A mutant of glutathione cysteine dipeptide ligase and its encoding gene, recombinant plasmid, recombinant microorganism and application

Abstract

The application belongs to the field of enzymes and particularly relates to a mutant of glutamyl cysteine dipeptide ligase, a coding gene of the mutant, a recombinant plasmid, a recombinant microorganism and application, wherein the amino acid sequence of the mutant is an amino acid sequence with at least one mutation of T84V, S27A, L128K and A691S in the wild amino acid sequence shown in SEQ ID NO. 1. Researches of the application show that the catalytic activity of the enzyme can be significantly enhanced by the mutation of the specific sites in the wild type sequence of SEQ ID NO. 1, and the reaction conversion yield can be effectively improved when the enzyme is used for the catalytic synthesis of gamma-glutamyl cysteine dipeptide.

Description

Technical Field

[0001] This invention belongs to the field of enzymes, specifically relating to a mutant of glutamylcysteine ​​dipeptide ligase and its encoding gene, recombinant plasmid, recombinant microorganism and its application fields. Background Technology

[0002] Reduced glutathione (GSH) has important physiological functions in living organisms, such as scavenging excess free radicals and neutralizing and detoxifying, and is now widely used in the medical and food industries.

[0003] γ-Glutamylcysteine ​​(GGC), an intermediate product in the synthesis of glutathione (GSH) in vivo, contains both γ-glutamyl and sulfhydryl groups, and its function is very similar to that of GSH, giving it strong stability and multiple physiological functions. For example, the most prominent group is the γ-peptide bond formed by the condensation of the γ-COOH of L-glutamate and the α-NH2 of L-cysteine. This group not only prevents itself from being hydrolyzed by various enzymes but also protects the protein, giving it strong stability. Furthermore, it plays an important role in maintaining the γ-glutamyl cycle, which is essential for maintaining normal cellular physiological functions. Compared to glutathione, GGC has its own unique advantages. Studies have shown that GSH cannot effectively enter cells and is easily oxidized extracellularly to the larger oxidized form of glutathione. Compared to directly providing GSH, GGC has a smaller molecular weight and can enter cells to directly synthesize GSH using intracellular glutathione synthase, making it more effective in increasing intracellular GSH levels.

[0004] Furthermore, studies have shown that the sulfhydryl groups in γ-glutamylcysteine ​​can protect the sulfhydryl groups of important proteins in the body from oxidation. It also binds to intracellular oxygen free radicals through its side chain, reducing them to metabolizable substances, thus accelerating metabolism and reducing the damage caused by oxygen free radicals to tissue cells. γ-glutamylcysteine ​​can improve oxidative damage to neurons and astrocytes, and can also inhibit the production of tumor necrosis factor stimulated by lipopolysaccharide, thereby reducing inflammatory responses. Due to its antioxidant and stabilizer functions, γ-glutamylcysteine ​​can also be used as a food additive to improve the taste and color of food, enhancing its quality. Therefore, γ-glutamylcysteine ​​has broad application prospects in the pharmaceutical, cosmetic, and food industries.

[0005] Regarding the synthesis of GGC, three main methods have been reported: yeast fermentation, chemical synthesis, and enzymatic synthesis. Yeast fermentation suffers from drawbacks such as long processing time, low product concentration, and high cost due to extraction difficulties. Chemical synthesis, using glutamic acid and cysteine ​​as raw materials, involves side-chain group protection, peptide condensation, and deprotection, but is cumbersome, energy-intensive, and has low safety. In contrast, enzymatic synthesis is a more efficient, economical, and environmentally friendly method.

[0006] In terms of enzymatic synthesis, literature reports that various enzymes can catalyze the synthesis of GGC, such as γ-glutamylcysteine ​​synthase (GSH I) and γ-glutamyltransferase (GGT). However, both of these enzymes have bottlenecks and drawbacks for industrial application. For example, GGT enzyme has transpeptidation, autotransferase, and hydrolysis activities, and the reaction system requires L-glutamine and cysteine ​​as substrates. After synthesizing glutamylcysteine ​​through enzymatic transpeptidation, it is further reduced by dithiothreitol to obtain the target product GGC. Its maximum conversion rate is only 24.5%, which is too low for industrial application. Similarly, the enzymatic synthesis using GSH I is also limited by low conversion yield and impurities, hindering its industrial application.

