CN110734904A

CN110734904A - method for producing glutamic acid decarboxylase and application thereof

Info

Publication number: CN110734904A
Application number: CN201911147542.0A
Authority: CN
Inventors: 陈晟; 吴敬; 邱玲
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-11-21
Filing date: 2019-11-21
Publication date: 2020-01-31

Abstract

The invention discloses methods for producing glutamate decarboxylase and application thereof, belonging to the technical field of biology.A recombinant bacillus subtilis capable of expressing the glutamate decarboxylase is successfully constructed by taking food-grade bacillus subtilis as a host, the recombinant bacillus subtilis can be used for producing the food-grade glutamate decarboxylase and food-grade gamma-aminobutyric acid, and a foundation is laid for the application of the glutamate decarboxylase and the gamma-aminobutyric acid in the fields of food and medicine.

Description

method for producing glutamic acid decarboxylase and application thereof

Technical Field

The invention relates to methods for producing glutamate decarboxylase and application thereof, belonging to the technical field of biology.

Background

The research shows that GABA has the effects of promoting sleep, enhancing memory, resisting anxiety, preventing and treating epilepsy, delaying brain aging, relieving blood vessels, regulating hormone secretion, improving fertility rate, relieving ammonia toxicity, enhancing liver function and the like, so that GABA is widely applied to the fields of medicine, food, chemical industry, agriculture and the like by .

At present, methods for producing gamma-aminobutyric acid include chemical synthesis, plant enrichment, and biological synthesis. The chemical synthesis method is mainly characterized in that phthalimide potassium and gamma-chlorobutyl cyanide react at a high temperature of 180 ℃ to produce GABA, but the GABA production cost is high due to the severe reaction conditions and the limitation of expensive natural raw materials, and the GABA produced by the chemical synthesis method is poor in safety, can only be used in the chemical field and cannot meet the requirements of the food and medicine fields.

The plant enrichment method is mainly to remove α -carboxyl of L-glutamic acid by using glutamic acid decarboxylase (GAD, EC 4.1.1.15) separated from plant tissue cells to produce GABA, but the glutamic acid decarboxylase exists in a low amount in plants and is difficult to extract and separate from plant tissues in a large amount, so the feasibility of producing GABA by using the plant enrichment method is too low to meet market demands.

The biosynthesis principle mainly utilizes the glutamic acid decarboxylase separated from microbial cells or the microorganism whole cell special capable of producing the glutamic acid decarboxylase to irreversibly remove α -carboxyl of L-glutamic acid to produce GABA, and the method has the advantages of mild reaction conditions, no need of expensive raw materials and low energy consumption, and the glutamic acid decarboxylase from the microorganisms and the microbial cells capable of producing the glutamic acid decarboxylase can be obtained in large quantity by simple cell culture.

However, most of the current microbial strains capable of producing glutamate decarboxylase are non-food safe strains such as Escherichia coli, for example, KANG et al obtain GAD △ 466 by mutating Escherichia coli-derived glutamate decarboxylase, the catalytic activity and the suitable pH range of GAD △ 466 are both improved compared with wild type, but the Escherichia coli host for expressing GAD △ 466 needs to add IPTG (expensive and toxic IPTG) as an inducer to induce Enzyme production during fermentation, and the Escherichia coli host for expressing GAD △ 466 is easy to produce endotoxin during fermentation, so GAD △ 466 produced by the strain is also not high in safety and has a distance of (see KaTJ, HO, PACK SP. buffer-free production of microorganism-amino butyric acid) from food and medicine fields (see in particular: KANG J, NAT, PACK SP. engineering-201recombinant Enzyme derived from Escherichia coli).

Therefore, it is urgently needed to find methods for producing food-grade gamma-aminobutyric acid to meet the demand of the food and medicine fields for gamma-aminobutyric acid.

Disclosure of Invention

[ problem ] to

The technical problem to be solved by the invention is to provide methods for producing food-grade gamma-aminobutyric acid.

