of which are γ-glutamyl peptides. In 2010, Ajinomoto's researchers discovered that 46 kinds of γ-glutamyl peptides, including γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly, γ-EVG), glutathione, and γ-glutamyl-serine (γ-Glu-Ser, γ-ES), can enhance the kokumi effect of basic tastes such as sweet, salty, and fresh by activating calcium receptors. Among them, γ-glutamyl-cysteine ​​(γ-Glu-Cys) and γ-EV have been reported to exert anti-inflammatory activity by activating CaSR.

[0003] At present, a variety of glutamylcysteine ​​ligases GCL have been tried for the synthesis of γ-Glu-Xaa flavor dipeptides, but overall, glutamylcysteine ​​ligases have strong substrate specificity, resulting in less substrate selectivity, low enzyme activity, high cost, and inability to synthesize more valuable γ-EV. Therefore, it is of great practical significance to develop GCL enzymes with low cost, wide substrate selectivity, and high catalytic efficiency and biosynthetic pathways for γ-Glu-Xaa flavor dipeptides. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a biosynthetic method for γ-Glu-Xaa flavor dipeptide.

[0005] The present invention provides a glutamylcysteine ​​ligase variant, wherein the glutamic acid at position 62 of the amino acid sequence shown in SEQ ID NO:25 is mutated into aspartic acid; specifically, the amino acid sequence of the glutamylcysteine ​​ligase variant is shown in SEQ ID NO:33.

[0006] The present invention provides the use of the glutamylcysteine ​​ligase variant and / or glutamylcysteine ​​ligase in the synthesis of γ-Glu-Xaa dipeptide;

[0007] The γ-Glu-Xaa dipeptide includes γ-Glu-Ser, γ-Glu-Ile, γ-Glu-Lys, γ-Glu-Glu, γ-Glu-His, γ-Glu-Gly, γ-G One or more of lu-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 glutamylcysteine ​​ligase is 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: NO: 13 (GCL07), SEQ ID: NO: 15 (GCL08), SEQ ID: NO: 17 (GCL09), SEQ ID: NO: 19 (GCL010), SEQ ID: NO: 21 (GCL11), SEQ ID: NO: 23 (GCL012) and / or SEQ ID: NO: 25 (GCL013).

[0009] The test results in the specific embodiments of the present invention show that the glutamylcysteine ​​ligase of 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 glutamylcysteine ​​ligase of 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 glutamylcysteine ​​ligase of 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 glutamylcysteine ​​ligase of the amino acid sequence shown in SEQ ID NO: 7 (GCL04) can be used for the synthesis of γ-Glu-Ser; the glutamylcysteine ​​ligase of the amino acid sequence shown in SEQ ID NO: 9 (GCL05) can be used for the synthesis of γ-Glu-Ser; The glutamylcysteine ​​ligase with the amino acid sequence shown in SEQ ID NO:13 (GCL07) can be used for the synthesis of γ-Glu-Gln; the glutamylcysteine ​​ligase with the amino acid sequence shown in SEQ ID NO:15 (GCL08) can be used for the synthesis of γ-Glu-Ser; the glutamylcysteine ​​ligase with the amino acid sequence shown in SEQ ID NO:17 (GCL09) can be used for the synthesis of γ-Glu-Val and γ-Glu-Met; the glutamylcysteine ​​ligase with the amino acid sequence shown in SEQ ID NO:19 (GCL010) can be used for the synthesis of γ-Glu-Val and γ-Glu-Lys; the glutamylcysteine ​​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 and γ-Glu-Thr;

[0010] The present invention provides a combined enzyme comprising at least one of glutamylcysteine ​​ligase (GCL enzyme) and / or the glutamylcysteine ​​ligase variants of the present invention and a polyphosphate kinase.

