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Method for producing gamma-aminobutyric acid by using recombinant Bacillus subtilis

A technology of Bacillus subtilis and aminobutyric acid, which is applied in the field of microbial catalytic production of γ-aminobutyric acid, which can solve the problems of low conversion rate, limited application of endotoxin removal, and acidic conditions required for conversion.

Active Publication Date: 2016-09-14
常州科畅生物科技有限公司
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Problems solved by technology

The transformation rates of wild strains such as lactic acid bacteria, red yeast rice and grape juice yeast are all low. Although the transformation efficiency of Escherichia coli is relatively high, the addition of antibiotics in the production process and the removal of endotoxins in the post-treatment process limit its application.
Mo Zhengjie and others tried to use the safe host cell Bacillus subtilis to transform GABA, but because there is no regeneration pathway of pyridoxal phosphate in Bacillus subtilis, the transformation efficiency is low and the transformation requires acidic conditions, and the pH value is controlled at 4.5

Method used

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  • Method for producing gamma-aminobutyric acid by using recombinant Bacillus subtilis
  • Method for producing gamma-aminobutyric acid by using recombinant Bacillus subtilis

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Embodiment Construction

[0019] 1. Construction of recombinant Bacillus subtilis genetic engineering bacteria

[0020] (1) Cloning of Escherichia coli gadA and pdxH genes

[0021] Genomic DNA of Escherichia coli DH5α was extracted using a bacterial genomic DNA extraction kit. Using the genomic DNA as a template, P1 and P2 as primers, PCR amplifies the gadA gene, and uses primers P3 and P4 as primers to amplify the pdxH gene. Using a PCR product recovery kit, purify the PCR amplified product gadA gene fragment and pdxH gene fragment. The purified PCR product was ligated with the pUCm-T vector, and the ligated reaction product was transformed into Escherichia coli DH5α. Spread the blue-white screening plate, select the white colony on the plate, extract the plasmid, carry out double enzyme digestion and identification of the plasmid, and screen to obtain the recombinant bacteria DH5α / pUCmT-gadA and DH5α / pUCmT-pdxH. Extract the plasmid and send it to Shanghai Sangon Bioengineering Co., Ltd. for sequen...

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Abstract

The invention relates to a method for producing gamma-aminobutyric acid by using recombinant Bacillus subtilis. The method uses Bacillus subtilis as the host cells to conduct expression of Escherichia coli decarboxylase gene (gadA) or co-expression of regeneration gene of coenzyme pyridoxal phosphate (pdxH); the conditions for transformation of glutamic acid for synthesis of gamma-aminobutyric acid by using recombinant B.subtilis 168 / pHT01-gadA-pdxH whole cells are further optimized, and the optimal conditions are as below: a transformation buffer system is 0.1MTris-HCl with pH value of 6.5, the transformation temperature is 35 DEG C, and an activator is 5mmol / LCa<2+> and 5 mmol / L Mg<2+>. Under the above conditions, the transformation is carried out for 24h, and the generated GABA reaches a concentration of 327g / L; and the residual L-glutamate in the transformation system is less than 1%. The recombinant strain obtained by the invention has high conversion efficiency, and has certain industrialization prospect.

Description

technical field [0001] The invention relates to a production of gamma-aminobutyric acid, in particular to a method for producing gamma-aminobutyric acid by microorganism catalysis. Background technique [0002] γ-aminobutyric acid is a naturally occurring non-protein amino acid, which is an important inhibitory neurotransmitter in the mammalian central nervous system. About 50% of the central nervous system synapses use GABA as a transmitter. In addition to the physiological effects of sedation, blood pressure reduction and anticonvulsion, GABA also shows good effects in promoting animal growth, regulating appetite and anti-stress, and has attracted more and more attention. Because GABA has good physiological effects, it has been widely used in food, medicine and cosmetics in Japan and the United States. In my country's food industry, the Ministry of Health approved GABA as a new resource food in the No. 12 announcement on September 27, 2009, and its related health care pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12R1/125
CPCC12P13/00
Inventor 许伟张六六
Owner 常州科畅生物科技有限公司
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