A GMAS mutant and its application in the synthesis of L-theanine

Abstract

The present invention discloses a GMAS mutant and its application in the synthesis of L-theanine, belonging to the field of biotechnology. In the present invention, error-prone PCR is used to randomly mutate the γ-glutamylcarboxamide synthetase derived from Methylovorus mays, and a mutant enzyme GMAS with improved enzyme activity is screened out. Y198S The specific activity is 24.4±1.6 U / mg, and the specific activity is increased by 35.8% compared with the original enzyme. The present invention also couples polyphosphate kinase PPK to improve the supply capacity of ATP. Further, by optimizing the substrate concentration of PPK enzyme, in the case of only adding 5 mM ATP, 2.5 U / mL PPK and 10 U / mL GMAS Y198S are coupled to catalyze 100 mM glutamate to produce 94.6±3.5 mM L-theanine, and the conversion rate can reach 94.6%, which improves the conversion rate of L-theanine and reduces the production cost.

Description

Technical Field

[0001] The present invention relates to a GMAS mutant and its application in synthesizing L-theanine, belonging to the field of biotechnology. Background Art

[0002] L-theanine naturally exists in tea and belongs to non-protein amino acids. L-theanine has many beneficial physiological functions, such as regulating the human brain nerves and reducing blood pressure. At the same time, L-theanine is also a safe food and drug additive. Therefore, a series of products based on L-theanine have further promoted the development of the pharmaceutical and health industries.

[0003] The production methods of L-theanine mainly include extraction method, chemical synthesis method and enzymatic catalysis. Since the content of L-theanine in dry tea leaves is low in the extraction method, the extraction amount is small and the purity is low, which cannot meet the large-scale production requirements. The chemical synthesis method is the main way to produce L-theanine at present. By controlling the reaction process and reaction conditions, L-glutamic acid and ethylamine are combined to form L-theanine, but there are also problems such as difficult separation and low product yield. Recently, the production of L-theanine by enzymatic catalysis has attracted the attention of many scholars. At present, there are mainly 4 enzymes from different sources that can be used as ideal biocatalysts and show different potentials. These 4 enzymes from bacterial sources include L-glutamine synthetase (GS, EC 6.3.1.2), γ-glutamylmethylamide synthetase (GMAS, EC 6.3.4.12), glutamyl transpeptidase (GGT, EC 2.3.2.2), L-glutaminase (EC 3.5.1.2). Compared with other enzymes, since γ-glutamylmethylamide synthetase shows a high binding affinity for ethylamine, the present invention selects γ-glutamylmethylamide synthetase to synthesize L-theanine.

[0004] γ-Glutamylmethylamide synthetase (GMAS) is a class of enzymes found in methanotrophs that can catalyze the synthesis of L-theanine. In the presence of ATP and Mg 2+It can catalyze the synthesis of theanine from glutamate and ethylamine in the presence of GMAS enzyme. The catalytic activity of GMAS enzyme towards ethylamine is much higher than that of other enzymes such as GS enzyme. The recombinant expression of GMAS from Methylovorus mays No.9 was carried out in Escherichia coli, and the enzyme production capacity of the recombinant Escherichia coli was 23 times that of the original Methylovorus mays No.9 strain. However, the enzyme properties of the recombinant GMAS were not significantly different from those of the native GMAS. This finding makes it possible to produce GMAS in large quantities for the industrial production of theanine. Since this enzyme requires the participation of ATP, directly adding ATP in the production process will lead to an increase in cost, which seems not very feasible. Polyphosphate kinase PPK can use the cheap and easily available substrate polyphosphate polyP as a substrate and can catalyze the conversion of ADP to ATP. By introducing an ATP regeneration pathway, ATP is catalytically generated from the substrate sodium hexametaphosphate and ADP to supply the ATP required for L-theanine production, providing a basis for the large-scale industrial production of theanine under extremely small amounts of ATP. Summary of the Invention

[0005] Technical Problem:

[0006] The technical problem to be solved by the present invention is to overcome the disadvantages of low enzyme activity and high cost in the production of L-theanine using γ-glutamylcarboxamide synthetase GMAS, to provide a mutant with increased specific activity of GMAS to improve the conversion rate of L-theanine, and at the same time to provide a method for coupling the PPK enzyme to regenerate the ATP system to reduce the catalytic cost of L-theanine.

[0007] Technical Solution:

[0008] The present invention provides a γ-glutamylcarboxamide synthetase mutant having the amino acid sequence shown in SEQ ID NO.5.

[0009] In one embodiment, the mutant is based on the parental sequence shown in SEQ ID NO.4, and tyrosine at position 198 is mutated to serine.

