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Glutamate decarboxylase mutant and application thereof in preparation of gamma-aminobutyric acid

A technology of glutamic acid decarboxylase and glutamic acid oxidase, which is applied in the field of bioengineering, can solve the problems of unknown enzyme activity and achieve the effects of avoiding food safety hazards, high comprehensive utilization of raw materials, and reducing production costs

Active Publication Date: 2020-09-08
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patent CN105255849B describes a glutamic acid decarboxylase mutant obtained after molecular transformation, the enzyme activity can reach 60.6U / mg at pH 4.8, but the enzyme activity in a near-neutral environment is unknown

Method used

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  • Glutamate decarboxylase mutant and application thereof in preparation of gamma-aminobutyric acid
  • Glutamate decarboxylase mutant and application thereof in preparation of gamma-aminobutyric acid
  • Glutamate decarboxylase mutant and application thereof in preparation of gamma-aminobutyric acid

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Experimental program
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Effect test

Embodiment 1

[0015] The construction of embodiment 1 glutamic acid decarboxylase mutant

[0016] By analyzing the alignment structure of the wild-type glutamic acid decarboxylase sequence and homologous sequences derived from other strains, combined with homology modeling and active site prediction, the 94th tyrosine Y of the amino acid sequence of SEQ ID NO:1 was determined And the 249th serine S is the mutation target. The site-directed mutagenesis strategy was adopted, and the point mutation primers were designed according to the amino acid site to be mutated, and the mutant sequence of glutamate decarboxylase was obtained by PCR.

[0017] Entrusted Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the glutamic acid decarboxylase gene of Bacillus megaterium, and subcloned it into the pET21 expression plasmid based on the NdeI / HindIII double restriction site, and the recombinant plasmid was named pET21-GAD. Use the Tiangen Rapid Site-directed Mutagenesis Kit to carry out mutations ...

Embodiment 2

[0021] The enzyme activity assay of embodiment 2 glutamic acid decarboxylase mutants

[0022] The recombinant Escherichia coli obtained above containing the pET21-GAD, pET21-GAD-Y94H and pET21-GAD-S249F plasmids respectively were subjected to protein induction and expression. The specific method was: pick single clones and inoculate them in 100mL LB liquid medium (5g / L yeast extract, 10g / L tryptone, 10g / L NaCl), for overnight culture. Subsequently, according to the starting OD 600 ≈ 0.1 Transfer the seed solution to 100mL LB liquid medium, and wait until the cell concentration OD 600 At about 0.6, add a final concentration of 0.4mM IPTG to induce the expression of the target protein, adjust the culture temperature to 28°C, and continue the induction culture for 12h. After the induction is completed, the bacterial liquid is subjected to high-pressure homogenization and crushing treatment, and the purified glutamic acid decarboxylase protein is obtained by using a laboratory ...

Embodiment 3

[0027] Example 3 Construction of recombinant strains containing glutamic acid decarboxylase mutants

[0028] The host bacterial strain mentioned in the present invention is a bacterial strain that can express the glutamic acid decarboxylase mutant, and the host bacterial strain can be selected from Corynebacterium, Escherichia or Bacillus, for example, glutamic acid rod Bacillus (Corynebacterium glutamicum), Escherichia coli (Escherichia coli) or Bacillus subtilis (Bacillus subtilis), etc. One of the preferred hosts provided in this example is Escherichia coli BL21 and Corynebacterium glutamicum ATCC 13032 respectively.

[0029] Competent Escherichia coli BL21 and Corynebacterium glutamicum ATCC13032 were prepared by conventional laboratory standard methods. The pET21-GAD, pET21-Y94H, pET21-S249F, pET21-Y94H / S249F plasmids were transformed into Escherichia coli BL21 competent, respectively, and E. coli recombinant bacteria BL21-GAD, BL21-Y94H, BL21-Y94H, BL21-S249F and BL21-...

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Abstract

The invention discloses a glutamate decarboxylase mutant with improved enzyme activity and application thereof. The glutamate decarboxylase coding gene from bacillus megatherium is subjected to site-specific mutagenesis, and the catalytic performance of the obtained mutant is obviously improved compared with that of a wild type mutant. A recombinant engineering strain is constructed on the basis of the mutant, gamma-aminobutyric acid is prepared by taking a large amount of fermentation product L-glutamic acid as a substrate through a whole-cell catalysis method, the maximum yield of gamma-aminobutyric acid reaches 625.6 g / L, the molar conversion rate in a catalytic system is close to 100%, and no by-product is produced. The glutamate decarboxylase mutant disclosed by the invention has a very wide application prospect in efficient production and preparation of gamma-aminobutyric acid.

Description

technical field [0001] The invention relates to a glutamic acid decarboxylase mutant and application thereof, belonging to the technical field of bioengineering. Background technique [0002] γ-aminobutyric acid is a naturally occurring non-protein amino acid, which is a strong neuroinhibitory amino acid. It has physiological effects of sedation, hypnosis, anticonvulsion, and hypotension. It has a wide range of applications in the fields of food, medical care, and beverage processing. Application prospect and market demand. According to rough statistics, the current total production capacity of various types of GABA in the world is about 60,000 tons per year, and the total market value of GABA-added related products in my country is about 2.5 billion to 3 billion yuan, and the market is still in a period of slow growth. The preparation methods of γ-aminobutyric acid mainly include chemical synthesis, microbial fermentation and biotransformation, etc., but the chemical synth...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N1/21C12P13/00C12R1/19C12R1/15
CPCC12N9/88C12P13/005C12Y401/01015
Inventor 刘君徐宁
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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