[ solution ]

In order to solve the technical problems, the invention provides methods for producing glutamate decarboxylase, which comprise the steps of inoculating recombinant Bacillus subtilis into a culture medium containing pyridoxal for culture to obtain recombinant Bacillus subtilis thallus containing glutamate decarboxylase, extracting the recombinant Bacillus subtilis thallus containing the glutamate decarboxylase to obtain the glutamate decarboxylase, and expressing a gene coding the glutamate decarboxylase by taking Bacillus subtilis as a host in the recombinant Bacillus subtilis.

In embodiments of the invention, the nucleotide sequence of the gene encoding glutamate decarboxylase is shown as SEQ ID No. 1.

In embodiments of the present invention, the Bacillus subtilis (Bacillus subtilis) is Bacillus subtilis (CCTCC NO: M2016536. the Bacillus subtilis (Bacillus subtilis) CCTCC NO: M2016536 is described in the patent application publication No. CN 106754466A.

In embodiments of the present invention, the recombinant Bacillus subtilis uses Bacillus subtilis CCTCC NO: M2016536 as host, and plasmid P_HpaΙΙ-P_amyQThe gene encoding glutamate decarboxylase is expressed for an expression vector.

In embodiments of the present invention, the pyridoxal is added to the medium in an amount of 0.1 to 0.5 mmol/L.

In embodiments of the present invention, the pyridoxal is added to the medium in an amount of 0.1 mmol/L.

The invention also provides the application of the method in the aspect of producing the glutamic acid decarboxylase.

The invention also provides methods for producing gamma-aminobutyric acid, which comprise the steps of adding glutamic acid decarboxylase into a reaction system containing glutamic acid for reaction to obtain a reaction liquid, extracting the reaction liquid to obtain gamma-aminobutyric acid, inoculating the recombinant Bacillus subtilis into a culture medium containing pyridoxal for culture to obtain a recombinant Bacillus subtilis thallus containing the glutamic acid decarboxylase, extracting the recombinant Bacillus subtilis thallus containing the glutamic acid decarboxylase to obtain the glutamic acid decarboxylase, and expressing a gene coding the glutamic acid decarboxylase by using the Bacillus subtilis as a host.

In embodiments of the present invention, the method comprises first subjecting glutamate decarboxylase to 40-50U/g_{Glutamic acid}The additive amount of (A) is added into a reaction system containing 100-400 g/L glutamic acid, the reaction is carried out under the conditions that the temperature is 37-40 ℃, the pH is 4.5-5 and the rotating speed is 150-200 rpm to obtain a reaction solution, and then the reaction solution is extracted to obtain the gamma-aminobutyric acid.

In embodiments of the invention, the method begins with glutamate decarboxylase at 40U/g_{Glutamic acid}The amount of (A) is added to a reaction system containing 400g/L glutamic acid, the reaction is carried out under the conditions of the temperature of 40 ℃, the pH value of 5 and the rotating speed of 150rpm to obtain a reaction solution, and then the reaction solution is extracted to obtain the gamma-aminobutyric acid.

The invention also provides the application of the method in the aspect of producing the gamma-aminobutyric acid.

The invention also provides recombinant Bacillus subtilis which takes Bacillus subtilis as a host to express a gene for coding glutamic acid decarboxylase.

In embodiments of the present invention, the recombinant Bacillus subtilis uses Bacillus subtilis CCTCC NO: M2016536 as host, and plasmid P_HpaΙΙ-P_amyQThe gene encoding glutamate decarboxylase is expressed for an expression vector. The invention also provides application of the recombinant bacillus subtilis in producing glutamic acid decarboxylase.

The invention also provides application of the recombinant bacillus subtilis in the aspect of producing gamma-aminobutyric acid.