[0007] To improve enzyme activity, existing technologies have also provided some mutation methods. For example, Chinese patent application document CN117757760A discloses a glutamylcysteine ​​synthase and its application. The glutamylcysteine ​​synthase is obtained by mutating glycine at position 262 to alanine, isoleucine at position 456 to leucine, alanine at position 470 to glutamine, and threonine at position 496 to tryptophan. For example, patent application CN116769738A discloses a mutant of glutamate-cysteine ​​synthase and its application in glutathione synthesis. It screened out the mutant sites at positions 150, 188, 235, 236, 241, 300, 301, 302 and 330 of wild-type glutamate-cysteine ​​synthase (EC :6.3.2.2) derived from Citrobacter koseri (strain ATCC BAA-895) by performing single-site mutations on amino acid residues at positions 150, 236, 302 and 330.

[0008] In summary, although existing technologies have reported some mutants of glutamylcysteine ​​dipeptide ligases, the wild-type sequences and mutation methods of different mutants vary, all of which significantly affect the overall structure and catalytic activity of the enzyme, thus substantially impacting GGC synthesis. Therefore, existing enzymatic synthesis methods for GGC require further development. Summary of the Invention

[0009] Based on the problems existing in the catalytic synthesis of GGC enzymes, this invention provides a mutant of glutamylcysteine ​​dipeptide ligase, which aims to improve the enzyme catalytic activity of GGC.

[0010] The second objective of this invention is to provide the application of the mutant in the enzyme-catalyzed synthesis of GGC.

[0011] A third objective of this invention is to provide a coding gene, a recombinant plasmid, and a recombinant microorganism that encodes the mutant.

[0012] A mutant of a glutamylcysteine ​​dipeptide ligase, wherein the amino acid sequence of the mutant is a mutated amino acid sequence containing at least one of the following mutated amino acid sequences: T84V, S27A, L128K, and A691S, in the wild-type amino acid sequence shown in SEQ ID NO. 1.

[0013] This invention innovatively involves single or multiple mutations of T84V, S27A, L128K, and A691S in the wild-type amino acid sequence shown in SEQ ID NO.1, which can improve the enzyme catalytic synthesis activity of GGC.

[0014] The mutants described in this invention are obtained by site-directed mutagenesis using the amino acid sequence of the glutamylcysteine ​​dipeptide ligase derived from Streptococcus sp. (SEQ ID NO.1, corresponding GenBank accession number: WP_314462384.1) as a template. Specifically, T84V refers to the mutation of threonine (T) at position 84 to valine (V); S27A refers to the mutation of serine (S) at position 27 to alanine (A); L128K refers to the mutation of leucine (L) at position 128 to lysine (K); and A691S refers to the mutation of alanine (A) at position 691 to serine (S).

[0015] In this invention, the mutant is a mutation in which the threonine at position 84 of the glutamylcysteine ​​dipeptide ligase shown in SEQ ID NO.1 is mutated to valine (T84V single-point mutant, SEQ ID NO.2); or the serine at position 27 is mutated to alanine (S27A single-point mutant, SEQ ID NO.3); or the threonine at position 84 and the serine at position 27 are mutated to valine and alanine, respectively (T84V / S27A two-point mutant, SEQ ID NO.4); or the threonine at position 84, the serine at position 27, the leucine at position 128, and the alanine at position 691 are mutated to valine, alanine, lysine, and serine, respectively (T84V / S27A / L128K / A691S four-point mutant, SEQ ID NO.5).

[0016] The present invention demonstrates that the mutant of SEQ ID NO.5 can unexpectedly further improve the enzymatic catalytic activity of GGC.