[0011] Furthermore, in the enzyme combination of the present invention,

[0012] The amino acid sequence of the glutamylcysteine ​​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] GCL enzyme has a strong substrate preference; in substrate selection, GCL enzymes in nature have good activity on substrates Glu and Cys, some have average activity on Glu and Ser, but generally have poor activity on Glu and Val, for example, GCL from Streptococcus agalactiae can generate γ-Glu-Cys and γ-Glu-Ser, and GCL from Clostridium acetobutylicum can only generate γ-Glu-Cys. The present invention obtains GCL enzymes (GCL013 and GCL12, of which GCL013 is better) with a wide substrate range and high conversion activity through screening. The GCL enzyme has the function of synthesizing three flavor dipeptides such as γ-Glu-Val (γ-EV), γ-Glu-Ser (γ-ES), and γ-Glu-Asn (γ-EN). Subsequently, after modification, a mutant with higher activity in synthesizing γ-Glu-Val (γ-EV) is obtained (the amino acid sequence is shown in SEQ ID NO: 33). The mutant is used together with other enzymes, such as polyphosphate kinase, for the bidirectional synthesis of γ-Glu-Xaa flavor dipeptide. The bidirectional synthesis has high conversion efficiency and low cost, and has broad industrial application prospects.

[0015] The present invention provides nucleic acids, which include nucleic acids encoding the glutamylcysteine ​​ligase variant of the present invention, and / or the combined enzyme of the present invention, and / or encoding glutamylcysteine ​​ligase;

[0016] The amino acid sequence of the glutamylcysteine ​​ligase is 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: 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 invention provides an expression unit comprising an expression element and a nucleic acid according to the present invention.

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

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

[0020] The nucleic acid described in the present invention can be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, 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. 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). Nucleic acid can be linear or circular in topology. Nucleic acid can be a 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 assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0021] In the present invention, the nucleic acid is optimized, and these optimizations include but are not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repeat sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning;

[0022] In a specific embodiment of the present invention, after codon optimization,

[0023] The nucleotide sequence encoding the glutamylcysteine ​​ligase variant GCL13 (E62D) is shown in SEQ ID NO: 34;

[0024] The nucleotide sequence of the nucleic acid encoding the glutamylcysteine ​​ligase (GCL12) having 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 glutamylcysteine ​​ligase (GCL13) having 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 (S1PPK2) is shown in SEQ ID NO:30.

[0027] The present invention also provides an expression unit, which refers to a DNA sequence starting from a promoter and ending at a terminator. A regulatory segment may also be included on both sides or between the promoter and the terminator, and the regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably connected to the nucleic acid sequence, such as an enhancer of a promoter, a poly (A) signal, etc.

[0028] The present invention provides a recombinant vector comprising the nucleic acid of the present invention or the expression unit of the present invention.

[0029] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0030] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0031] Further, the vector sources of the present invention include plants, animals, bacteria, fungi, phages, or viruses, and the present invention does not limit this. The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule, which contains the desired coding sequence and the appropriate nucleic acid sequence or element that is essential for the expression of the operably connected coding gene in a specific host organism. The nucleic acid sequence or element necessary for the expression in the model animal or mammalian cell includes a promoter, a ribosome binding site and possible other sequences. It is known that prokaryotes 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, integrate into the genome itself. In this specification, "plasmid" and "vector" can sometimes be interchanged and used, because plasmids are the most commonly used vector forms at present. However, the present invention is intended to include such other forms of expression vectors, which play an equivalent role, which are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors and / or only potential genome inserts. In a specific embodiment, the nucleic acid encoding the fusion protein provided by the present invention can be constructed in various prokaryotic (bacterial) expression vectors, for example, pET series vectors, more specifically pET28a.

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

[0033] The host cell provided by the present invention may be derived from plants, animals, bacteria, fungi, bacteriophages or viruses, which are not limited by the present invention. The present invention uses a vector constructed by recombinant DNA technology to transform or transfect the host cell, so that the transformed host cell has the ability to replicate the protein-encoding vector or express the desired protein. In a specific embodiment of the present invention, the host cell is derived from bacteria, specifically Escherichia coli, more specifically Escherichia coli BL21 (DE3).

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

[0035] 1), the combined enzyme of the present invention;

[0036] II), the nucleic acid of the present invention;

[0037] III), the recombinant vector of the present invention;

[0038] IV), the host cell of the present invention;

[0039] V), culturing the host cell of the present invention to obtain a culture.

[0040] wherein Xaa represents an amino acid residue.

