Biotransformation Law Recombinant Glutamic Acid Decarboxylase Synthesis Of Gamma-aminobutyric Acid

y-aminobutyric acid (GABA), a free non-protein amino acid, acts as a major inhibitory neurotransmitter in the central nervous system. GABA has several physiological functions such as neurotransmission, induction of hypotensive effects, diuretic effects, treatment of epilepsy and tranquilizer effects, regulation of hormone secretion, activation hepatorenal, etc. Preparation and application of GABA were received by people's concern and attention. The glutamate decarboxylase (GAD, EC 4.1.1.15) catalyze L-glutamate by decarboxylation reaction to generate GABA. It is rate-limiting enzyme for biocatalysis GABA. Although the Escherichia coli GAD and GABA synthesis have been profoundly investigated, how to further obtain the recombinant GAD with high GABA-forming activity and produce GABA from enzyme-catalyzed synthesis with low cost and high efficiency remain poorly understood. The major experiment results of dissertation were as follows:1,We amplified the gene coding for glutamate decarboxylase from the genome of E.coli K-12 by PCR. The recombinant plasmid pET28a-gadA was constructed, and then transformed to E.coli BL21. Escherichia coli GAD was highly expressed (about 70-75% of total protein) as soluble protein containing pET28a-gadA, which was induced by IPTG and lactose in LB medium with the calculated molecular weight of 53 KDa.2,The reaction conditions for synthesizing recombinant GABA were also investigated. Incubated at 37℃for 4 h in 1 mL reaction system(31 g/L glutamate,1.15 U crude extract, pH3.8). The yield of GABA was 19.57 g/L, and the conversion rate of sodium glutamate as to GABA was 93%. It has provided a good prospect for GABA production.3,Effect of divalent cations and pyridoxal phosphate(PLP) coenzyme on GABA-forming activity of the recombinant E.coli GAD were also investigated. The optimal concentration (7.5 mM) of PLP can improve one time the activity of recombinant enzyme(198%). At optimal pH of 3.8, the relative activity was markedly higher activated by Ca2+ (174%),Mn2+(164%) than that by other seven bivalent cations, but the co-effect of Ca2+ and Mn2+ exhibited antagonism effect when added simultaneously. The results demonstrated that E.coli GAD is likely the PLP-dependent enzyme and Ca2+/CaM binding protein that can be activated by PLP and Ca2+. Finally, the 1 L reaction system (150 g/L glutamate,0.6 mM Ca2+,0.15 mM PLP,200 mM HAc-NaAc buffer of pH 3.8 at 37℃,30 mL crude GAD/12 U/mL) was tested. Glutamate was added each 2 h, GABA content was about 94 g/L after 24 h reaction, and the conversion of glutamate was about 90%.4,By entrapping Escherichia coli glutamate decarboxylase into sodium alginate(25 g/L) and carrageenan gel(10 g/L) beads, the activity of immobilized GAD(IGAD) remained 85% during the initial five batches and the activity still remained 50% at the tenth batch, these results indicated that the recombinant Escherichia coli GAD was feasible for the future industrial production of GABA.