[0017] The present invention also includes the application of the mutant described herein, using it for the enzymatic synthesis of γ-glutamylcysteine.

[0018] The application described in this invention involves subjecting a mixture containing glutamate, cysteine, water-soluble magnesium salt, ATP, and an enzyme catalyst to an enzymatic reaction to produce γ-glutamylcysteine; the enzyme catalyst comprises the mutant described in this invention.

[0019] In the application described in this invention, the glutamate is sodium glutamate;

[0020] Cysteine ​​salt is cysteine ​​hydrochloride;

[0021] The water-soluble magnesium salt is magnesium chloride;

[0022] In the mixture, the amount of the mutant added is 1-1.2 kU / L. The concentration of ATP is 25-500 mmol / L; the concentration of glutamate is 120-200 mmol / L; the concentration of cysteine ​​is 150-200 mmol / L; the concentration of water-soluble magnesium salt is 50-150 mmol / L; further, the concentration of ATP is 30-350 mmol / L; the concentration of glutamate is 140-160 mmol / L; the concentration of cysteine ​​is 155-175 mmol / L; and the concentration of water-soluble magnesium salt is 90-110 mmol / L.

[0023] In the application described in this invention, the temperature of the enzyme-catalyzed reaction is 20~35℃, and more preferably 28~32℃; the pH is 6~7.5, and more preferably 6.5~7.

[0024] The present invention also provides a coding gene for a mutant of the aforementioned glutamylcysteine ​​dipeptide ligase.

[0025] The present invention also provides a recombinant plasmid loaded with the aforementioned gene.

[0026] The vector for the recombinant plasmid can be the pET-30a(+) plasmid.

[0027] The present invention also provides a recombinant microorganism capable of expressing the aforementioned mutant; it may further be a bacterium or a fungus. The bacterium, for example, is *Escherichia coli*.

[0028] The recombinant microorganism described herein carries the recombinant plasmid.

[0029] In this invention, the recombinant microorganisms can be used for fermentation to obtain a fermentation broth; the fermentation broth can be centrifuged to collect the bacterial cells; the bacterial cells can be broken up and centrifuged to obtain a cell lysis supernatant; the cell lysis supernatant can be extracted to obtain the mutant.

[0030] In this invention, the cells of recombinant microorganisms after fermentation can be directly mixed with a substrate solution containing ATP, glutamate, cysteine, and water-soluble magnesium salt to carry out enzymatic synthesis and obtain GGC.

[0031] Beneficial effects

[0032] The glutamylcysteine ​​dipeptide ligase mutants described in this invention exhibit excellent enzyme activity, particularly the four-site mutant T84V / S27A / L128K / A691S, which increases the enzyme's catalytic activity by 2.06 times. Under the same fermentation cell load, the optimal mutant showed a 2.14-fold increase in conversion rate compared to the wild type, with a product GGC concentration reaching 34.26 mg / mL.

[0033] The glutamylcysteine ​​dipeptide ligase mutant of the present invention significantly improves the conversion yield while improving the catalytic activity of the enzyme, which helps to reduce production costs and improve production efficiency, and has the prospect of industrial application. Attached Figure Description

[0034] Figure 1 SDS-PAGE images of protein expression in different mutants of gshAB;

[0035] Figure 2 This is the liquid phase detection spectrum of GGC. Detailed Implementation

[0036] Example 1: Construction of a recombinant strain containing glutamylcysteine ​​dipeptide ligase

[0037] The amino acid sequence of glutamylcysteine ​​dipeptide ligase derived from Streptococcus sp. (SEQ ID NO.1, corresponding GenBank accession number: WP_314462384.1) was downloaded from GenBank and submitted to General Biotech (Anhui) Co., Ltd. for whole genome sequence synthesis (using E. coli preferred codons).