[0041] Further, the γ-Glu-Xaa dipeptide includes γ-Glu-Ser, γ-Glu-Ile, γ-Glu-Lys, γ-Glu-Glu, γ-Glu-His, γ-Glu-Gly, One or more of 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 invention provides a method for preparing a γ-Glu-Xaa dipeptide, which comprises synthesizing the γ-Glu-Xaa dipeptide using at least one of the following i) to v):

[0043] i), the combined enzyme of the present invention;

[0044] ii), the nucleic acid of the present invention;

[0045] iii), the recombinant vector of the present invention;

[0046] iv), the host cell of the present invention;

[0047] v), culturing the host cell of the present invention to obtain a culture.

[0048] The present invention obtains a GCL enzyme with high activity in synthesizing γ-EV through screening and mutation, and its amino acid sequence is shown in SEQ ID NO:15, and its substrate selection range is wide and the catalytic efficiency is high; based on this enzyme, a synthesis process for biosynthesizing γ-EV and other γ-glutamyl dipeptides is developed, and the synthesis process has simple steps, low cost, and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0050] Figure 2 Shows the structural cluster analysis of glutamylcysteine ​​ligase (GCL);

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

[0052] Figure 4 Shows GCL enzyme activity screening;

[0053] Figure 5 The mass spectrometry results of the γ-ES product peak synthesized by GCL13 enzyme are shown;

[0054] Figure 6 The mass spectrometry results of the γ-EV product peak synthesized by GCL13 enzyme are shown;

[0055] Figure 7 shows the expression electrophoresis diagram of GCL-E62D enzyme;

[0056] Figure 8 The mass spectrometry results of the γ-EN product peak synthesized by GCL13 enzyme are shown;

[0057] Fig. 9 The reaction formula for the synthesis of γ-ES by GCL enzyme is shown;

[0058] Fig.10 The reaction formula for the synthesis of γ-EV by GCL enzyme is shown. DETAILED DESCRIPTION

[0059] The present invention provides a biosynthetic method for γ-Glu-Xaa flavor dipeptide, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

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

[0061]

[0062] The 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] Amino acid sequence of GCL08: 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 (Betaproteobacteria 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] The amino acid sequence of SlPPK2 (Sulfurovum lithotrophicum) is: MKKNIYKKELYKLQVELVKFQKYVIEENVAVCLVLEGRDTAGKDGTIKRFTTEHLSPREARTVALGVPSDKEKKSWYFQRYVPHLPSAGEIVFFNRSWYNRAGVEKVMGFCTKKQYKAFMEEVGSFEQMLTHSNIRFFKYYLDITKKEQKKRLEARKTDPLKQWKLSPIDAKAQKMWDAYSKARDDMFNKTSFIYAPWYVVHTDDKKEARINIMKHFLSLNDYPDKDKALLVYDHDVICKFDPVCYEKEMIAP (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 invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0097] Example 1 Screening of GCL enzyme

[0098] 1. Screening of existing enzyme libraries

[0099] Eight candidate enzymes in Table 1 were selected, and reverse primers were designed respectively. The corresponding GCL enzyme fragments were amplified from the original template in combination with universal primer T7, digested with NdeI and XhoI restriction endonucleases, connected to the pET28a vector, and transformed into the BL21 (DE3) expression host.

[0100] Table 1

[0101]

[0102]

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

[0104] Take the stored glycerol bacteria and streak them on LB plates (Kan) and culture them in a 37°C incubator overnight. Then pick 3 single colonies of appropriate size from each plate and culture them in 10mL LB liquid culture medium (Kan) at 37°C and 200rpm for 6-8h until turbidity. Take 1mL of it to make glycerol bacteria for storage, and transfer the rest to 400mL LB liquid culture medium (Kan) and culture it at 37°C and 200rpm for 2-3h until OD reaches 0. 600 =0.6-1.0, add 0.1mM IPTG and transfer to 20℃200rpm to induce expression for 14-18h. Collect the cells by low-temperature centrifugation and weigh them, add 9mL of disruption buffer (20mM K2HPO4 / KH2PO4, 100mM KCl, pH 7.6) according to 1g of wet cells, resuspend, ultrasonically disrupt, take samples and centrifuge at high speed, prepare samples of supernatant and precipitate respectively, and detect expression by protein electrophoresis.