[ advantageous effects ]

(1) The invention successfully constructs and obtains the recombinant bacillus subtilis capable of expressing glutamate decarboxylase (the nucleotide sequence is shown as SEQ ID NO. 1) by taking food-grade bacillus subtilis as a host; the recombinant bacillus subtilis can be used for producing food-grade glutamate decarboxylase and food-grade gamma-aminobutyric acid, and lays a foundation for the application of the glutamate decarboxylase and the gamma-aminobutyric acid in the fields of food and medicine.

(2) The invention provides methods for producing food-grade glutamate decarboxylase, which comprises the steps of adding pyridoxal in the process of producing the food-grade glutamate decarboxylase by taking recombinant bacillus subtilis as a production strain to improve the yield of the food-grade glutamate decarboxylase, and carrying out shake flask induction fermentation for 48 hours by using the method, so that the total enzyme activity of the glutamate decarboxylase in fermentation liquor can reach 28.14U/mL, which is 1.72 times that of the product without the pyridoxal.

(3) The invention provides methods for producing food-grade gamma-aminobutyric acid, which is to use the food-grade glutamic acid decarboxylase produced by the method for producing food-grade glutamic acid decarboxylase of the invention with 40U/g_{Glutamic acid}Is added into a reaction system with high substrate concentration and containing 400g/L of glutamic acid to react so as to produce food-grade gamma-aminobutyric acid; the method is used for reacting for 48 hours, so that the yield of the gamma-aminobutyric acid in the reaction liquid is up to 275.6g/L, and the conversion rate is up to 98.43%.

Drawings

FIG. 1: recombinant plasmid P_HpaΙΙ-P_amyQ-enzyme digestion verification of gadB; wherein, M: DL-10000bp DNAmarker, 1: and (3) recombinant plasmid enzyme digestion products.

FIG. 2: recombinant Bacillus subtilis B.subtilis/P_HpaΙΙ-P_amyQ-growth curve of gadB in fermentation medium; wherein GAD-0 is recombinant Bacillus subtilis B.subtilis/P_HpaΙΙ-P_amyQGrowth curve of gadB in pyridoxal-free fermentation Medium, GAD-L being recombinant bacillus subtilis/P_HpaΙΙ-P_amyQFermentation of-gadB in pyridoxalGrowth curves in medium.

FIG. 3: recombinant Bacillus subtilis B.subtilis/P_HpaΙΙ-P_amyQ-enzyme production profile of gadB in fermentation medium; wherein GAD-0 is recombinant Bacillus subtilis B.subtilis/P_HpaΙΙ-P_amyQEnzyme production profile of gadB in pyridoxal-free fermentation medium, GAD-L being recombinant bacillus subtilis b_HpaΙΙ-P_amyQ-enzyme production profile of gadB in fermentation medium containing pyridoxal.

FIG. 4: recombinant Bacillus subtilis B.subtilis/P_HpaΙΙ-P_amyQ-results of SDS-PAGE electrophoretic analysis of cell disruption supernatants obtained by gadB shake flask induced fermentation; wherein, M: molecular weight Marker in standard protein, 1: cell disruption supernatant of control group, 2: cell disruption supernatant of experimental group, 3: fermentation supernatant of control group, 4: fermentation supernatants of experimental groups.

FIG. 5: PL concentration vs. recombinant Bacillus subtilis B.subtilis/P_HpaΙΙ-P_amyQ-effect of the production of glutamate decarboxylase by gadB.

FIG. 6: influence of the amount of enzyme added on the conversion of GAD-0 and GAD-L to GABA.

FIG. 7: influence of substrate concentration on the conversion of GAD-0 and GAD-L to GABA.

FIG. 8: effect of reaction time on the conversion of GAD-0 and GAD-L to GABA.

