Glutamate decarboxylase 생산을 위한 재조합 E. coli 의 배양에 관한 연구

Abstract

γ-Aminobutyric acid (GABA) and glutamate are thought to be the most important inhibitory and excitatory amino acid neurotransmitters in the central nervous system (CNS).GABA, a four-carbon non-protein amino acid, acts as the major inhibitory neurotransmitter of the central nervous system. GABA is generated from glutamate by the action of glutamic acid decarboxylase(GAD). GAD has been purified from several brain tissues and it is generally accepted that the diametric protein possesses a relative molecular mass of around 110,000 Da. In this study, the fermentation technique was applied to produce GAD using recombinant E. coli(BL21). The effects of ammonium sulfate, magnesium sulfate, glucose concentration, three different feeding methods, agitation speed, aeration rate, and pH on the production GAD were investigated in this work also. The optimal medium composition was glucose 40 g/L, (NH4)2SO4 6 g/L, KH2PO4 3 g/L, K2HPO4 3 g/L, yeast extract 1 g/L and monosodium glutamate(MSG) 20 g/L. MSG induced the expression of GAD. GAD activity and specific GAD activity was 248.6 units, 4.2 units/cell mass under optimal culture conditions. The fed-batch fermentation was performed to increase the cell mass (3.7 g/L) and GAD activity (337.3 units). The production of GAD rate was increased with the oxygen with increased agitation speed and aeration. The cell mass, GAD activity and specific GAD activity was 3.9 g/L, 764 units and 5.7 unit/cell mass at 300-350 rpm and 7 vvm, respectively. Since GAD were fermentation pH-dependent, pH was controlled at pH 5.5 to enhance GAD productivity. With pH-controlled conditions, the cell mass, GAD activity and specific GAD activity was 3.7/L, 834.7 and 6.6 unit/ cell mass, respectively. Finally a concentration of 1 M of GABA was produced from glucose 10 hr enzyme reaction. In summary, this fermentation system would be considered to be suitable for the product of GAD at the condition of glucose 40 g/L, (NH4)2SO4 6 g/L, KH2PO4 3 g/L, K2HPO4 3 g/L, yeast extract 1 g/L MSG 20 g/L, 300-350 rpm, 7vvm aeration, and pH 5.5.