[0105] like Figure 1 As shown, 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 molecular construction stage verification and was not expressed in shake flask culture.

[0106] Take 0.1mL of the broken liquid and add 0.9mL of the reaction solution (10mM Glu, 10mM Xaa (representing different amino acids), 15mMATP, 20mM MgCl2, pH 8.0), and place in a water bath at 38°C. Samples were taken for high performance liquid chromatography (HPLC) detection. The reduction of ATP after 1h of reaction is shown in Table 2. "-" means that the reduction is less than 1mM and is not counted. The initial activity of the fusion-evolved GCL enzyme in synthesizing the following γ-Glu-Xaa dipeptide was 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. Among them, GCL04, GCL05, and GCL08 can only synthesize γ-Glu-Ser, and GCL07 can only synthesize γ-Glu-Gln.

[0107] Table 2

[0108]

[0109] 3. Structural cluster analysis of enzymes

[0110] Based on Table 1, GCL01 to GCL08 are all unable to synthesize γ-Glu-Val (γ-EV); in order to explore the activity of independently evolved GCL enzymes in synthesizing γ-Glu-Xaa dipeptide, the UniProt code of EcGCL enzymes was input into the structural clustering analysis website https: / / cluster.foldseek.com / , as shown in Figure 2 As shown, EcGCL has been classified into a structural cluster consisting of 224 protein members, from which 6 candidate enzymes in Table 3 were selected for full gene synthesis, constructed in pET28a vector, and transformed into BL21 (DE3) expression host.

[0111] Table 3

[0112] UniProt Encoding 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 independently evolved GCL enzymes

[0114] Streak the stored glycerol bacteria on LB plates (Kan) and culture them in a 37°C incubator overnight. Then pick 3 single colonies of appropriate size from each plate and culture them in 10 mL LB liquid culture medium (Kan) at 37°C and 200 rpm for 6-8 hours until turbidity. Take 1 mL of the culture medium to prepare glycerol bacteria for storage, and transfer the rest to 400 mL LB liquid culture medium (Kan) and culture at 37°C and 200 rpm for 2-3 hours until OD reaches 0. 600=0.6-1.0, add 0.1mM IPTG and transfer to 20℃200rpm to induce expression for 14-18h. Collect the cells by low-temperature centrifugation and weigh them, add 9mL of disruption buffer (20mM K2HPO4 / KH2PO4, 100mM KCl, pH 7.6) according to 1g of wet cells, resuspend, ultrasonically disrupt, take samples and centrifuge at high speed, prepare samples of supernatant and precipitate respectively, and detect expression by protein electrophoresis.

[0115] like Figure 3 As shown, GCL12 and GCL13 were mainly expressed in the supernatant, GCL10 and GCL11 were expressed in both the supernatant and the precipitate, GCL09 was expressed at a low level, and EcGCL was found to be expressed in the precipitate during the transfection stage. The addition of a solubilizing tag did not improve the situation, and shake flask culture expression was not performed.

[0116] 0.1 mL of the broken liquid was added to 0.9 mL of the reaction solution (10 mM Glu, 10 mM Xaa, 15 mM ATP, 20 mM MgCl2, pH 8.0) and incubated in a 38°C water bath. Samples were taken for high performance liquid chromatography (HPLC) detection. The amount of ATP reduction after 1 h of reaction is shown in Table 4. "-" means that the reduction is less than 1 mM and is not counted. The amount of ATP reduction after 1 h of reaction for GCL12 and GCL13 with a wide substrate range is shown in Table 4. Figure 4 As shown, GCL13 enzyme may have a higher conversion rate than GCL12 enzyme in synthesizing three γ-glutamyl dipeptides, namely γ-Glu-Val (γ-EV), γ-Glu-Ser (γ-ES), and γ-Glu-Asn (γ-EN).

