CN116218796A - GMAS mutant and application thereof in theanine synthesis - Google Patents

Abstract

The invention discloses a GMAS mutant and application thereof in theanine synthesis, and belongs to the technical field of biology. The invention carries out random mutation on the Methylovorus mays-source gamma-glutamine synthetase by error-prone PCR, screens to obtain mutant enzyme GMAS with improved enzyme activity _Y198S The specific enzyme activity is 24.4+/-1.6U/mg, and the specific enzyme activity is improved by 35.8 percent compared with the original enzyme. The invention also improves the ATP supply capacity by coupling polyphosphate kinase PPK. Further by optimizing PPK enzymeSubstrate concentration, 2.5U/mL PPK and 10U/mL GMAS with the addition of only 5mM ATP _Y198S The coupling catalysis of 100mM glutamic acid produces 94.6+ -3.5 mM L-theanine, the conversion rate can reach 94.6%, the conversion rate of L-theanine is improved, and the production cost is reduced.

Description

GMAS mutant and application thereof in theanine synthesis

Technical Field

The invention relates to a GMAS mutant and application thereof in theanine synthesis, belonging to the technical field of biology.

Background

L-theanine naturally occurs in theanine and belongs to the non-protein amino acid. L-theanine has many beneficial physiological functions such as regulating human brain nerves, lowering blood pressure, etc.; meanwhile, the L-theanine is also a safe food and drug additive. Thus, the method is applicable to a variety of applications. A series of products using L-theanine as raw material further promote the development of medicine and health care industry.

The production method of L-theanine mainly comprises an extraction method, a chemical synthesis method and enzymatic catalysis. The dry tea leaves have low L-theanine content due to the extraction method. The extraction quantity is small, the purity is low, and the large-scale production requirement can not be met; the chemical synthesis method is a main way for producing L-theanine at present, and combines L-glutamic acid and ethylamine to form L-theanine by controlling the reaction process and reaction conditions, but the method also has the problems of difficult separation and low product yield. Recently, the use of enzymatic catalysis for the production of L-theanine has attracted much attention from many scholars, and currently there are mainly 4 enzymes of different origins which can be used as ideal biocatalysts and which show different potential. These 4 bacterially derived enzymes include L-glutamine synthetase (GS, EC 6.3.1.2), gamma-glutamyl-methyl-amide synthetase (GMAS, EC 6.3.4.12), glutamyl-transpeptidase (GGT, EC 2.3.2.2), L-glutaminase (EC 3.5.1.2). Since gamma-glutamyl-methyl-amide synthetase shows high binding force to ethylamine as compared to other enzymes, gamma-glutamyl-methyl-amide synthetase was selected for the synthesis of L-theanine according to the present invention.

Gamma-glutamylformamide synthetase (GMAS) is a class of enzymes found in methanogens that catalyze the synthesis of theanine, in ATP and Mg ²⁺ The presence of the catalyst can catalyze glutamic acid and ethylamine to synthesize theanine. The catalytic activity of GMAS enzyme on ethylamine is far higher than that of GS enzyme and other enzymes. Recombinant expression of Methylovorus mays No. 9-derived GMAS in E.coli, the recombinant E.coli enzyme producing ability was that of the original methylThe 23-fold strain of rus mays No.9, but the fact that recombinant GMAS has no significant difference in enzyme properties from the inherent GMAS, has made possible the mass production of GMAS for the industrial production of theanine. Since the enzyme requires the participation of ATP, the direct addition of ATP during the production process results in an increase in cost, which seems to be less viable. The polyphosphate kinase PPK can utilize a cheap and easily available substrate polyphosphate as a substrate, and can catalyze ADP to be converted into ATP. Introducing an ATP regeneration path, catalyzing and generating ATP through substrates sodium hexametaphosphate and ADP, supplying ATP required by L-theanine production, and providing a foundation for large-scale industrial production of theanine under the condition of a very small amount of ATP.

Disclosure of Invention

Technical problems:

the invention aims to solve the technical problems of low enzyme activity and high cost in the process of producing L-theanine by using gamma-glutamine synthetase GMAS, provides a mutant with improved specific enzyme activity of the GMAS, improves the conversion rate of the L-theanine, and simultaneously provides a method for reducing the catalytic cost of the L-theanine by coupling a PPK enzyme regeneration ATP system.

The technical scheme is as follows:

the invention provides a gamma-glutamine synthetase mutant, which has an amino acid sequence shown as SEQ ID NO. 5.

In one embodiment, the mutant is one in which tyrosine 198 is mutated to serine based on the parent sequence set forth in SEQ ID NO. 4.