[0038] The synthesized gene, with a His tag at its C-terminus, was constructed into the prokaryotic expression vector pET30a(+). The prokaryotic expression vector contains Nde I restriction sites at the 5' end and Xho I restriction sites at the 3' end. After purification, the constructed plasmid pET30a(+)-gshAB was transformed into the *E. coli* expression strain BL21(DE3) using the CaCl2 heat shock method. The bacterial culture was plated on LB agar plates containing kanamycin sulfate (50 µg / mL) and incubated overnight at 37 °C. The colonies that grew on the plates were the prokaryotic recombinant expression strain *E. coli* BL21(DE3) / pET30a(+)-gshAB containing gamma-glutamylcysteine ​​dipeptide ligase.

[0039] Using a sterile pipette tip, carefully pick a single colony of the prokaryotic expression recombinant strain of glutamylcysteine ​​dipeptide ligase from the above-mentioned LB solid medium plate, and inoculate it into an Erlenmeyer flask containing 20 mL of LB liquid medium. Incubate overnight at 37 ℃ with shaking at 200 r / min. The next day, inoculate the shake-flask culture at a rate of 1% into an Erlenmeyer flask containing 100 mL of TB liquid medium. Incubate at 37 ℃ with shaking at 220 r / min, and measure the OD value of the culture medium every 1 h. When the OD value of the culture medium reaches 1.5, add lactose to a final concentration of 1% (m / v), and continue incubation at 25 ℃ with shaking for 4 h to 6 h, then stop incubation.

[0040] Collect the above-mentioned fermentation cells, wash them 2-3 times with sterile water, add physiological saline, and break the cells by sonication or homogenization. After solid-liquid separation, take an appropriate amount of supernatant for GGC synthesis testing.

[0041] Add the reaction raw materials to a test tube according to the following reaction system:

[0042] In a 10 mL reaction system, 0.15 M sodium glutamate, 0.16 M cysteine ​​hydrochloride, 0.1 M magnesium chloride hexahydrate, and 0.3 M ATP were added to a test tube and dissolved in pure water. The mixture was kept at a constant temperature of 25 °C, and the pH was adjusted to 6.8 with liquid alkali. 1 mL of the fermentation lysate of the above recombinant engineered bacteria was added, and the reaction time was 2 h. After sampling, the amount of GGC generated was detected by HPLC.

[0043] HPLC detection conditions:

[0044] Instrument model: Agilent 1200;

[0045] The ultraviolet detector has a wavelength of 220 nm;

[0046] The chromatographic column was C18 (4.6 × 250 mm, 5 μm);

[0047] Mobile phase: Methanol / water (containing 0.1% acetic acid) = 5 / 95.

[0048] The column temperature was 25 ℃, the injection volume was 10 μL, and the flow rate was 1.0 mL / min.

[0049] The activity of glutamylcysteine ​​dipeptide ligase is defined as follows: at 25 °C and pH 6.8, the amount of enzyme that produces 1 μmol of GGC per minute in the reaction system per unit volume is defined as one unit of enzyme activity, denoted as 1 U.

[0050] Example 2: Mutation screening of glutamylcysteine ​​dipeptide ligase

[0051] Compared to Example 1, the difference lies in the use of the pET30a(+)-gshAB recombinant plasmid as the PCR template, and the use of conventional T7F / T7R as universal primers (primer sequences: T7F: 5'-TAATACGACTCACTATAGGG-3', T7R: 5'-GCTAGTTATTGCTCAGCGG-3') for error-prone PCR amplification of the gshAB gene. The Mg content in the PCR amplification reaction system was adjusted. 2+ Mn 2+ The concentrations of dCTP and dTTP oligonucleotides were adjusted to ensure that the base mismatch rate of the mutant library was only 0.2%, guaranteeing that only 1 to 2 amino acids were mutated in each mutant. The error-prone PCR reaction system is shown in Table 1.