[0117] Table 4

[0118]

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

[0120] Weigh 0.736 g (50 mM) of glutamic acid (Glu), 0.525 g (50 mM) of serine (Ser), 2.756 g (50 mM) of adenosine triphosphate disodium (ATP-Na2), and 0.407 g (20 mM) of magnesium chloride hexahydrate (MgCl2), dissolve them in 80 mL of pure water, adjust the pH to 8.0 and make the volume to 90 mL, preheat at 38°C, add 10 mL of GCL13 crude enzyme solution obtained by resuspending and ultrasonically disrupting the wet cells: disruption buffer at a ratio of 1:4.

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

[0122] Example 3 Bidirectional reaction of GCL13 enzyme in synthesizing γ-ES

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

[0124] Weigh 0.917 g (15 mM) of sodium hexametaphosphate (6P), 0.736 g (50 mM) of glutamic acid (Glu), 0.525 g (50 mM) of serine (Ser), 0.110 g (2 mM) of adenosine triphosphate disodium (ATP-Na2), and 0.407 g (20 mM) of magnesium chloride hexahydrate (MgCl2), and dissolve them in 70 mL of pure water. Adjust the pH to 8.0 and make the volume to 80 mL. After preheating at 38°C, add 10 mL of GCL13 crude enzyme solution and 10 mL of SlPPK2 crude enzyme solution obtained by resuspending and ultrasonically disrupting the wet cells: disruption buffer at a ratio of 1:4.

[0125] 100 μl of the reaction solution was taken, quenched with 700 μl of 60% acetonitrile, and detected by high performance liquid chromatography (HPLC). After 2 h of reaction, Glu and Ser were consumed by 99% and 100%, respectively.

[0126] Example 4 Laboratory Scale-up of GCL13 Enzyme Synthesis of γ-ES

[0127] Weigh 18.34 g (30 mM) of sodium hexametaphosphate (6P), 14.71 g (100 mM) of glutamic acid (Glu), 10.51 g (100 mM) of serine (Ser), 1.10 g (2 mM) of adenosine triphosphate disodium (ATP-Na2), and 4.07 g (20 mM) of magnesium chloride hexahydrate (MgCl2), and dissolve them in 700 mL of pure water, adjust the pH to 8.0 and make the volume to 80 mL, preheat at 38°C, add 100 mL of GCL13 crude enzyme solution and 100 mL of SlPPK2 crude enzyme solution obtained by resuspending and ultrasonically disrupting the wet cells: disruption buffer at a ratio of 1:4.

[0128] Take 100μl of the reaction solution, add 700μl of 60% acetonitrile to quench, and detect by high performance liquid chromatography (HPLC). After 4h of reaction, Glu and Ser are consumed by 97% and 97% respectively. After 5h of reaction, adjust the pH of the reaction solution to 3.0, centrifuge and take the supernatant, filter and prepare freeze-dried to obtain a γ-ES sample with a purity of 97.14% and a content of 70.05% (NMR quantitative results).

[0129] Example 5 Unidirectional reaction of GCL13 enzyme to synthesize γ-EV

[0130] Weigh 0.736 g (50 mM) of glutamic acid (Glu), 0.586 g (50 mM) of valine (Val), 2.756 g (50 mM) of adenosine triphosphate disodium (ATP-Na2), and 0.407 g (20 mM) of magnesium chloride hexahydrate (MgCl2), dissolve them in 80 mL of pure water, adjust the pH to 8.0 and make the volume to 90 mL, preheat at 38°C, add 10 mL of GCL13 crude enzyme solution obtained by resuspending and ultrasonically disrupting the wet cells: disruption buffer at a ratio of 1:4.

[0131] Take 100 μl of the reaction solution, add 700 μl of 60% acetonitrile to quench, and detect by high performance liquid chromatography (HPLC). After 6 hours of reaction, Glu, Val and ATP are consumed by 99%, 82% and 84% respectively, and the product peaks are 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 in synthesizing γ-EV

[0133] The ATP regeneration system consisting of polyphosphate kinase SlPPK2 from Sulfurovum lithotrophicum and sodium hexametaphosphate was also introduced.