Detailed Description

Pyridoxal (PL), Pyridoxine (Pyridoxine), pyridoxamine (pyridoxamine) and pyridoxal (pyridoxamine) referred to in the examples below were purchased from shanghai vitar chemicals, inc; bacillus subtilis CCTCC NO: M2016536, referred to in the following examples, is described in the patent application publication No. CN 106754466A; plasmid P referred to in the examples below_HpaΙΙ-P_amyQIs prepared by mixing two promoters P_HpaΙΙ-P_amyQInserted into plasmid pHY300PLK to construct, and the construction process is described in the literature "Zhang kang, Bacillus subtilis strain transformation, promoter optimization and high-efficiency preparation research of pullulanase [ D ]]Jiangnan university, 201In 8, plasmid pHY300PLK was purchased from Youbao; coli JM109/pET-24a (+) -gadB referred to in the following examples is described in the patent application publication No. CN 104531652A; escherichia coli (Escherichia coli) JM109 referred to in the following examples was purchased from Takara Bio Inc.; HindIII restriction enzymes referred to in the following examples were purchased from TaKaRa Co., Ltd; primer Star referred to in the examples^TMHS DNA polymerase, DL-10000bp DNAmarker, ampicillin and tetracycline are purchased from Baozi biological Limited; the Exnase II seamless ligation kit referred to in the examples below was purchased from tokyo nuozokenza biotechnology ltd; the plasmid extraction kit and the agarose gel recovery kit related in the following examples were purchased from Tiangen Biotechnology, Inc.; the molecular weight Marker and the protein gel preparation kit in the standard protein, which are referred to in the following examples, are purchased from Biyuntian biotechnology Co.

The media involved in the following examples are as follows:

LB liquid medium: yeast powder 5.0 g.L^-1Tryptone 10.0 g.L^-1、NaCl 10.0g·L^-1。

LB solid medium: yeast powder 5.0 g.L^-1Tryptone 10.0 g.L^-1、NaCl 10.0g·L ^-115 g.L agar powder^-1。

TB liquid medium: yeast powder 24 g.L^-1Tryptone 12 g. L ^-15 g.L of glycerin^-1、K₂HPO₄16.43 g·L^-1、KH₂PO₄2.31 g·L^-1。

The detection methods referred to in the following examples are as follows:

the detection method of glutamate decarboxylase enzyme activity comprises the following steps:

mu.L of substrate solution (0.1M sodium glutamate Hydroxyland 0.15mM PLP in 50mM Na, pH 4.5)₂HPO₄Citric acid buffer, storing at 4 ℃ in the dark), reacting 40. mu.L of enzyme solution at 37 ℃ for 4min, adding 600. mu.L of 0.2M, pH boric acid buffer 10, boiling for 10min to terminate the reaction, and measuring the content of gamma-aminobutyric acid in the reaction solution by using an HPLC method.

Definition of enzyme Activity the enzyme amount required for 1min catalytic substrate conversion to 1. mu. mol GABA was enzyme activity units (1U).

The enzyme activity calculation formula is as follows: the enzyme activity (U/mL) is the amount of γ -aminobutyric acid produced by conversion (μ g/mL)/conversion time (min).

The method for detecting the content and the conversion rate of gamma-aminobutyric acid (GABA) comprises the following steps:

placing the reaction solution in boiling water bath for 10min, then 12000 r.min^-1Centrifuging for 5min, sucking supernatant, and detecting the content and conversion rate of gamma-aminobutyric acid (GABA) in the reaction solution by adopting an HPLC-OPA pre-column derivatization method;

the HPLC chromatographic conditions are as follows: an Agilent 1200HPLC chromatograph, an Agilent autosampler, a GL InertsilODS-3 liquid phase column, and an Agilent ultraviolet detector; mobile phase A: 4.52g of anhydrous sodium acetate, 995mL of purified water is added, after stirring and dissolving, 200 muL of triethylamine and 5mL of tetrahydrofuran are added, then the pH value is adjusted to 7.2 by using acetic acid, and after mixing, the mixture is filtered by using a 0.22 muM inorganic cellulose filter membrane for standby; mobile phase B: 4.52g of anhydrous sodium acetate, 200mL of purified water is added, the pH value is adjusted to 7.2 by acetic acid after stirring and dissolving, 400mL of chromatographically pure methanol and 400mL of acetonitrile are added, the pH value is adjusted to 7.2 by acetic acid, and the mixture is filtered by a 0.22 mu m organic nylon filter membrane for standby; gradient elution with flow rate of 0.8mL/min and column temperature of 40 deg.C;

calculating the yield of the generated GABA according to the area of the absorption peak and the area of the peak of the GABA standard sample;

GABA conversion (%). the number of moles actually produced GABA/the theoretical number of moles converted to GABA x 100.

