CN106566823B

CN106566823B - Cloning and application of glutamate decarboxylase gene

Info

Publication number: CN106566823B
Application number: CN201510654051.0A
Authority: CN
Inventors: 刘君; 程海娇; 徐宁; 马延和
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2015-10-10
Filing date: 2015-10-10
Publication date: 2021-04-30
Anticipated expiration: 2035-10-10
Also published as: CN106566823A

Abstract

The invention relates to cloning and application of a novel glutamate decarboxylase gene, wherein glutamate decarboxylase obtained from bacillus is subjected to induced expression in escherichia coli, and the property of the glutamate decarboxylase is researched, so that the glutamate decarboxylase has high activity at the pH value of 4.0-6.0, the optimum temperature is 45-60 ℃, Vmax is 150-200U/mg, and Km is 7-9 mmol/L. Recombinant Escherichia coli containing the enzyme is subjected to whole-cell transformation, 500g/L of L-glutamic acid is added by fed-batch, and the mixture is transformed in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with the temperature of 37 ℃ and the pH value of 7.0 for 12 hours to finally generate 347.8 g/L of gamma-aminobutyric acid, wherein the molar conversion rate is 99.4%. Compared with the existing glutamate decarboxylase, the novel glutamate decarboxylase has higher catalytic efficiency and wider pH range, and is more suitable for industrial application.

Description

Cloning and application of glutamate decarboxylase gene

Technical Field

The invention relates to a glutamic acid decarboxylase and application thereof in production of gamma-aminobutyric acid, belonging to the field of biocatalysis.

Background

Glutamate decarboxylase is a pyridoxal 5' -phosphate (PLP) -dependent enzyme that catalyzes the decarboxylation of L-glutamic acid to gamma-aminobutyric acid (GABA) and carbon dioxide. The product gamma-aminobutyric acid is an important inhibitory neurotransmitter, has various physiological functions of resisting anxiety, regulating blood pressure, treating epilepsy and the like, and has important application value in the industries of food, medicine and the like. At present, the main methods for producing gamma-aminobutyric acid include a chemical synthesis method, a plant enrichment method and a microbial fermentation method. The microbial fermentation method is to utilize GAD contained in organisms to biologically catalyze the decarboxylation of L-glutamic acid or L-glutamate to generate GABA. The method has the advantages of mild reaction conditions, short reaction period, no environmental pollution and the like, and thus the method is receiving more and more attention.

By usingThe GABA producing bacteria by fermentation method include yeast, Monascus and lactobacillus, etc., and the corresponding yield is 4.3g/L^[1]、9.18g/L^[2]And 10.78g/L^[3]The maximum yield of the lactobacillus optimized by mutagenesis and fermentation reaches 100g/L^[4]. The glutamic acid decarboxylase of the lactobacillus brevis is heterogeneously expressed in corynebacterium glutamicum, and the yield of GABA obtained is 30.18g/L^[5]. The glutamic acid decarboxylase used for producing GABA by the fermentation methods has low catalysis efficiency, the optimum pH value is slightly acidic, and the pH value of the culture medium is reduced to be below 4.5 or 4.5 so as to enable the culture medium to play a catalysis function. Expressing the glutamic acid decarboxylase gene of escherichia coli or lactobacillus brevis in escherichia coli, utilizing a whole-cell transformation method to produce GABA, wherein the yield can reach 280-300g/L respectively^[6]And 278.3g/L^[7]Although the catalytic efficiency is high, the pH of the conversion environment is still required to be reduced to 4.5 or below 4.5, and the equipment loss is large.

The currently discovered glutamate decarboxylase has low activity generally, the optimum pH of the enzyme is less than 5.0, and the enzyme activity is reduced sharply when the pH is more than 6.0, which greatly limits the application of the glutamate decarboxylase in industry.