The invention also provides a gene for encoding the gamma-glutamine synthetase mutant.

The invention also provides a recombinant plasmid carrying the gene.

In one embodiment, the plasmid includes, but is not limited to, a pET series plasmid.

The invention also provides a method for improving the enzyme activity of the gamma-glutamine synthetase, which is to mutate the 198 th tyrosine of the amino acid shown in SEQ ID NO.4 into serine.

The invention also provides a recombinant escherichia coli, which expresses the gamma-glutamine synthetase mutant.

In one embodiment, the recombinant E.coli further expresses a polyphosphate kinase.

In one embodiment, the polyphosphate kinase has the amino acid sequence set forth in SEQ ID NO. 2.

In one embodiment, the polyphosphate kinase is expressed using a pET28a (+) plasmid as an expression vector.

In one embodiment, the expression of the gamma-glutamylformamide synthetase takes a pET28a (+) plasmid as an expression vector.

The invention also provides application of the gamma-glutamine synthetase in producing L-theanine.

In one embodiment, the use is to react at 35-37 ℃ with L-glutamic acid as a substrate and the gamma-glutamine synthetase mutant and polyphosphate kinase as a catalyst.

In one embodiment, the reaction system further comprises a polyphosphate at a final concentration of 5 to 10mM of a polyphosphate ₆ 。

The invention also provides the application of the gamma-glutamine synthetase mutant or the method in the production of L-theanine-containing products.

The beneficial effects are that:

1. the invention uses the Methylovorus mays-source gamma-glutamine synthetase as a starting sequence, randomly mutates key enzyme GMAS by error-prone PCR, determines the enzyme activity after mutation, and screens to obtain mutant enzyme GMAS with obviously improved specific enzyme activity _Y198S The specific enzyme activity is 24.4+/-1.6U/mg, which is improved by 35.8% compared with the original enzyme.

2. The invention realizes the high-efficiency cyclic regeneration of ATP by introducing polyphosphate mutant kinase PPK. And the substrate polyP is made by optimizing the substrate concentration of the PPK enzyme ₆ At a concentration of 10mM, when only 5mM ATP was added, 94.6.+ -. 3.5mM L-theanine was produced, and the conversion rate was 94.6%, thereby improving the conversion rate of L-theanine.

The invention breaks through the defects of low enzyme activity and high cost in the process of producing L-theanine by using gamma-glutamine synthetase GMAS by mutating key enzymes in the process of catalyzing L-theanine and coupling polyphosphate kinase for regenerating ATP, improves the conversion rate of producing L-theanine, and provides thought for further producing L-theanine in biocatalysis reaction.

Drawings

Fig. 1: enzyme activity of GMAS mutant enzyme.

Fig. 2: optimum substrate concentration for polyphosphate kinase PPK.

Fig. 3: the enzyme method catalyzes the production of L-theanine.

Detailed Description

The media referred to in the examples below:

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

The method for measuring specific enzyme activity of GMAS comprises the following steps: tris-HCl (pH 7.0, 100 mM) contains 50mM sodium glutamate, 50mM ethylamine hydrochloride, 25mM MgCl ₂ 50mM ATP and pure enzyme. GMAS on ATP and Mg ²⁺ In the presence of catalytic glutamic acid to synthesize theanine, the reaction was terminated in a boiling water bath at 30℃for 5min, and the amount of inorganic phosphorus produced was measured at 825 nm. GMAS enzyme Activity definition (U): the amount of enzyme that generates 1. Mu. Mol of inorganic phosphorus per minute is one enzyme activity unit.

Calculation of conversion: the conversion rate was calculated as the amount of substrate glutamic acid to theanine, the amount of glutamic acid (mM)/total amount of glutamic acid added (mM). Times.100% required for the production of theanine.

Example 1: mutation and screening of gamma-glutamylformamide synthetase GMAS

The method comprises the following specific steps:

(1) Error-prone PCR

Designing a primer P1/P2, randomly mutating a GMAS gene fragment (shown as SEQ ID NO. 3) by using an error-prone PCR kit, wherein the amplification conditions are as follows: pre-denaturation at 95 ℃,5min: denaturation at 95 ℃,30s, annealing at 58 ℃,30s, extension at 72 ℃,60s,30 cycles; finally, the extension is carried out for 5min at 72 ℃. Designing a primer P3/P4, and amplifying by taking the pET28a plasmid as a template to obtain the linearization plasmid. The amplification conditions were: pre-denaturation at 95 ℃,5min: denaturation at 95 ℃,30s, annealing at 58 ℃,30s, extension at 72 ℃,120s,30 cycles; finally, the extension is carried out for 5min at 72 ℃. And purifying and recovering the PCR product by using a gel recovery kit, and detecting the concentration of the recovered product. The obtained mutant gene fragments were purified and then respectively ligated with linearized plasmid pET28a by homologous recombination enzyme ClonExpress II One Step Cloning Kit (Norflu) and transformed into E.coli BL21 (DE 3) competent cells, which were plated on LB solid medium containing kanamycin resistance at a concentration of 50. Mu.g/mL, and cultured at 37℃for 12 hours. The grown transformants were used for subsequent enzyme activity assays. The primer sequences involved are as follows:

P1：5’-atgaagtccctggaagaagca-3’；

P2：5’-caaccgctacgtgcagttctactaa-3’；

(2) Screening of GMAS mutant enzymes

Inoculating the transformant into 10ml of LB liquid medium, culturing at 37 ℃ for 12 hours, inoculating 1% of the inoculum size into 50ml of LB liquid medium, culturing until OD ₆₀₀ About 0.8, and 0.05mM IPTG was added thereto at a final concentration and incubated at 16℃for 16 hours. Washing cells with PBS for three times, suspending the collected bacteria again with PBS, breaking cells with an ultrasonic breaker, purifying the crude enzyme liquid with a protein purification nickel column to obtain pure enzyme.

Mu.l of pure enzyme was added to Tris-HCl containing 50mM glutamic acid and 50mM TP, and after 30 minutes of reaction, the reaction was terminated by boiling water bath for 5 minutes, and absorbance at 825nm was measured. The mutant enzyme with the most significant improvement of enzyme activity compared with the original enzyme was selected, and its corresponding transformant was inoculated into 10ml of LB liquid medium, cultured at 37℃for 12 hours, and the extract was sent to Jin Weizhi company for sequencing. Sequencing results show that the mutation point of the mutant enzyme with the highest enzyme activity is 198-residue tyrosine Y and is mutated into residue serine S, and structural analysis of the protein shows that the mutation point is positioned at the active center of the GMAS protein, and after tyrosine is mutated into serine, the active pocket of the protein is enlarged, so that more substrates can enter the active center, and the enzyme activity is improved. The mutant enzyme was designated GMAS _Y198S Specific enzyme activity of GMAS before mutation is 18.0+ -2.4U/mg, and GMAS after mutation _Y198S The specific enzyme activity of the mutant enzyme GMAS is 24.4+/-1.6U/mg, and the specific enzyme activity of the original enzyme GMAS is taken as 100 percent, compared with the original enzyme GMAS _Y198S The relative enzyme activity of (2) is improved by 35.8%.

Example 2: recombinant bacterium BL21/pET28a-GMAS, BL21/pET28a-GMAS _Y198S Construction of BL21/pET28a-PPK

The method comprises the following specific steps:

(1) Over-expression plasmid pET28a-GMAS and pET28a-GMAS _Y198S Construction of pET28a-PPK

And respectively taking the pET28a plasmid as a template, designing primers P3/P4 and P5/P6, and carrying out PCR amplification to obtain the linearized plasmid. The amplification conditions were: pre-denaturation at 95 ℃,5min: denaturation at 95 ℃,30s, annealing at 58 ℃,30s, extension at 72 ℃,2.5min,30 cycles; finally, the extension is carried out for 5min at 72 ℃. GMAS, GMAS _Y198S The PPK gene fragment (shown as SEQ ID NO. 1) is respectively connected with a linearization plasmid pET28a through homologous recombinase ClonExpress II One Step Cloning Kit (nuuzan) after purification, and is transformed into E.coli BL21 (DE 3) competent cells to obtain a transformant, the transformant is coated in LB solid medium containing the kanamycin with the concentration of 50 mu g/mL, the culture is carried out for 12 hours at 37 ℃, positive colonies are picked, and single colony PCR verification is carried out on the single colony through Taq DNA polymerase by taking P7/P8 as a primer; after inoculating positive single colony with target stripe size into a small bottle containing LB liquid culture medium for culturing for 12h, extracting plasmid, and sending to gold and other intelligent company for sequencing correctly, then obtaining pET28a-GMAS and pET28a-GMAS _Y198S The pET28a-PPK plasmid is successfully constructed. The primer sequences involved are as follows:

P3：5’-ctacgtgcagttctactaaaagcttgcggccgcactcgag-3’；

P4：5’-aaatgggtcgcggatccgaattcatgaagtccctggaagaagca-3’；

P5：5’-cagaaaagtcctccgattaaaagcttgcggccgcactcgagcac-3’；

P6：5’-caaatgggtcgcggatccgaattcatggcaaccgatttttctaa-3’；

P7：5’-gtagaggatcgagatctcgatccc-3’；

P8：5’-tgaaaggaggaactatatccggat-3’；

(2) Preparation of E.coli BL21 competence

Coli e.coli BL21 was streaked on a non-resistant LB plate,placing and culturing in a 37 ℃ incubator, picking colonies, inoculating the colonies into a 10mL LB small bottle, culturing for 12h, transferring 1% inoculum size into a 50mL LB culture medium bottle, and preparing the large intestine competence when the thallus concentration reaches 0.4-0.6. Pre-cooling the related reagents and instruments in advance, placing the solution A, the solution B, the 1.5mL EP tube and the 50mL centrifuge tube in the prepared kit on ice, and controlling the temperature of the centrifuge to 4 ℃. Packaging 50mL of the bacterial liquid in a sterile operation table, and centrifuging (8000 r.min ^-1 5 min), the supernatant was removed. 5mL of solution A was sucked for blowing suspension, and centrifuged (8000 r.min ^-1 5 min), the supernatant was removed. 5mL of solution B is sucked for blowing suspension, and the suspension is split into EP pipes which are pre-cooled in advance, and each pipe is filled with 100 mu L.

(3) Recombinant bacterium BL21/pET28a-GMAS, BL21/pET28a-GMAS _Y198S Construction of BL21/pET28a-PPK

The pET28a-GMAS and pET28a-GMAS obtained in the step (1) are subjected to _Y198S Transforming pET28a-PPK plasmid into BL21 competent cells to obtain a transformant, coating the transformant on a LB solid medium containing the resistance of the kanamycin with the concentration of 50 mu g/mL, culturing at 37 ℃ for 12 hours, picking up positive colonies, and carrying out colony PCR verification on single colonies by using P7/P8 as a primer through Taq DNA polymerase; positive single colonies with the target band size were inoculated into a vial containing LB liquid medium for 12h, and after the culture, plasmids were extracted and sent to the gold-only intelligent company for sequencing. The correct sequencing indicates that the recombinant strain BL21/pET28a-GMAS, BL21/pET28a-GMAS _Y198S BL21/pET28a-PPK construction was successful.

Example 3: optimum substrate concentration of polyphosphate kinase PPK

The preparation method of the PPK pure enzyme comprises the following steps:

inoculating single colony of recombinant strain BL21/pET28a-PPK of example 2 into 10ml LB liquid medium, culturing at 37deg.C for 12 hr, inoculating 1% of the strain into 50ml LB liquid medium, culturing to OD ₆₀₀ About 0.8, and 0.05mM IPTG was added thereto at a final concentration and incubated at 16℃for 16 hours. Washing cells with PBS for three times, suspending the collected bacteria again with PBS, crushing cells with an ultrasonic crusher, crushing Escherichia coli for 1s, stopping for 3s, and crushing for 15min. Centrifugation 10000rpm,20min, purifying the crude enzyme liquid by a protein purification nickel column to obtain pure enzyme.

To optimize the ATP regeneration system, the substrate concentration of the key enzyme PPK was optimized. 1mL of reaction system: tris-HCl (pH 8, 200 mM), ADP 10g/L, polyP ₆ (5 mM, 10mM, 20mM, 30mM, 40mM, respectively, based on the final concentration), mgCl ₂ 5g/L and pure enzyme (final concentration 2.5U/mL), at 37℃for 30min, boiling the reaction in boiling water for 5min to terminate the reaction, and the absorbance of the supernatant was measured at 340 nm.

The results show (FIG. 2) that polyphosphate kinase PPK was at 5mM polyP ₆ The specific enzyme activity is 356.4 +/-13.2U/mg at the concentration and is 10mM of polyP ₆ The highest specific enzyme activity was 605.2.+ -. 17.1U/mg at 20mM polyP ₆ At this concentration, the specific enzyme activity of PPK was reduced to 287.5.+ -. 8.2U/mg,30mM poly P ₆ At a concentration, the specific enzyme activity of PPK is 179.3+/-7.3U/mg, 40mM of polyP ₆ At the concentration, the specific enzyme activity of PPK is 163.4+/-6.8U/mg. It follows that the PPK enzyme follows the substrate polyP ₆ The concentration of PPK is 10mM of polyP, which is the optimum substrate concentration, since the concentration of PPK is increased and then decreased compared with the enzyme activity ₆ Thus, 10mM of polyP was selected ₆ The concentrations were used for subsequent experiments.