Example 1: construction of recombinant plasmid

Taking out the recombinant plasmid pET-24a (+) -gadB in the recombinant Escherichia coli E.coli JM109/pET-24a (+) -gadB by using a plasmid extraction kit; the recombinant plasmid pET-24a (+) -gadB is taken as a template, forward and reverse primers are respectively designed according to the design principle of a seamless cloning homology arm, the gadB gene (the nucleotide sequence is shown as SEQ ID NO. 1) is amplified, an amplification product is detected by 0.1 percent agarose gel electrophoresis, the length is 1428bp, the theoretical value is the same as that, and the primer sequence is as follows:

forward primer F1: 5'-GGAGTGTCAAGAATGGACCAGAAGCTGTTAACGGA-3' (the nucleotide sequence is shown as SEQ ID NO. 2),

reverse primer R1: 5'-TTTATTACCAAGCTTTCAGGTGTGTTTAAAGCTGTTC-3' (the nucleotide sequence is shown in SEQ ID NO. 3);

with plasmid P_HpaΙΙ-P_amyQRespectively designing forward and reverse primers according to the design principle of seamless cloning homology arm as a template, and amplifying P_HpaΙΙ-P_amyQThe expression vector, the amplification product was detected by 0.1% agarose gel electrophoresis, the length was 5731bp, the same as the theoretical value, and the primer sequences were as follows:

forward primer F2: 5'-TCAAATAAGGAGTGTCAAGAATG-3' (the nucleotide sequence is shown as SEQ ID NO. 4),

reverse primer R2: 5'-GGTGTTTTTTTATTACCAAGCTT-3' (the nucleotide sequence is shown in SEQ ID NO. 5);

amplified galdB gene and plasmid P were ligated with ExnaseII ligase_HpaΙΙ-P_amyQE.coli (Escherichia coli) JM109 was transformed after the ligation; spreading the transformed Escherichia coli (Escherichia coli) JM109 in an LB solid culture medium containing 50 mug/mL ampicillin, and performing inverted culture at 37 ℃ for 10-12 h; selecting positive transformants, inoculating the positive transformants to an LB liquid culture medium containing 50 mu g/mL ampicillin, carrying out inverted culture at 37 ℃ for 12-16 h, extracting plasmids, carrying out enzyme digestion verification, carrying out sequencing, and successfully sequencing to obtain recombinant plasmid P_HpaΙΙ-P_amyQ-gadB。

Example 2: construction of recombinant Bacillus subtilis

The recombinant plasmid P obtained in example 1 was used_HpaΙΙ-P_amyQ-GAdB transformed Bacillus subtilis (Bacillus subtilis) CCTCC NO: M2016536; coating an LB solid culture medium containing 20 mu g/mL tetracycline on the transformed Bacillus subtilis CCTCCNO (Bacillus subtilis) M2016536, and performing inverted culture at 37 ℃ for 10-12 h; selecting positive transformants, inoculating the positive transformants to an LB liquid culture medium containing 20 mu g/mL tetracycline, carrying out inverted culture at 37 ℃ and 200rpm for 8-10 h, extracting plasmids, carrying out enzyme digestion verification (the verification result is shown in figure 1), and obtaining the recombinant bacillus subtilis B.subtilis/P after verification is correct_HpaΙΙ-P_amyQ-gadB。