Reference documents:

1. zheng hong goose, Zhao Wei, Changyan Hig response surface method optimizes the fermentation process [ J ] of gamma-aminobutyric acid produced by Candida yeast Y6. food science 2015,36(09): 130-.

2. The leaf inkstone response surface method optimizes the process condition [ J ] of producing the gamma-aminobutyric acid by the monascus X27 through liquid fermentation, Chinese food and oil academy, 2010,25(9): 107-.

3. Culture medium optimization of gamma-aminobutyric acid lactic acid bacteria in traditional dairy products [ J ] food industry science and technology, 2009,7: 124-.

4. Selection and mutagenesis of lactic acid bacteria producing gamma-aminobutyric acid [ J ] Nuclear agrimony, 2006,20(5): 379-.

5.F Shi.J Jiang.Y Li,et al.Enhancement of c-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis[J].J Ind Microbiol Biotechnol,2013,40:1285–1296.

6.A Plohov,M Gusytiner,T Yampolskaya,et al.Preparation of γ-aminobutyric acid using E.coli cells with high activity of glutamate decarboxylase[J].Appl Biochem Biotech,2000,88:257-265.

7. The construction of a recombinant Escherichia coli/pET-28 a-lpad and the optimization of the transformation conditions for efficiently transforming gamma-aminobutyric acid [ J ] the biological engineering report, 2012,28(1): 65-75.

Disclosure of Invention

The invention mainly aims to find a glutamic acid decarboxylase and construct an expression strain. After the glutamic acid decarboxylase is induced, expressed and purified, the enzymological properties are determined. The enzyme property research shows that the optimum temperature of the enzyme is 45-60 ℃, and the enzyme is relatively stable at 30 ℃; the optimum pH value is 4.0-6.0, and the most stable pH value is 7.0; vmax is 150-200U/mg, Km is 7-9 mmol/L.

In a whole cell transformation experiment, the total feeding amount of the L-glutamic acid is 500g/L, 347.92g/L of gamma-aminobutyric acid is finally generated after 12h of transformation, and the molar conversion rate is 99.41%.

Drawings

FIG. 1 is a SDS-PAGE image of BmGAD before and after purification;

FIG. 2 shows the optimum temperature (A) and temperature stability (B) of BmGAD;

FIG. 3 shows the optimum pH (A) and pH stability (B) of BmGAD;

FIG. 4 of the specification is a graph showing the calculation of enzyme kinetic constants by the Lineweaer-Burk mapping method;

description figure 5 shows the determination of GABA content in the conversion system by HPLC method.

Detailed description of the invention

The present invention will be described below with reference to specific examples, but these examples are not intended to limit the form, scope and effect of the present invention.

The glutamate decarboxylase gene is from China center for culture Collection of microorganisms, and the number of the strain is 10055. Has 1404 nucleotide sequences and encodes a protein with the size of 53 kDa. The similarity between the glutamate decarboxylase gene of Bacillus megaterium and the glutamate decarboxylase genes of Escherichia coli and lactic acid bacteria is about 50% and 30% respectively through sequence comparison.

Construction of recombinant strains

Designing a PCR primer by using a glutamic acid decarboxylase gene sequence of a bacillus megaterium model strain, obtaining a gene fragment of BmGAD by using a genome of bacillus megaterium as a template through PCR, constructing a recombinant plasmid pET21b-Bmgad by using pET21b as a vector and Nde I and Hind III as enzyme cutting sites, transforming E.coli DH5 alpha by using the recombinant plasmid, transforming E.coli BL21 after PCR, enzyme cutting verification and sequencing, and constructing a recombinant strain E.coli BL21(pET21 b-Bmgad).

Recombinant bacteria induced expression purification

E.coli BL21(pET21b-Bmgad) preserved at-80 ℃ is inoculated into an LB culture medium containing ampicillin resistance (final concentration 100 mu g/ml), activated overnight at 37 ℃, then a new LB culture medium containing ampicillin resistance (final concentration 100 mu g/ml) is transferred according to the inoculation amount of 2%, when the culture OD value is 0.6-0.8, 0.2-0.6 mmol/L of IPTG is added, and induction is carried out for 6-8 h at 25 ℃. And ultrasonically crushing the collected bacteria, and purifying by Ni column affinity chromatography to obtain pure enzyme BmGAD.