[0052] Table 1: Error-prone PCR reaction systems

[0053] Error-prone PCR reaction conditions: Pre-denaturation at 95 ℃ for 5 min; followed by denaturation at 94 ℃ for 30 s, annealing at 56 ℃ for 1 min, and extension at 72 ℃ for 1.5 min, for a total of 25 cycles; final extension at 72 ℃ for 10 min. 2 μL of the above error-prone PCR product was sampled and detected by agarose gel electrophoresis. After successful detection, the product was purified using a PCR product purification kit. At 37 ℃, the purified PCR product and the prokaryotic expression vector pET30a(+) were double-digested with Nde I and Xho I restriction endonucleases, respectively. The digested products were recovered from the gel (the recovered purified PCR product fragment was approximately 2274 bp, and the recovered pET30a(+) fragment was approximately 5400 bp). The mixture was then combined with the pET30a(+) prokaryotic expression vector at a molar ratio of 3:1, and T4 DNA ligase was added for overnight ligation at 16 ℃. The next day, the ligation product was transferred into E. coli BL21(DE3) by electroporation to construct an engineered bacterium, thus obtaining a large-capacity random mutant library.

[0054] The high-throughput screening method for clones by selecting random mutant libraries is as follows:

[0055] Add 15 μL of premixed substrate mixture (0.15 M monosodium glutamate, 0.16 M cysteine ​​hydrochloride, 0.1 M magnesium chloride hexahydrate, 0.3 M ATP, and 100 mM Tris-HCl buffer at pH 6.8) to a 96-well microplate, along with 10 μL of cell lysis supernatant. Incubate at 25°C for 30 min. Then add 25 μL of 1% ammonium molybdate solution and 50 μL of 12% ascorbic acid, and incubate at room temperature for 5 min for color development. Next, add 100 μL of 2% acetic acid and 2% sodium citrate, incubate at room temperature for 15 min, and then read the values ​​at 655 nm using a microplate reader. The unmutated strain in the first row serves as the control. Wells showing significant changes in microplate readings are selected for subsequent shake-flask replication verification. Viability is detected by HPLC and sequenced for analysis. The results are shown in Table 2.

[0056] Table 2: Different glutamylcysteine ​​dipeptide ligase mutants

[0057] SDS-PAGE images of different mutant gshAB proteins are shown below. Figure 1 Where M represents the standard molecular weight of the protein. Lane 1 shows the results of the original wild-type gshAB; lane 2 shows the results of gshAB-1; lane 3 shows the results of gshAB-2; lane 4 shows the results of gshAB-3; lane 5 shows the results of gshAB-4.

[0058] As shown in the preliminary screening results in Table 2, the mutation scheme of the present invention can significantly enhance enzyme activity, especially the special four-point mutant, which can achieve better mutation results.

[0059] Example 3: Optimization of reaction conditions for GGC-catalyzed synthesis 1

[0060] According to Example 2, the optimal mutant gshAB-4 was selected for optimization of reaction conditions for GGC-catalyzed synthesis.

[0061] (1) pH of different reactions. In a 10 mL reaction system, sodium glutamate (0.15 M), cysteine ​​hydrochloride (0.16 M), magnesium chloride hexahydrate (0.1 M), and ATP (0.3 M) were added to a test tube and dissolved in pure water. The system was kept at a constant temperature of 25 °C, and the pH was set to 6.0, 6.5, 7.0, and 7.5. 1 mL of the fermentation lysate of the above recombinant engineered bacteria was added (GGC enzyme activity was about 10 U; a reasonable operational error of ±0.1 U was allowed for the enzyme activity). The reaction time was 2 h. The amount of GGC generated was detected by HPLC. The results are shown in Table 3.

[0062] Table 3: GGC production of gshAB-4 mutant under different reaction pH conditions

[0063] (2) Different reaction temperatures. In a 10 mL reaction system, 0.15 M sodium glutamate, 0.16 M cysteine ​​hydrochloride, 0.1 M magnesium chloride hexahydrate, and 0.3 M ATP were added to a test tube and dissolved in pure water. The pH was adjusted to 7.0 with liquid alkali. 1 mL of the fermentation lysate of the above recombinant engineered bacteria (GGC enzyme activity was about 10 U) was added. The reaction was carried out at 20℃, 30℃, and 35℃ for 2 h. The amount of GGC generated was detected by HPLC. The results are shown in Table 4.