Example 3: production of glutamate decarboxylase

The recombinant Bacillus subtilis B.subtilis/P obtained in example 2 was used_HpaΙΙ-P_amyQ-gadB is inoculated into LB liquid culture medium containing 20 mug/mL tetracycline in an inoculation amount of 0.2% (v/v), and inverted culture is carried out at 37 ℃ and 200rpm for 8-10 h to obtain seed liquid; transferring a seed solution into a TB liquid culture medium containing 20 mu g/mL tetracycline in an inoculation amount of 5% (v/v) by taking Pyridoxal (PL) not added as a control, culturing at 37 ℃ and 200rpm for 2-3 h, adding Pyridoxal (PL) with the concentration of 0.1mM, continuing to shake flask for induced fermentation at 33 ℃ and 200rpm for 48h to obtain a fermentation liquid of an experimental group, transferring the seed solution into the TB liquid culture medium containing 20 mu g/mL tetracycline in an inoculation amount of 5% (v/v), culturing at 37 ℃ and 200rpm for 2-3 h, continuing to shake flask for induced fermentation at 33 ℃ and 200rpm for 48h to obtain a fermentation liquid of a control group; centrifuging the fermentation liquor of the experimental group and the fermentation liquor of the control group to obtain fermentation supernatant and sediment of the experimental group and fermentation supernatant and sediment of the control group; collecting the precipitate of the experimental group and the precipitate of the control group, ultrasonically crushing, and centrifuging to obtain cell crushing supernatant of the experimental group and cell crushing supernatant of the control group (recombinant Bacillus subtilis/P)_HpaΙΙ-P_amyQThe growth and enzyme production curves of gadB in the fermentation medium without and with pyridoxal are shown in FIGS. 2-3, respectively).

Detecting the total enzyme activity of the glutamate decarboxylase in the fermentation liquid of the experimental group and the fermentation liquid of the control group (namely the sum of the enzyme activity of the glutamate decarboxylase in the fermentation supernatant and the enzyme activity of the glutamate decarboxylase in the cell disruption supernatant), wherein the detection result is as follows: the total enzyme activity of the glutamate decarboxylase in the fermentation liquor of the experimental group is 28.14U/mL, the total enzyme activity of the glutamate decarboxylase in the fermentation liquor of the control group is 16.34U/mL, and the total enzyme activity of the glutamate decarboxylase in the fermentation liquor of the experimental group is 1.72 times that of the total enzyme activity of the glutamate decarboxylase of the control group.

Protein electrophoresis analysis was performed on the fermentation supernatant of the experimental group, the fermentation supernatant of the control group, the cell disruption supernatant of the experimental group, and the cell disruption supernatant of the control group, and the analysis results showed that bands corresponding to the theoretical molecular weight of were present at 53kDa in the fermentation supernatant of the experimental group, the fermentation supernatant of the control group, the cell disruption supernatant of the experimental group, and the cell disruption supernatant of the control group (see fig. 4 in particular).

Example 4: effect of Co-factors on glutamate decarboxylase production

Based on example 3, Pyridoxal (PL) in the experimental group was replaced with Pyridoxine (Pyridoxine) or pyridoxamine (pyridoxamine), respectively, to obtain fermentation broth a and fermentation broth B; centrifuging the fermentation liquor A and the fermentation liquor B to obtain a fermentation supernatant A, a fermentation supernatant B, a precipitate A and a precipitate B; and collecting the precipitate A and the precipitate B, carrying out ultrasonic disruption, and then centrifuging to obtain a cell disruption supernatant A and a cell disruption supernatant B.

Detecting the total enzyme activity of the glutamate decarboxylase in fermentation liquor A (namely the sum of the enzyme activity of the glutamate decarboxylase in the fermentation supernatant A and the enzyme activity of the glutamate decarboxylase in the cell disruption supernatant A) and fermentation liquor B (namely the sum of the enzyme activity of the glutamate decarboxylase in the fermentation supernatant B and the enzyme activity of the glutamate decarboxylase in the cell disruption supernatant B), wherein the detection result is as follows: the total enzyme activity of the glutamate decarboxylase in the fermentation liquid A is 15.55U/mL, and the enzyme activity of the glutamate decarboxylase in the fermentation liquid B is 20.11U/mL. As can be seen, only Pyridoxal (PL) in example 2 significantly increased the B.subtilis/P of recombinant Bacillus subtilis_HpaΙΙ-P_amyQ-production of glutamate decarboxylase by gadB.

