Heterologous Expression And Optimization Of Glutamate Decarboxylase From Lactobacillus Plantarum ZJ3443

y-aminobutyric acid (GABA) is an important nonprotein amino acid, which has been detected as the main inhibitory neurotransmitter in the mammalian central nervous system. It can be applied in functional foods and health-care foods to enhance memory, regulate the blood fattiness, and reduce the risk of cancer and diabetes. However, the yield of the GABA is still limited due to its low content in natural organisms. To get high titer of GABA, many researchs has been spending effort on producing the glutamate decarboxylase (GAD), which is the enzyme that catalyzes the biosynthesis of GABA from glutamic acid. In this study, the GAD gene from Lactobacillus plantarum was cloned and recombinantly expressed in Escherichia coli and Bacillus subtilis.1. Based on the GAD gene informations from NCBI database, the GAD gene from L. plantarum ZJ3443was cloned by PCR. This gad gene is1410bp, coding469amino acids without a signal peptide. The GC content of the gad gene was47.7%. The gad gene was cloned to the plasmid pET-22b(+) and trasnsformed to E. coli BL21(DE3). SDS-PAGE analysis exhibited that the GAD was successfully expressed with the intracellular GAD specific activity of5.5U/mg. Further HPLC analysis showed that the GABA could be synthesized by the recombinant cells from glutamic acid.2. On the condition of being cultured in shaker flasks with TB medium, and initially induced at OD6oo=2with0.6mM IPTG at30℃, the highest catalytic efficiency of recombinant cells was achieved. Under the above condition, the time required for the biocatalysis of0.1M glutamic acid by the recombinant cells collected from10mL cultured medium was reduced from130min to80min.3. The recombinant LOX from the recombinant E. coli was purified by cell lysis, ammonium sulfate precipitation, dialysis and Ni2+affinity chromatography. The specific activity of the purified GAD was43.12U/mg. The optimal temperature and pH of the purified LOX were40℃and4.5, respectively. The purified GAD was relatively stable in pH3and4, but it is of thermal unstability. Most of the tested ions have little effects on the catalytic activity of the purified GAD. However, the EDTA, Zn2+, SDS can inhibite GAD’s activity whilie the Ca2+can enhance its activity. The Km and Vmax value of the purified GAD was6.55mM and2.827mM/min, respectively.4. The GAD gene was cloned to the plasmid pMA5and the resulted plasmid was transformed to Bacillus subtilis WB600. The GAD was expressed as intracellular active enzyme in B. subtilis. The cell growth and intracellular enzyme activity were increased by the supplement of20g/L glycerol and10g/L urea. When the bioconversion was performed at37℃and pH4.5, the conversion rate of0.1M glutamic acid reached85%after22h.