Studied On Production Of γ-aminobutyric Acid By Microbial Fermentation

y-aminobutyric acid(GABA) is putative inhibitory neurotransmitter. It is concerned with metabolic activity and has high biological activity. GABA can stave off brain senescence, reduce blood pressure, cure neurological disability, resist arrhythmia etc. It is used widely in medicine and food field and becomes a hot spot. GABA fermentation culture medium, culture conditions and immobilization conditions were optimized on the basis of GABA strains identification. Engineering bacteria was constructed to maximize the content of GABA by the method of molecular biology.A strain was isolated from picked Chinese cabbage and it has a good property of producing GABA. The content of GABA was increased by mutation breeding. The results showed that:8strains were isolated and the strain Lp-Lw-131had the best property of producing GABA by the method of thin-layer chromatography. The strain was identified Lactobacillus plantarum by morphological observation and biochemical tests. This strain was irradiated with ultraviolet and then mutagenized with diethyl sulfate (DES). UV irradiation time was90s and the distance was30cm. DES mutation time was20min and the concentration of DES-ethanol solution was25%. The content of GABA was1.815g/L. After12successive generations, the content of GABA was stable. The strain was numbered Lp-Lw-131-34.Based on the single factor experiment, the composition of the culture medium and the culture conditions were optimized by the method of response surface. The best composition was:MgSO4·7H2O49.42mmol/L, CaCl26.18mmol/L, MSG57.09g/L; the best culture conditions were:inoculum concentration3.00%, pH6.00, culture time47.40h. After the optimization, the content of GABA was2.24g/L,23.42%higher than the original content. The original content was1.815g/L.In order to increase the content of GABA, immobilization reactions were studied. The result showed that:the best composition of embedding medium was sodium alginate1.5%,CaCl20.6%; the best embedding time was2.5h; the best reaction conditions were:cells concentration4.0%, MSG concentration20g/L, temperature37℃, pH4.0; the content of GABA was1.224g/L. The content of GABA was14.326g/L after15reactions of immobilization cells. Enzyme activity declined steadily.There is GAD gene in the strain Lp-Lw-131-34. GAD is used to produce GABA from MSG. Engineering bacteria was constructed to increase the content of GABA by the method of molecular biology and GAD gene sequence was analysed by bioinformatics. The cloned Lp-Lw-131-34GAD gene nucleotide sequence was analysed and translated. The result showed that: the similarity between amplification DNA sequence and the known DNA sequence of the four Lactobacillus plantarum strains was respectively97.7%,98.3%,98.7%and98.8%. Molecular weight:54020.3; theoretic pI:5.58; the totality of electronegative residue:61;the totality of electropositive:40; N-terminal of the sequence:Clu; the total average hydrophily:-0.271; the protein was hydrophilic; there was no signal peptide; there were two N-glycosylation sites. Structural domain result showed that the protein had decarboxylase activity. Tertiary structure was calculated; the similarity between the protein sequence and the reported sequence was42.74%. The cloned gene was transformed to the carrier BL21and the content of GABA was0.523g/L.