[0064] Table 4: GGC production of gshAB-4 mutant under different reaction temperatures

[0065] In summary, the optimal reaction conditions for the synthesis of GGC catalyzed by the optimal mutant gshAB-4 are pH 7.0 and reaction temperature 30℃, which can achieve the maximum conversion rate of GGC, reaching 59.49%.

[0066] Example 4: Optimization of conditions for the catalytic synthesis of GGC

[0067] Following the optimized conditions of Example 3 (gshAB-4 mutant group), a 10 mL reaction system was set up. 0.15 M sodium glutamate, 0.16 M cysteine ​​hydrochloride, 0.1 M magnesium chloride hexahydrate, and 0.03 M ATP were added to the test tube and dissolved in pure water. The system was kept at a constant temperature of 30 °C, and the pH was set to 7.0. 1 mL of the fermentation lysate of the recombinant engineered bacteria (approximately 10 U enzyme activity) was added. To achieve ATP recycling, an acetyl phosphate energy supply system (ATP recycling system) was used. 1.2 mL of acetyl phosphate solution and 0.2 mL of acetate kinase (5000 U / mL) were added to the reaction system. The reaction time was 2 h, and the amount of GGC generated was detected by HPLC. A reaction system with wild-type recombinant engineered bacteria lysate was selected as a control. The results are shown in Table 5. HPLC results are shown in Table 5. Figure 2 .

[0068] Table 5: GGC synthesis catalyzed by the gshAB-4 mutant (ATP cycle)

[0069] Note: The enzyme activity of wild-type gshAB and gshAB-4 is the same;

[0070] As shown in Table 5, the mutation described in this invention can significantly enhance the catalytic activity and selectivity of GGC.

[0071] SEQ ID NO.1: MTINQLLQKLESTSPILQANFGIERESLRVDRQGQLAHTPHPSCLGARSFHPYIQTDFCEFQMELITPVAKSTTEARRLLGAITDVTGRSISQDEVLWPLSMPPRIKAEEIQVAQLENEFERHYRNYLAEKYGTKLQAISGIHYNMELGKDLVEALFQESDQIDIIAFKNALYLKLAQNYLRYRWVITYLFGAAPIAEQGFFDQEVPEPVRSFRNSDHGYVNKEEIQVSFDNLEDYVSDIETYIANGDLIAEKEFYSAVRFRGQKANRSFLDKGITYLEFRNFDLNPFERIGISQTTMDTVHLLILAFLWLDSPENVDHVLAQGHALNEKIALSHPLEPLPDQAIAETKDIIKALDQLVQHFGLGDYHQDLVKQVKATFADPKQTLSAQLLPYIKDKSLADFALNKALAYQDYDWTAHYALKGYEEMELSTQMLLFDAIQKGINFDILDEQDQFLKLWHKDHVEYVKNGNMTSKDNYVVPLAMANKTVTKKILADAGFPVPAGDEFTSLEQGLAYYPLIKNKQIVVKPKSTNFGLGISIFQEPASLENYQKALEIAFAEDTAVLVEEFIPGTEYRFFILDGHCKAVLLRVAANVVGDGKHTIRELVAQKNANPLRGHDHRSPLEIIALGDIEQLMLAQQGYTPDDVLPDGKKVNLRRNSNISTGGDSIDVTETMDSSYQELAAAMATSMGAWACGVDLIIPDETQIASKENPHCTCIELNFNPSMYMHTYCAEGPGQAITPKILDKLFPELVALKHQN。