Determination of enzymatic Properties of BmGAD

The catalytic generation of 1 mu mol of gamma-aminobutyric acid per minute is defined as an enzyme activity unit U, and the specific enzyme activity U/mg. The reaction system was 3mL disodium hydrogen phosphate-citric acid buffer (containing 50mmol/L sodium glutamate, 0.1mmol/L PLP). The enzyme activity is measured by a colorimetric method, and the protein concentration is measured by a Coomassie brilliant blue method.

Optimum temperature and temperature stability: the optimum temperature was measured by selecting a reaction buffer at 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ and pH5. The temperature stability is measured at 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, and the residual enzyme activity is measured every 1h, and the total sampling time is 6 h.

Determination of optimum pH and pH stability: the optimum pH value of the enzyme was determined by conducting reactions at pH4.0, pH4.5, pH5.0, pH5.5, pH6.0, pH6.5 and pH7.0, respectively, at 50 ℃. The temperature stability is measured by dissolving BmGAD in buffer solutions of pH4.0, pH4.5, pH5.0, pH5.5, pH6.0, pH6.5, and pH7.0, respectively, incubating at 30 deg.C for 3h, and measuring the residual enzyme activity.

Determination of the effect of metal ions on the enzyme activity: respectively adding Na with the final concentration of 2mmol/L into the reaction system⁺、Ca²⁺、Co²⁺、Mn²⁺、Zn²⁺、Fe²⁺、Fe³⁺、Cu²⁺And determining the effect of different metal ions on the enzyme activity.

Determination of kinetic constants: reaction buffers with the concentration of 1mmol/L, 3mmol/L, 5mmol/L, 9mmol/L, 15mmol/L, 20mmol/L, 30mmol/L, 45mmol/L, 70mmol/L and 90mmol/L of sodium glutamate and the concentration of Vmax and Km are respectively prepared and obtained by a Lineweaer-Burk mapping method.

Transformation experiment of recombinant bacteria

The conversion buffer solution is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH7.0, the bacterial concentration is 15g/L, the PLP concentration is 0.2mmol/L, and the conversion temperature is 37 ℃. The total input amount of substrate glutamic acid is 500g/L, fed-batch addition is adopted, the initial concentration is 100g/L, 50g/L of glutamic acid is respectively added in 0.75h, 1.5h, 2.5h, 3.5h, 5h, 6.5h, 8h and 10h, and sampling is carried out at 12h to determine the final conversion rate. The conversion test results were determined by liquid chromatography.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> novel glutamate decarboxylase and application thereof

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<170> PatentIn version 3.3

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<213> Bacillus megaterium

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tttagttcat cggttcaagt ggtatgggag aagttcgcaa actattggga cgtagagcct 540

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cttatctggg atttccgctt gccgcgagta aagtccatta acgtgtcagg acacaagtat 840

ggtttagttt accctggctt gggatgggtg atttggagag aaaaagagga cttgcctgaa 900

gatcttattt tccgcgtttc ttatttaggg ggcaacatgc caacttttgc gctcaacttc 960

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aaactgaaag aagactatac accaaactgg actctttatg atttgtctag acaactgcgt 1200

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ttcaaacaag ccgttgaatt tcttaactcg ttggatagac ctgttcttaa agacacgaaa 1380

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Claims

1. The application of glutamate decarboxylase in converting L-glutamic acid or glutamate to generate gamma-aminobutyric acid is characterized in that the glutamate decarboxylase is derived from bacillus, and the amino acid sequence of the glutamate decarboxylase is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein the nucleotide sequence encoding glutamate decarboxylase is represented by SEQ ID No. 2.