[0134] Weigh 0.917 g (15 mM) of sodium hexametaphosphate (6P), 0.736 g (50 mM) of glutamic acid (Glu), 0.586 g (50 mM) of valine (Val), 0.110 g (2 mM) of adenosine triphosphate disodium (ATP-Na2), and 0.407 g (20 mM) of magnesium chloride hexahydrate (MgCl2), and dissolve them in 70 mL of pure water. Adjust the pH to 8.0 and make the volume to 80 mL. After preheating at 38°C, add 10 mL of GCL13 crude enzyme solution and 10 mL of SlPPK2 crude enzyme solution obtained by resuspending and ultrasonically disrupting the wet cells: disruption buffer at a ratio of 1:4.

[0135] 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 68% and 62% respectively.

[0136] Example 7 Rational design of GCL13 enzyme

[0137] In order to broaden the substrate pocket of the GCL13 enzyme to accommodate Val, which is larger than Cys and Ser, a GCL13-E62D mutant was designed, in which the glutamic acid at position 62 was mutated to aspartic acid. The corresponding plasmid pET28a-GCL3-E62D was constructed using the Quick-Change site-directed mutagenesis kit (Agilent) with pET28a-GCL3 as a template and GCL13-E62D-up and GCL13-E62D-dn as primers. After successful sequencing, it was transformed into a BL21 (DE3) expression host. The GCL13-E62D mutant was cultured and expressed according to Example 1 ( Figure 7 (Figure 5A shows the expression electrophoresis of GCL-E62D enzyme).

[0138] Example 8 Bidirectional reaction of GCL13-E62D enzyme in synthesizing γ-EV

[0139] Weigh 0.917 g (15 mM) of sodium hexametaphosphate (6P), 0.736 g (50 mM) of glutamic acid (Glu), 0.586 g (50 mM) of valine (Val), 0.110 g (2 mM) of adenosine triphosphate disodium (ATP-Na2), and 0.407 g (20 mM) of magnesium chloride hexahydrate (MgCl2), and dissolve them in 70 mL of pure water. Adjust the pH to 8.0 and make the volume to 80 mL. After preheating at 38°C, add 10 mL of GCL13-E62D crude enzyme solution and 10 mL of SlPPK2 crude enzyme solution obtained by resuspending and ultrasonically disrupting the wet cells: disruption buffer at a ratio of 1:4.

[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 hours of reaction, Glu and Val were consumed by 93% and 85% respectively, and the substrate consumption rate was >80%, which has the potential for industrial production.

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

[0142] Weigh 0.736 g (50 mM) of glutamic acid (Glu), 0.661 g (50 mM) of asparagine (Asn), 2.756 g (50 mM) of adenosine triphosphate disodium (ATP-Na2), and 0.407 g (20 mM) of magnesium chloride hexahydrate (MgCl2), dissolve them in 80 mL of pure water, adjust the pH to 8.0 and make the volume to 90 mL, preheat at 38°C, add 10 mL of GCL13 crude enzyme solution obtained by resuspending and ultrasonically disrupting the wet cells: disruption buffer at a ratio of 1:4.

[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 hours of reaction, Glu was consumed by 62%; Asn could not be quantified because its elution peak overlapped with adenosine monophosphate AMP.

[0144] Example 10 Bidirectional reaction of GCL13 enzyme in synthesizing γ-EN

[0145] The ATP regeneration system consisting of polyphosphate kinase SlPPK2 from Sulfurovum lithotrophicum and sodium hexametaphosphate was also introduced.

[0146] Weigh 0.917 g (15 mM) of sodium hexametaphosphate (6P), 0.736 g (50 mM) of glutamic acid (Glu), 0.110 g (2 mM) of asparagine (Asn) (50 mM), 0.407 g (20 mM) of adenosine triphosphate disodium (ATP-Na2), and 0.407 g (20 mM) of magnesium chloride hexahydrate (MgCl2), and dissolve them in 70 mL of pure water. Adjust the pH to 8.0 and make the volume to 80 mL. After preheating at 38°C, add 10 mL of GCL13 crude enzyme solution and 10 mL of SlPPK2 crude enzyme solution obtained by resuspending and ultrasonically disrupting wet cells: disruption buffer at a ratio of 1:4.

[0147] Take 100 μl of the reaction solution, add 700 μl of 60% acetonitrile to quench, and detect by high performance liquid chromatography (HPLC). After 2 hours of reaction, Glu consumption was 72%, and the product peak was collected for mass spectrometry peptide sequence analysis. Figure 8 The product was confirmed to be γ-EN.