[0072] SEQ ID NO.2: MTINQLLQKLESTSPILQANFGIERESLRVDRQGQLAHTPHPSCLGARSFHPYIQTDFCEFQMELITPVAKSTTEARRLLGAIVDVTGRSISQDEVLWPLSMPPRIKAEEIQVAQLENEFERHYRNYLAEKYGTKLQAISGIHYNMELGKDLVEALFQESDQIDIIAFKNALYLKLAQNYLRYRWVITYLFGAAPIAEQGFFDQEVPEPVRSFRNSDHGYVNKEEIQVSFDNLEDYVSDIETYIANGDLIAEKEFYSAVRFRGQKANRSFLDKGITYLEFRNFDLNPFERIGISQTTMDTVHLLILAFLWLDSPENVDHVLAQGHALNEKIALSHPLEPLPDQAIAETKDIIKALDQLVQHFGLGDYHQDLVKQVKATFADPKQTLSAQLLPYIKDKSLADFALNKALAYQDYDWTAHYALKGYEEMELSTQMLLFDAIQKGINFDILDEQDQFLKLWHKDHVEYVKNGNMTSKDNYVVPLAMANKTVTKKILADAGFPVPAGDEFTSLEQGLAYYPLIKNKQIVVKPKSTNFGLGISIFQEPASLENYQKALEIAFAEDTAVLVEEFIPGTEYRFFILDGHCKAVLLRVAANVVGDGKHTIRELVAQKNANPLRGHDHRSPLEIIALGDIEQLMLAQQGYTPDDVLPDGKKVNLRRNSNISTGGDSIDVTETMDSSYQELAAAMATSMGAWACGVDLIIPDETQIASKENPHCTCIELNFNPSMYMHTYCAEGPGQAITPKILDKLFPELVALKHQN。

[0073] SEQ ID NO.3: MTINQLLQKLESTSPILQANFGIEREALRVDRQGQLAHTPHPSCLGARSFHPYIQTDFCEFQMELITPVAKSTTEARRLLGAITDVTGRSISQDEVLWPLSMPPRIKAEEIQVAQLENEFERHYRNYLAEKYGTKLQAISGIHYNMELGKDLVEALFQESDQIDIIAFKNALYLKLAQNYLRYRWVITYLFGAAPIAEQGFFDQEVPEPVRSFRNSDHGYVNKEEIQVSFDNLEDYVSDIETYIANGDLIAEKEFYSAVRFRGQKANRSFLDKGITYLEFRNFDLNPFERIGISQTTMDTVHLLILAFLWLDSPENVDHVLAQGHALNEKIALSHPLEPLPDQAIAETKDIIKALDQLVQHFGLGDYHQDLVKQVKATFADPKQTLSAQLLPYIKDKSLADFALNKALAYQDYDWTAHYALKGYEEMELSTQMLLFDAIQKGINFDILDEQDQFLKLWHKDHVEYVKNGNMTSKDNYVVPLAMANKTVTKKILADAGFPVPAGDEFTSLEQGLAYYPLIKNKQIVVKPKSTNFGLGISIFQEPASLENYQKALEIAFAEDTAVLVEEFIPGTEYRFFILDGHCKAVLLRVAANVVGDGKHTIRELVAQKNANPLRGHDHRSPLEIIALGDIEQLMLAQQGYTPDDVLPDGKKVNLRRNSNISTGGDSIDVTETMDSSYQELAAAMATSMGAWACGVDLIIPDETQIASKENPHCTCIELNFNPSMYMHTYCAEGPGQAITPKILDKLFPELVALKHQN。

[0074] SEQ ID NO.4: MTINQLLQKLESTSPILQANFGIEREALRVDRQGQLAHTPHPSCLGARSFHPYIQTDFCEFQMELITPVAKSTTEARRLLGAIVDVTGRSISQDEVLWPLSMPPRIKAEEIQVAQLENEFERHYRNYLAEKYGTKLQAISGIHYNMELGKDLVEALFQESDQIDIIAFKNALYLKLAQNYLRYRWVITYLFGAAPIAEQGFFDQEVPEPVRSFRNSDHGYVNKEEIQVSFDNLEDYVSDIETYIANGDLIAEKEFYSAVRFRGQKANRSFLDKGITYLEFRNFDLNPFERIGISQTTMDTVHLLILAFLWLDSPENVDHVLAQGHALNEKIALSHPLEPLPDQAIAETKDIIKALDQLVQHFGLGDYHQDLVKQVKATFADPKQTLSAQLLPYIKDKSLADFALNKALAYQDYDWTAHYALKGYEEMELSTQMLLFDAIQKGINFDILDEQDQFLKLWHKDHVEYVKNGNMTSKDNYVVPLAMANKTVTKKILADAGFPVPAGDEFTSLEQGLAYYPLIKNKQIVVKPKSTNFGLGISIFQEPASLENYQKALEIAFAEDTAVLVEEFIPGTEYRFFILDGHCKAVLLRVAANVVGDGKHTIRELVAQKNANPLRGHDHRSPLEIIALGDIEQLMLAQQGYTPDDVLPDGKKVNLRRNSNISTGGDSIDVTETMDSSYQELAAAMATSMGAWACGVDLIIPDETQIASKENPHCTCIELNFNPSMYMHTYCAEGPGQAITPKILDKLFPELVALKHQN。