[0010] The present invention also provides a gene encoding the γ-glutamylcarboxamide synthetase mutant.

[0011] The present invention also provides a recombinant plasmid carrying the gene.

[0012] In one embodiment, the plasmid includes but is not limited to pET series plasmids.

[0013] The present invention also provides a method for increasing the enzyme activity of γ-glutamylcarboxamide synthetase, which is to mutate tyrosine at position 198 of the amino acid shown in SEQ ID NO.4 to serine.

[0014] The present invention also provides a recombinant Escherichia coli expressing the γ-glutamine synthetase mutant.

[0015] In one embodiment, the recombinant Escherichia coli also expresses polyphosphate kinase.

[0016] In one embodiment, the polyphosphate kinase has the amino acid sequence shown in SEQ ID NO.2.

[0017] In one embodiment, the expression of the polyphosphate kinase uses the pET28a(+) plasmid as an expression vector.

[0018] In one embodiment, the expression of the γ-glutamine synthetase uses the pET28a(+) plasmid as an expression vector.

[0019] The present invention also provides the application of the γ-glutamine synthetase in the production of L-theanine.

[0020] In one embodiment, in the application, L-glutamic acid is used as a substrate, and the γ-glutamine synthetase mutant and polyphosphate kinase are used as catalysts, and the reaction is carried out at 35-37°C.

[0021] In one embodiment, the reaction system further contains polyphosphate, and the polyphosphate is polyP6 with a final concentration of 5-10 mM.

[0022] The present invention also provides the application of the γ-glutamine synthetase mutant or the method in the production of products containing L-theanine.

[0023] Beneficial effects:

[0024] 1. The present invention uses the γ-glutamine synthetase from Methylovorus mays as a starting sequence, randomly mutates the key enzyme GMAS through error-prone PCR, measures the enzyme activity after mutation, and screens a mutant enzyme GMAS with a significantly improved specific enzyme activity. Y198S The specific enzyme activity is 24.4±1.6 U / mg, which is 35.8% higher than the original enzyme.

[0025] 2. The present invention realizes the efficient recycling regeneration of ATP by introducing the polyphosphate mutant kinase PPK. By optimizing the substrate concentration of the PPK enzyme, when the substrate polyP6 has a concentration of 10 mM and only 5 mM ATP is added, 94.6±3.5 mM of L-theanine is produced, and the conversion rate reaches 94.6%, improving the conversion rate of L-theanine.

[0026] By mutating the key enzymes in the catalytic process of L-theanine and coupling the polyphosphate kinase that regenerates ATP, the present invention overcomes the disadvantages of low enzyme activity and high cost in the production of L-theanine using γ-glutamylcarboxamide synthetase GMAS, improves the conversion rate of L-theanine production, and provides ideas for further production of L-theanine in biocatalytic reactions. Description of the Drawings

[0027] Figure 1 : Enzyme activity of the GMAS mutant enzyme.

[0028] Figure 2 : Optimal substrate concentration of polyphosphate kinase PPK.

[0029] Figure 3 : Yield of L-theanine produced by enzymatic catalysis. Detailed Description of the Invention

[0030] The culture media involved in the following examples:

[0031] LB medium (1 L): 10 g NaCl, 10 g tryptone, 5 g yeast extract.

[0032] Method for measuring the specific enzyme activity of GMAS: Tris-HCl (pH 7.0, 100 mM) contains 50 mM sodium glutamate, 50 mM ethylamine hydrochloride, 25 mM MgCl2, 50 mM ATP and pure enzyme. GMAS catalyzes the synthesis of theanine from glutamate in the presence of ATP and Mg 2+ and terminates the reaction by boiling water bath at 30 °C for 5 min. The amount of inorganic phosphorus generated is measured at 825 nm. Definition of GMAS enzyme activity (U): The amount of enzyme that generates 1 μmol of inorganic phosphorus per minute is defined as one enzyme activity unit.

[0033] Calculation of conversion rate: Calculate the conversion rate based on the amount of theanine generated from the substrate glutamate, (the amount of glutamate required to produce the theanine yield (mM) / total amount of glutamate added (mM)) × 100%.