[0148] Example 11 One-way reaction of GCL12 enzyme in synthesizing γ-ES

[0149] Weigh 0.736 g (50 mM) of glutamic acid (Glu), 0.525 g (50 mM) of serine (Ser), 2.756 g (50 mM) of adenosine triphosphate disodium (ATP-Na2), and 0.407 g (20 mM) of magnesium chloride hexahydrate (MgCl2), dissolve them in 80 mL of pure water, adjust the pH to 8.0 and make the volume to 90 mL, preheat at 38°C, add 10 mL of GCL12 crude enzyme solution obtained by resuspending and ultrasonically disrupting the wet cells: disruption buffer at a ratio of 1:4.

[0150] 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 Ser were consumed by 99% and 88% respectively.

[0151] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A glutamylcysteine ​​ligase variant, characterized in that The amino acid sequence is as shown in SEQ ID: NO: 25, in which the glutamic acid at position 62 is mutated into aspartic acid.

2. Use of the glutamylcysteine ​​ligase variant and / or glutamylcysteine ​​ligase according to claim 1 in the synthesis of γ-Glu-Xaa dipeptide; The γ-Glu-Xaa dipeptide includes γ-Glu-Ser, γ-Glu-Ile, γ-Glu-Lys, γ-Glu-Glu, γ-Glu-His, γ-Glu-Gly, γ-G One or more of lu-Gln, γ-Glu-Ala, γ-Glu-Val, γ-Glu-Asn, γ-Glu-β-Ala, γ-Glu-Met and / or γ-Glu-Thr; The amino acid sequence of the glutamylcysteine ​​ligase is shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID:13, SEQ ID:15, SEQ ID:17, SEQ ID:19, SEQ ID:21, SEQ ID:23 and / or SEQ ID:

25.

3. A combined enzyme, characterized in that The method comprises at least one of glutamylcysteine ​​ligase and / or the glutamylcysteine ​​ligase variant according to claim 1 and polyphosphate kinase.

4. The combined enzyme according to claim 3, characterized in that: The amino acid sequence of the glutamylcysteine ​​ligase is shown in SEQ ID: NO: 23 (GCL012) and / or SEQ ID: NO: 25 (GCL013); The amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO:

29.

5. A nucleic acid, characterized in that Comprising a nucleic acid encoding the glutamylcysteine ​​ligase variant of claim 1, and / or the combined enzyme of claim 3 or 4, and / or encoding glutamylcysteine ​​ligase; The amino acid sequence of the glutamylcysteine ​​ligase is shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID:13, SEQ ID:15, SEQ ID:17, SEQ ID:19 and / or SEQ ID:

21.

6. A recombinant vector, characterized in that Containing the nucleic acid according to claim 5.

7. A host cell, characterized in that Transfect or transform the recombinant vector as described in claim 6.

8. Use of at least one of the following I) to V) in the synthesis of γ-Glu-Xaa dipeptide: 1), the combined enzyme according to claim 3 or 4; II), the nucleic acid according to claim 5; III), the recombinant vector according to claim 6; IV), the host cell according to claim 7; V), a culture obtained by culturing the host cell according to claim 7. wherein Xaa represents an amino acid residue.

9. The use according to claim 8, characterized in that: The γ-Glu-Xaa dipeptide includes γ-Glu-Ser, γ-Glu-Ile, γ-Glu-Lys, γ-Glu-Glu, γ-Glu-His, γ-Glu-Gly, γ-G One or more of lu-Gln, γ-Glu-Ala, γ-Glu-Val, γ-Glu-Asn, γ-Glu-β-Ala, γ-Glu-Met and / or γ-Glu-Thr.

10. A method for preparing a γ-Glu-Xaa dipeptide, characterized in that: The method comprises synthesizing the γ-Glu-Xaa dipeptide by using at least one of the following i) to v): i), the combined enzyme according to claim 3 or 4; ii), the nucleic acid according to claim 5; iii) the recombinant vector according to claim 6; iv), the host cell according to claim 7; v) culturing a culture obtained by culturing the host cell according to claim 7.

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