[0075] SEQ ID NO.5: MTINQLLQKLESTSPILQANFGIEREALRVDRQGQLAHTPHPSCLGARSFHPYIQTDFCEFQMELITPVAKSTTEARRLLGAIVDVTGRSISQDEVLWPLSMPPRIKAEEIQVAQLENEFERHYRNYKAEKYGTKLQAISGIHYNMELGKDLVEALFQESDQIDIIAFKNALYLKLAQNYLRYRWVITYLFGAAPIAEQGFFDQEVPEPVRSFRNSDHGYVNKEEIQVSFDNLEDYVSDIETYIANGDLIAEKEFYSAVRFRGQKANRSFLDKGITYLEFRNFDLNPFERIGISQTTMDTVHLLILAFLWLDSPENVDHVLAQGHALNEKIALSHPLEPLPDQAIAETKDIIKALDQLVQHFGLGDYHQDLVKQVKATFADPKQTLSAQLLPYIKDKSLADFALNKALAYQDYDWTAHYALKGYEEMELSTQMLLFDAIQKGINFDILDEQDQFLKLWHKDHVEYVKNGNMTSKDNYVVPLAMANKTVTKKILADAGFPVPAGDEFTSLEQGLAYYPLIKNKQIVVKPKSTNFGLGISIFQEPASLENYQKALEIAFAEDTAVLVEEFIPGTEYRFFILDGHCKAVLLRVAANVVGDGKHTIRELVAQKNANPLRGHDHRSPLEIIALGDIEQLMLAQQGYTPDDVLPDGKKVNLRRNSNISTGGDSIDVTETMDSSYQELAAAMATSMGSWACGVDLIIPDETQIASKENPHCTCIELNFNPSMYMHTYCAEGPGQAITPKILDKLFPELVALKHQN。

Claims

1. A mutant of a glutamylcysteine ​​dipeptide ligase, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.

5.

2. An application of the mutant according to claim 1, characterized in that, γ-glutamylcysteine ​​is prepared by enzymatic reaction of a mixture containing glutamate, cysteine, water-soluble magnesium salt, ATP and an enzyme catalyst; wherein the enzyme catalyst comprises the mutant described in claim 1.

3. The application of the mutant as described in claim 2, characterized in that, The glutamate is monosodium glutamate; Cysteine ​​salt is cysteine ​​hydrochloride; The water-soluble magnesium salt is magnesium chloride; In the mixture, the amount of the mutant added is 1~1.2 kU / L; the concentration of ATP is 25~500 mmol / L; the concentration of glutamate is 120~200 mmol / L; the concentration of cysteine ​​is 150~200 mmol / L; and the concentration of water-soluble magnesium salt is 50~150 mmol / L.

4. The application of the mutant as described in claim 2, characterized in that, The temperature for enzyme-catalyzed reactions is 20-35℃, and the pH is 6-7.

5.

5. A gene encoding a mutant of the glutamylcysteine ​​dipeptide ligase of claim 1.

6. A recombinant plasmid comprising the encoding gene of claim 5.

7. A recombinant microorganism expressing the mutant of claim 1.

8. The recombinant microorganism as described in claim 7, characterized in that, It contains the recombinant plasmid as described in claim 6.

Images