Catalytic Synthesis γ-Amino-Butyric Acid (Gaba) With Microbial Cell

y-aminobutyric acid(GABA) is putative inhibitory neurotransmitter in the vertebrate nervous system.It is widely used in medicine and food field. A method of fermentation using microorganism to produce GABA is to use glutamic acid decarboxylase in microorganism to produce GABA. A Lactobacillus plantarum strain with the ability of producing GABA was screened from the nature and identified preliminary;GAD gene was extracted and amplified, then the prodcut was connected with a carrier and transformed to a Escherichia coli strain, Electropositive transformers were sequenced and analyzed;The best medium composition and conditions were achieved after the study of fermentation technology.then the Lactobacillus plantarum cells were immobilized and the qualities of the immobilized cells were studied.Two strains were isolated from picked Chinese cabbage and then purified. The strains were numbered LpL328, ST328and the former was proved to have a property to produce y-amino butyric acid (GABA). LpL328strain was identified Lactobacillus plantarum by microscopic observation and biochemical properties. The strain was inoculated into fermentation medium, then the cells were treated by two methods:one was collecting the cells and making them into acetone powder; the other was dealing the cells with nothing and GABA was produced by reaction of the cells with substrate sodium glutamate directly. The content of GABA was2.16g/L and2.20g/L respectively. TYG was a good medium for producing GABA but MRS was not. PLP was a necessary coenzyme for the production of GABA.The GAD gene was encoded from Lactobacillus plantarum LpL328.The product was cut and connected with the plasmid treating with the same method, the product was transformed to the E. coli BL21competence cells. Electropositive transformers were screened by mb-on method. Gene was encoded from the transformers and the sequence was analyzed. The result was:sequence homology of the PCR product DNA and Ipgad distributed by NCBI(GenBank Accession No. CP002222.1)was99.79%;molecular weight of LpL328GAD was53574.9,pl5.58;the protein was hydrophilic; there was no signal peptide so the protein was not secretion protein or a protein working in organelles,this kind of protein was synthesized in cell matrix and carried to organelles:there were two N-glycosylation sites; structural domain result showed that the protein had decarboxylase activity;motif result showed that there were four sites matching with the designated sequence pattern.The composition of the medium was optimized by the method of single factor experiment and the best composition was:glucoselOg/L, yeast extract10g/L, sodium succinate5g/L, and sodium glutamate8g/L. On the base of the optimized medium composition, the fermentation conditions were optimized by the method of single factor experiment and orthogonal experiment. The best conditions were:culture time36h, initial pH6.0, inoculum concentration2%.After the optimization of medium composition and fermentation conditions, the content of GABA was2.91g/L,35%higher than the original ones.The strains were cultured on the optimized conditions and medium composition, the cells were collected and embedded in sodium alginate and calcium chloride. The ability of immobilization cells for GABA production was studied by the method of single factor experiment and orthogonal experiment. The factors included cell concentration, substrate concentration,temperature and pH. After the experiment, the best conditions were:cell concentration6%, substrate concentration20g/L, temperature37℃and pH4.7.The content of GABA was0.96g/L.The conclusion of continuous experiment was that the residual enzyme activity of immobilization cells was68.3%.The content of GABA was7.065g/L after7reactions of immobilization cells.