Example 5: effect of cofactor concentration on glutamic acid decarboxylase production

On the basis of example 3, the concentration of Pyridoxal (PL) in the experimental group was replaced with 0.01mM, 0.03mM, 0.05mM, 0.07mM, 0.1mM, 0.2mM, or 0.3mM to obtain a fermentation broth; centrifuging the fermentation liquor to obtain fermentation supernatant and precipitate; collecting the precipitate, ultrasonically crushing, and centrifuging to obtain cell crushing supernatant.

The total enzyme activity of the glutamate decarboxylase in the fermentation liquid (namely the sum of the enzyme activity of the glutamate decarboxylase in the fermentation supernatant and the enzyme activity of the glutamate decarboxylase in the cell disruption supernatant) is detected, and the detection result is shown in figure 5.

As can be seen from FIG. 5, the concentration of Pyridoxal (PL) was 0.1mM, which increased the B.subtilis/P of recombinant Bacillus subtilis_HpaΙΙ-P_amyQ-gadB the effect of producing glutamate decarboxylase was best.

Example 6: production of gamma-aminobutyric acid

The cell disruption supernatant of the experimental group obtained in example 3 was designated as GAD-L, and the cell disruption supernatant of the control group obtained in example 3 was designated as GAD-0; GAD-L at 40U/g_{Glutamic acid}Adding the additive (B) into a reaction system containing 100g/L glutamic acid, and reacting at 40 ℃, pH 5 and rotation speed of 150rpm to obtain a reaction solution A; GAD-0 is added at 50U/g_{Glutamic acid}The amount of (B) was added to a reaction system containing 100g/L of glutamic acid, and the reaction was carried out at 40 ℃ and pH 5 at a rotation speed of 150rpm to obtain a reaction solution B.

And detecting the content and the conversion rate of the gamma-aminobutyric acid in the reaction liquid.

The detection result is as follows: the content of gamma-aminobutyric acid in the reaction liquid A is 70g/L, and the conversion rate is 100%; the content of gamma-aminobutyric acid in the reaction liquid B is 70g/L, and the conversion rate is 100%.

Example 7: effect of enzyme addition on the yield of gamma-aminobutyric acid

On the basis of example 6, the enzyme addition amounts of GAD-0 and GAD-L were respectively changed to 20, 30, 40, 50, 60U/g_{Glutamic acid}To obtain a reaction solution.

The content and conversion rate of gamma-aminobutyric acid in the reaction solution were measured, and the results are shown in FIG. 6.

As can be seen from FIG. 6, when the enzyme addition amount of GAD-L was 40U/g_{Glutamic acid}In the process, the content of gamma-aminobutyric acid in a reaction solution obtained by the GAD-L reaction is 70g/L, and the conversion rate is 100%; when the enzyme adding amount of GAD-0 is 50U/g_{Glutamic acid}In this case, the reaction mixture obtained by the GAD-0 reaction had a gamma-aminobutyric acid content of 70g/L and a conversion of 100%, and it was found that the optimum enzyme amounts of GAD-0 and GAD-L were 50U/g, respectively_{Glutamic acid}And 40U/g_{Glutamic acid}Then, the enzyme adding amount is continuously increased, the conversion rate is unchanged, and the reaction time is shortened.

Example 8: effect of substrate concentration on the yield of gamma-aminobutyric acid

A reaction solution was obtained by replacing the glutamic acid concentration with 400g/L in addition to example 6.

The content and conversion rate of gamma-aminobutyric acid in the reaction solution were measured, and the results are shown in FIG. 7.