[0034] Example 1: Mutation and Screening of γ-Glutamylcarboxamide Synthetase GMAS

[0035] The specific steps are as follows:

[0036] (1) Error-Prone PCR

[0037] Design primers P1 / P2, and use an error-prone PCR kit to randomly mutate the GMAS gene fragment (shown in SEQ ID NO.3). The amplification conditions are as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 60 s, for 30 cycles; final extension at 72°C for 5 min. Design primers P3 / P4, use the pET28a plasmid as a template, and amplify to obtain a linearized plasmid. The amplification conditions are as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 120 s, for 30 cycles; final extension at 72°C for 5 min. Use a gel recovery kit to purify and recover the PCR products, and detect the concentration of the recovered products. The obtained mutant gene fragments are respectively ligated to the linearized plasmid pET28a through the homologous recombinase ClonExpress II OneStep Cloning Kit (Vazyme), transformed into E. coli BL21(DE3) competent cells, and spread on an LB solid medium containing 50 μg / mL kanamycin resistance, and cultured at 37°C for 12 h. The grown transformants are used for subsequent enzyme activity assays. The primer sequences involved are as follows:

[0038] P1: 5’-atgaagtccctggaagaagca-3’;

[0039] P2: 5’-caaccgctacgtgcagttctactaa-3’;

[0040] (2) Screening of GMAS mutant enzymes

[0041] Inoculate the transformants into 10 ml of LB liquid medium and culture at 37°C for 12 hours. Inoculate at an inoculum size of 1% into 50 ml of LB liquid medium and culture until the OD 600 is approximately 0.8, add IPTG with a final concentration of 0.05 mM, and culture at 16°C for 16 h. Wash the cells three times with PBS, resuspend the collected cells with PBS again, break the cells with an ultrasonic cell disruptor, and purify the crude enzyme solution through a protein purification nickel column to obtain a pure enzyme.

[0042] Add 50 μl of pure enzyme to Tris-HCl containing 50 mM glutamate and 50 mM ATP. After reacting for 30 min, terminate the reaction by boiling water bath for 5 min, and measure the absorbance value at 825 nm. Compared with the original enzyme, select the mutant enzyme with the most significant increase in enzyme activity, inoculate its corresponding transformant into 10 ml of LB liquid medium, culture at 37 °C for 12 hours, extract the plasmid and send it to Genewiz for sequencing. The sequencing results show that the mutation site of the mutant enzyme with the highest enzyme activity is the tyrosine Y at residue 198 mutated to serine S. Through the structural analysis of this protein, it is found that this mutation site is located in the active center of the GMAS protein. After tyrosine is mutated to serine, the active pocket of this protein expands, which is beneficial for more substrates to enter the active center, thus increasing the enzyme activity. Name this mutant enzyme GMAS Y198S , the specific enzyme activity of GMAS before mutation was 18.0 ± 2.4 U / mg, and after mutation, GMAS Y198S had a specific enzyme activity of 24.4 ± 1.6 U / mg. Taking the specific enzyme activity of the original enzyme GMAS as 100%, compared with the original enzyme GMAS, the relative enzyme activity of this mutant enzyme GMAS Y198S increased by 35.8%.

[0043] Example 2: Construction of recombinant bacteria BL21 / pET28a-GMAS, BL21 / pET28a-GMAS Y198S 、BL21 / pET28a-PPK

[0044] The specific steps are as follows:

[0045] (1) Construction of overexpression plasmids pET28a-GMAS, pET28a-GMAS Y198S 、pET28a-PPK

[0046] Using the pET28a plasmid as a template respectively, design primers P3 / P4, P5 / P6, and perform PCR amplification to obtain linearized plasmids. The amplification conditions are: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2.5 min, for 30 cycles; final extension at 72 °C for 5 min. For GMAS, GMAS Y198S, the PPK gene fragment (as shown in SEQ ID NO.1) was purified and then ligated to the linearized plasmid pET28a using the homologous recombinase ClonExpress II One Step Cloning Kit (Novoprotein), and then transformed into E. coli BL21(DE3) competent cells to obtain transformants. The transformants were spread on an LB solid medium containing 50 μg / mL kanamycin and cultured at 37 °C for 12 h. Positive colonies were picked and verified by colony PCR of single colonies using Taq DNA polymerase with P7 / P8 as primers. After culturing positive single colonies with the size of the target band in a vial containing LB liquid medium for 12 h, plasmids were extracted. If the sequencing by Genewiz was correct, then the plasmids pET28a-GMAS and pET28a-GMAS Y198S , and the plasmids pET28a-PPK were successfully constructed. The primer sequences involved are as follows:

[0047] P3: 5’-ctacgtgcagttctactaaaagcttgcggccgcactcgag-3’;

[0048] P4: 5’-aaatgggtcgcggatccgaattcatgaagtccctggaagaagca-3’;

[0049] P5: 5’-cagaaaagtcctccgattaaaagcttgcggccgcactcgagcac-3’;

[0050] P6: 5’-caaatgggtcgcggatccgaattcatggcaaccgatttttctaa-3’;