Example 4: enzymatic production of L-theanine

The method comprises the following specific steps:

BL21/pET28a-GMAS, BL21/pET28a-GMAS _Y198S The BL21/pET28a-PPK strain is induced and purified. Inducing and expressing protein according to the step (2) of the example 1, collecting thalli, crushing for 15min by an ultrasonic crusher, and purifying to obtain GMAS and GMAS _Y198S And PPK enzyme. The L-theanine production system comprises the following components in percentage by final concentration: 20mL Tris-HCl (100 mM, pH 8.0), 100mM L-glutamic acid, 100mM ethylamine, 10mM poly P ₆ ，5mM ATP，20mM MgCl ₂ And enzyme (final concentration of 2.5U/mL PPK, final concentration of 10U/mL GMAS or GMAS) _Y198S ) Theanine yield was determined after 6h reaction at 37 ℃.

The control system is: 20mL Tris-HCl (100 mM, pH 8.0), 100mM L-glutamic acid, 100mM ethylamine, 10mM poly P ₆ ，150mM ATP，20mM MgCl ₂ And enzyme (final concentration 10U/mL GMAS or GMAS) _Y198S )。

In a catalytic system with 150mM ATP, the original enzyme GMAS can produce 80.5+ -3.2 mM theanine, the conversion rate of substrate glutamic acid is 80.5%, and the mutant enzyme GMAS _Y198S 96.3+/-2.1 mM theanine can be produced, the conversion rate is 96.3%, the theanine yield is improved by 18.8% compared with the original enzyme GMAS, which means that mutation of tyrosine at the 198 site of the GMAS enzyme into serine improves the catalytic activity of the GMAS, and the theanine yield and the conversion rate are further improved.

To reduce the production cost of theanine, the catalytic system for ATP regeneration coupled with PPK enzyme can produce 78.6+ -2.9 mM theanine by adding GMAS and PPK enzyme after only adding 5mM ATP, and adding GMAS _Y198S Can produce 94.6+/-3.5 mM theanine with PPK, and has a conversion rate of 94.6 percent, GMAS _Y198S Coupling with PPK can result in slightly lower theanine yields with the addition of 150mM ATP system. The mutation of residues near the active center of the GMAS enzyme can increase the quantity of substrates entering the active center, improve the catalytic activity of the GMAS, effectively improve the theanine production capacity of the GMAS, and couple a PPK regeneration system, enhance the sustainable capacity of ATP, effectively improve the theanine yield and reduce the substrate cost in catalysis. Thus, the mutant enzyme GMAS _Y198S The coupling with PPK can improve the output of theanine, remarkably improve the sustainability of ATP regeneration, improve the substrate conversion rate of theanine and save the cost of high energy consumption catalytic reaction.

Comparative example 1:

the specific embodiment is the same as in example 1, except that screening also yielded mutant Y198M, G238H, S246T, G231Y, and the results of the enzyme activity measurement according to the method of example 1 showed that the specific enzyme activities of the mutants were 20.3.+ -. 1.3U/mg, 20.9.+ -. 2.1U/mg, 21.8.+ -. 2.3U/mg, and 22.1.+ -. 1.4U/mg, respectively, each lower than mutant Y198S.

TABLE 1 mutant and specific enzyme Activity

/>

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A gamma-glutamylformamide synthetase mutant, which is characterized by having an amino acid sequence shown in SEQ ID No. 5.

2. A gene encoding the gamma-glutamylsynthetase mutant according to claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. The recombinant plasmid of claim 3, wherein the plasmid comprises a pET series plasmid.

5. A method for improving the enzyme activity of gamma-glutamine synthetase is characterized in that the 198 th tyrosine of the amino acid shown in SEQ ID NO.4 is mutated into serine.

6. A recombinant escherichia coli, wherein a pET28a (+) plasmid is used as an expression vector to express the gamma-glutamine synthetase mutant of claim 1.

7. A method for producing L-theanine, which is characterized in that L-glutamic acid is used as a substrate, and the gamma-glutamine synthetase mutant and polyphosphate kinase as defined in claim 1 are used as catalysts for reaction at 35-37 ℃.

8. The method of claim 7, wherein the polyphosphate kinase comprises the amino acid sequence set forth in SEQ ID No. 2.

9. The method according to claim 7 or 8, wherein the reaction system further comprises a polyphosphate; the polyphosphate is polyP with a final concentration of 5-10 mM ₆ 。

10. Use of a gamma-glutamyl carboxamide synthase mutant as claimed in claim 1 or a method as claimed in any of claims 7 to 9 for the production of a product comprising L-theanine.

Images