As can be seen from FIG. 7, the reaction mixture obtained by the GAD-L reaction had a content of 275.60g/L of gamma-aminobutyric acid and a conversion of 98.43%; the reaction solution obtained by the GAD-0 reaction has the content of gamma-aminobutyric acid of 273.61g/L and the conversion rate of 97.72 percent, and the GAD-L is more suitable for a reaction system with high substrate concentration.

Example 9: effect of reaction time on Gamma-aminobutyric acid production

Reaction times were changed to 5, 10, 15, 20, and 24 hours in addition to example 6 to obtain a reaction solution.

The content and conversion rate of gamma-aminobutyric acid in the reaction solution were measured, and the results are shown in FIG. 8.

As can be seen from FIG. 8, the content and conversion rate of gamma-aminobutyric acid in the reaction solution obtained by the reaction of GAD-0 and GAD-L are increased continuously within 5-20 h, the conversion rate of gamma-aminobutyric acid in the reaction solution obtained by the reaction of GAD-0 and GAD-L reaches 100% at 20h, and after 20h, the conversion rate is not changed with the progress of the reaction.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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.

Sequence listing

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<400>2

ggagtgtcaa gaatggacca gaagctgtta acgga 35

<210>3

<211>37

<212>DNA

<213> Artificial sequence

<400>3

tttattacca agctttcagg tgtgtttaaa gctgttc 37

<210>4

<211>23

<212>DNA

<213> Artificial sequence

<400>4

tcaaataagg agtgtcaaga atg 23

<210>5

<211>23

<212>DNA

<213> Artificial sequence

<400>5

ggtgtttttt tattaccaag ctt 23

Claims

The method for producing the glutamate decarboxylase is characterized in that the method comprises the steps of inoculating recombinant Bacillus subtilis into a culture medium containing pyridoxal for culture to obtain recombinant Bacillus subtilis thallus containing the glutamate decarboxylase, extracting the recombinant Bacillus subtilis thallus containing the glutamate decarboxylase to obtain the glutamate decarboxylase, and expressing a gene coding the glutamate decarboxylase by using Bacillus subtilis as a host through the recombinant Bacillus subtilis.
2. The method for producing glutamate decarboxylase of claim 1, wherein the nucleotide sequence of the gene encoding glutamate decarboxylase is shown as SEQ ID No. 1.
3. The method for producing glutamic acid decarboxylase as claimed in claim 1 or 2, wherein the Bacillus subtilis is Bacillus subtilis CCTCC NO: M2016536.
4. The process for producing glutamic acid decarboxylase as claimed in any of claims 1-3, wherein the recombinant Bacillus subtilis is Bacillus subtilis (CCTCC NO: M2016536) and plasmid P is used as host_HpaΙΙ-P_amyQThe gene encoding glutamate decarboxylase is expressed for an expression vector.
5. Use of the method of any of claims 1-4 to for the production of glutamate decarboxylase.
6. methods for producing gamma-aminobutyric acid, wherein the method comprises the steps of adding glutamate decarboxylase into a reaction system containing glutamic acid for reaction to obtain a reaction solution, extracting the reaction solution to obtain gamma-aminobutyric acid, inoculating recombinant Bacillus subtilis into a culture medium containing pyridoxal for culture to obtain recombinant Bacillus subtilis thallus containing glutamate decarboxylase, extracting the recombinant Bacillus subtilis thallus containing glutamate decarboxylase to obtain glutamate decarboxylase, and expressing a gene encoding the glutamate decarboxylase by using Bacillus subtilis as a host.
7. Use of the method of claim 6 for the production of gamma-aminobutyric acid.
8, kinds of recombinant Bacillus subtilis, characterized in that the recombinant Bacillus subtilis uses Bacillus subtilis as host to express gene coding glutamic acid decarboxylase.
9. Use of the recombinant Bacillus subtilis of claim 8 for the production of glutamate decarboxylase.
10. Use of the recombinant Bacillus subtilis of claim 8 for the production of gamma-aminobutyric acid.