[0051] P7: 5’-gtagaggatcgagatctcgatccc-3’;

[0052] P8: 5’-tgaaaggaggaactatatccggat-3’;

[0053] (2) Preparation of E. coli BL21 competent cells

[0054] Streak Escherichia coli BL21 on an antibiotic-free LB plate, incubate in an incubator at 37°C, pick colonies after incubation, inoculate into a 10 mL LB vial and culture for 12 h. Transfer with an inoculation amount of 1% to a 50 mL LB medium bottle. When the cell concentration reaches 0.4 - 0.6, prepare for the preparation of E. coli competent cells. Pre-cool relevant reagents and instruments in advance. Prepare solution A, solution B in the kit, 1.5 mL EP tubes and 50 mL centrifuge tubes, place them on ice, and control the temperature of the centrifuge to 4°C. Aliquot the 50 mL bacterial solution in a sterile workbench, centrifuge (8000 r·min -1 , 5 min), and discard the supernatant. Pipette 5 mL of solution A and blow-suck to suspend, centrifuge (8000 r·min -1 , 5 min), and discard the supernatant. Pipette 5 mL of solution B and blow-suck to suspend, aliquot into pre-cooled EP tubes prepared in advance, with 100 μL in each tube.

[0055] (3) Construction of recombinant strains BL21 / pET28a-GMAS, BL21 / pET28a-GMAS Y198S , BL21 / pET28a-PPK

[0056] Transform the pET28a-GMAS, pET28a-GMAS Y198S , pET28a-PPK plasmids obtained in step (1) into BL21 competent cells to obtain transformants, and spread the transformants on an LB solid medium containing 50 μg / mL kanamycin resistance. Incubate at 37°C for 12 h, pick positive colonies, and perform colony PCR verification on single colonies using P7 / P8 as primers with Taq DNA polymerase; inoculate the positive single colonies with the size of the target band into a vial containing LB liquid medium and culture for 12 h, then extract the plasmid and send it to Genewiz for sequencing. Correct sequencing indicates the successful construction of the recombinant strains BL21 / pET28a-GMAS, BL21 / pET28a-GMAS Y198S , BL21 / pET28a-PPK.

[0057] Example 3: Optimal substrate concentration of polyphosphate kinase PPK

[0058] Preparation method of PPK pure enzyme:

[0059] Inoculate a single colony of the recombinant strain BL21 / pET28a-PPK successfully constructed in Example 2 into 10 ml of LB liquid medium, incubate at 37°C for 12 hours, transfer with an inoculation amount of 1% to 50 ml of LB liquid medium, and culture until OD 600Approximately 0.8, add IPTG with a final concentration of 0.05 mM, and culture at 16 °C for 16 h. Wash the cells three times with PBS, resuspend the collected bacterial cells with PBS again, break the cells with an ultrasonic crusher, break the Escherichia coli for 1 s and stop for 3 s, and break for a total of 15 min. Centrifuge at 10,000 rpm for 20 min, and purify the crude enzyme solution through a protein purification nickel column to obtain pure enzyme.

[0060] To optimize the ATP regeneration system, the substrate concentration of the key enzyme PPK was optimized. 1 mL reaction system: Tris-HCl (pH 8, 200 mM), ADP 10 g / L, polyP6 (at final concentrations of 5 mM, 10 mM, 20 mM, 30 mM, 40 mM respectively), MgCl2 5 g / L and pure enzyme (final concentration of 2.5 U / mL), react at 37 °C for 30 min, terminate the reaction by boiling in boiling water for 5 min, and measure the absorbance value of the supernatant at 340 nm.

[0061] The results showed ( Figure 2 ), the specific enzyme activity of polyphosphate kinase PPK was 356.4 ± 13.2 U / mg at a polyP6 concentration of 5 mM, the specific enzyme activity was the highest at 10 mM polyP6, which was 605.2 ± 17.1 U / mg. At a polyP6 concentration of 20 mM, the specific enzyme activity of PPK decreased to 287.5 ± 8.2 U / mg. At a polyP6 concentration of 30 mM, the specific enzyme activity of PPK was 179.3 ± 7.3 U / mg. At a polyP6 concentration of 40 mM, the specific enzyme activity of PPK was 163.4 ± 6.8 U / mg. It can be seen that with the increase of the substrate polyP6 concentration, the specific enzyme activity of PPK first increased and then decreased. Therefore, the optimal substrate concentration of PPK is 10 mM polyP6. Therefore, 10 mM polyP6 concentration was selected for subsequent experiments.

