固定化したアルギナーゼ,ウレアーゼと遊離のグルタメートデヒドロゲナーゼの連続反応によるL-アルギニンの定量

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Arginase (EC 3.5.3.1), urease (EC 3.5.1.5) and glutamate dehydrogenase (EC 1.4.1.3) were coimmobilized in ethanol-insolubilized fibroin membrane, glutaraldehyde-crosslinked gelatin membrane or gelatin membrane photocrosslinked with diazidostilbene derivatives. Urease was greatly inactivated by glutaraldehyde. Glutamate dehydrogenase was readily inactivated by any immobilization treatment. Activity yields of arginase and urease immobilized in fibroin membrane (0.1mm thick at wet state) were 18% and 46%, showing better results than the other membranes. The immobilized enzymes were stable against repeated runs and storage. Thus, co-immobilized arginase and urease in the fibroin membrane and free glutamate dehydrogenase were used for L-arginine determination. In a six ml of reaction mixture, 50mM phosphate buffer (pH7.8), 2mM manganese chloride, 0.4mM α-ketoglutarate, 0.4mM NADH, 80mg fibroin membrane entrapping 5.4mg arginase (0.22U) and 0.7mg urease (0.79U), 1mg free glutamate dehydrogenase (5.58U) dialized against distilled water for 1h, and L-arginine were included. L-arginine concentration was varied to 100μM, 50μM and 20μM. Reaction was conducted by gentle stirring at 30℃. Absorption change at 340 nm was the measure of NADH oxidation. Reference experiment was conducted without L-arginine. A plateau level of NADH consumption was attained within 4h and the initial content of L-arginine was obtained with error below 6%.
1981-11-05