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Production method and strain of gamma-aminobutyric acid (GABA)

A technology of aminobutyric acid and production method, applied in the field of genetic engineering, can solve the problems of complex GABA process and high production cost, and achieve the effect of simplifying production steps and reducing production cost

Inactive Publication Date: 2011-08-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the process of producing GABA in the prior art is complicated and the production cost is high

Method used

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  • Production method and strain of gamma-aminobutyric acid (GABA)
  • Production method and strain of gamma-aminobutyric acid (GABA)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Acquisition of the glutamic acid decarboxylase gene gadB1

[0019] A γ-aminobutyric acid-producing lactic acid bacterium was screened out from the series of lactic acid bacteria in our laboratory by paper chromatography. Through microscopic examination and 16SrDNA identification, it was confirmed that the homology with the 16SrDNA of various Lactobacillus brevis published in GenBank was 99%, thus confirming that the bacterial strain is Lactobacillus brevis. The bacterial strain has been preserved in China Type Culture Collection Center on December 27, 2010, address: Wuhan, China, Wuhan University, preservation number: CCTCC NO: M 2010367, taxonomic name: Lactobacillus brevis Lb85 ( Lactobacillus brevis Lb85).

[0020] Primers were designed based on the glutamic acid decarboxylase gene LVIS_1847 of Lactobacillus brevis ATCC 367, which has been published in the full genome sequence, and the genome of the screened GABA-producing Lactobacillus brevis was used a...

Embodiment 2

[0025] Example 2: Construction of recombinant Corynebacterium glutamicum pDXW8-gadB1 / ATCC13032:

[0026] ① Digest gadB1 and pDXW-8 with the same two restriction endonucleases NheI and HindIII to recover 1.43kb and 9.48kb fragments respectively, and then connect the two fragments with T4 DNA ligase, and Transformed into Escherichia coli JM109 to complete the construction of the pDXW8-gadB1 recombinant plasmid ( figure 1 );

[0027] ② The recombinant plasmid pDXW8-gadB1 was introduced into Corynebacterium glutamicum ATCC13032 by electroporation to obtain recombinant Corynebacterium glutamicum pDXW8-gadB1 / ATCC13032.

[0028] Competent preparation of Corynebacterium glutamicum ATCC13032. Cultivate a single colony of Corynebacterium glutamicum ATCC13032 in liquid LBG medium (10g / L peptone, 5g / L yeast powder, 10g / L NaCl, 5g / L glucose) at 30°C overnight, and then transfer to Inoculated in 30mL competent medium (10g / L peptone, 5g / L yeast powder, 10g / L NaCl, 25g / L glycine, 0.1% Twe...

Embodiment 3

[0029] Embodiment three: recombinant Corynebacterium glutamicum fermentation produces GABA

[0030] Medium: seed medium (glucose 2.5%, urea 0.6%, corn steep liquor 3.0%, K 2 HPO 4 ·3H 2 O 0.15%, MgSO 4 ·7H 2 O 0.05%, pH 7.0~7.5); fermentation medium (glucose 16%, urea 0.4%, corn steep liquor 0.3%, K 2 HPO 4 ·3H 2 O 0.2%, MgSO 4 ·7H 2 O 0.08%, MnSO 4 2mg / L, FeSO 4 2mg / L, pH 7.0~7.5)

[0031] Culture conditions: After the recombinant Corynebacterium glutamicum was shaken in the seed medium for 12 hours at 30°C, it was transferred to the fermentation medium with a 10% inoculum size for fermentation, and 1 mM IPTG was added for induction at 12 hours of fermentation, and in the subsequent Within 24 hours, the pH value of the culture medium was controlled in a neutral range by intermittently adding urea, and then the fermentation was continued until 60 hours, and the content of γ-aminobutyric acid in the fermentation broth was determined.

[0032] The content of gamma-...

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Abstract

The invention discloses a production method and strain of gamma-aminobutyric acid (GABA), belonging to the technical field of gene engineering. The method comprises the following steps of: introducing a glutamate decarboxylase gene into a glutamic acid production bacterium to construct a gene engineering bacterium; and removing a carboxyl from a self-accumulated glutamic acid by using glutamate decarboxylase secreted by the gene engineering bacterium to synthesize the GABA. In the invention, fermentation of glutamic acid is combined with transformation of the GABA, so that the production steps of the GABA are simplified; moreover, glucose, urea and the like are taken as culturing raw materials, so that the production cost of the method disclosed by the invention is remarkably lowered compared with a biological transformation method in which L-glutamic acid or L-sodium glutamate serving as a precursor needs to be exogenously added.

Description

technical field [0001] The invention relates to a production method of gamma-aminobutyric acid and its production strain, belonging to the technical field of genetic engineering. Background technique [0002] γ-Aminobutyric acid (GABA for short) is produced by decarboxylation of glutamic acid (Glu) catalyzed by glutamate decarboxylase (GAD for short). γ-aminobutyric acid is an important inhibitory neurotransmitter in the mammalian central nervous system. It has important physiological functions. The reported physiological activities include regulating blood pressure, promoting mental stability, promoting brain blood flow, enhancing brain vitality, nourishing nerve cells, increasing growth hormone secretion, strengthening liver and kidney, preventing obesity, and promoting alcohol metabolism ( Sober up), improve menopausal syndrome and other effects. [0003] At present, there have been related reports on the research on further increasing the production of GABA by overexpr...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12N15/60C12N15/77C12N1/21C12R1/01
Inventor 史锋李佑新李永富王小元
Owner JIANGNAN UNIV
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