[0062] Example 4: Enzymatic catalysis for the production of L-theanine

[0063] The specific steps are as follows:

[0064] Induce and purify the BL21 / pET28a-GMAS, BL21 / pET28a-GMAS Y198S and BL21 / pET28a-PPK strains. Induce the expression of proteins according to the steps (2) of Example 1, collect the bacterial cells, break them with an ultrasonic crusher for 15 min, and obtain GMAS and GMAS after purification Y198Sand PPK enzyme. The L-theanine production system contains, by final concentration: 20 mL of Tris-HCl (100 mM, pH 8.0), 100 mM L-glutamic acid, 100 mM ethylamine, 10 mM polyP6, 5 mM ATP, 20 mM MgCl2 and enzyme (final concentration 2.5 U / mL PPK, final concentration 10 U / mL GMAS or GMAS Y198S ), and the theanine yield was measured after reacting at 37 °C for 6 h.

[0065] The control system was: 20 mL of Tris-HCl (100 mM, pH 8.0), 100 mM L-glutamic acid, 100 mM ethylamine, 10 mM polyP6, 150 mM ATP, 20 mM MgCl2 and enzyme (final concentration 10 U / mL GMAS or GMAS Y198S ).

[0066] In the catalytic system with 150 mM ATP, the original enzyme GMAS could produce 80.5 ± 3.2 mM of theanine, and the conversion rate of the substrate L-glutamic acid was 80.5%. The mutant enzyme GMAS Y198S could produce 96.3 ± 2.1 mM of theanine, and the conversion rate was 96.3%. The theanine yield increased by 18.8% compared with the original enzyme GMAS, indicating that the mutation of tyrosine at position 198 of the GMAS enzyme to serine improved the catalytic activity of GMAS and further increased the theanine yield and conversion rate.

[0067] To reduce the production cost of theanine, in the catalytic system of ATP regeneration coupled with PPK enzyme, after only adding 5 mM ATP, adding GMAS and PPK enzyme could produce 78.6 ± 2.9 mM of theanine, while adding GMAS Y198S and PPK could produce 94.6 ± 3.5 mM of theanine, and the conversion rate was 94.6%. The coupling of GMAS Y198S and PPK could produce a slightly lower theanine yield than that in the system with 150 mM ATP added. It shows that the mutation of the residues near the active center of the GMAS enzyme can increase the number of substrates entering the active center, improve the catalytic activity of GMAS, and effectively improve the ability of GMAS to produce theanine. And for the coupled PPK regeneration system, the enhanced ATP sustainability can also effectively increase the theanine yield and reduce the substrate cost in the catalysis. Therefore, the coupling of the mutant enzyme GMAS Y198S and PPK can increase the theanine yield, significantly improve the sustainability of ATP regeneration, increase the substrate conversion rate of theanine, and save the cost of high-energy-consuming catalytic reactions.

[0068] Comparative Example 1:

[0069] The specific implementation is the same as that of Example 1, except that mutants Y198M, G238H, S246T, and G231Y were also obtained through screening. The enzyme activity was measured according to the method of Example 1. The results showed that the specific enzyme activities of the mutants were 20.3±1.3 U / mg, 20.9±2.1 U / mg, 21.8±2.3 U / mg, and 22.1±1.4 U / mg respectively, all of which were lower than that of mutant Y198S.

[0070] Table 1 Mutants and Specific Enzyme Activities

[0071]

[0072]

[0073] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A γ-glutamylcarboxamide synthetase mutant, characterized in that, The amino acid sequence is as shown in SEQ ID NO.

5.

2. A gene encoding the γ-glutamine synthetase mutant according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2.

4. The recombinant plasmid according to claim 3, wherein The plasmid includes pET series plasmids.

5. A method for improving the enzyme activity of γ-glutamylcarboxamide synthetase, characterized in that, The tyrosine at position 198 of the amino acid shown in SEQ ID NO. 4 is mutated to serine.

6. A recombinant Escherichia coli, characterized in that, Using the pET28a(+) plasmid as an expression vector, the γ-glutamine synthetase mutant according to claim 1 was expressed.

7. A method for producing L-theanine, characterized in that, Using L-glutamic acid as a substrate, with the γ-glutamine synthetase mutant according to claim 1 and polyphosphate kinase as catalysts, the reaction was carried out at 35-37 °C.

8. The method according to claim 7, wherein The amino acid sequence of the polyphosphate kinase is as shown in SEQ ID NO.

2.

9. The method according to claim 7 or 8, characterized in that, The reaction system also contains polyphosphate; the polyphosphate is polyP6 with a final concentration of 5-10 mM.

10. Use of the γ-glutamine synthetase mutant according to claim 1 or the method according to any one of claims 7-9 in the production of a product containing L-theanine.

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