Operation and stability of enzyme reactor containing poly(ethylene glycol)-bound NAD and thermostable dehydrogenases

点击翻译按钮获取中文摘要

Continuous utilization of dehydrogenase activity requires the retention and regeneration of coenzymes such as NAD and NADP. We have prepared poly(ethylene glycol)-bound NAD (PEG-NAD) as a macromolecular NAD derivative for enzyme reactors. PEG-NAD has good cofactor activity for various dehydrogenases. In particular, Thermus thermophilus malate dehydrogenase and Bacillus stearothermophilus lactate dehydrogenase show high activities for PEG-NAD. Therefore, we used these thermostable enzymes for our enzyme reactor.The two enzymes and PEG-NAD were retained in the reactor with an ultrafiltration membrane, and the substrates (pyruvate and malate) were fed continuously. The continuous coupled two-enzyme reaction was operated for 7 - 21 d at different temperatures. Half-lives of the L-lactate-production rate of the reactor were 9.7, 6.2, 5.4 and 3.6 d at 30, 40, 45 and 50゜C, respectively. The decrease in the activity of the enzymes and PEG-NAD in the reactor during continuous operation was also measured. The enzymes were more stable than PEG-NAD, and the stability of PEG-NAD limit the stability of the reactor. The stability of the enzymes and PEG-NAD in the reactor was different from that in the buffer solution.Poly(ethylene glycol)-bound NADP (PEG-NADP) was also prepared by selective alkylation of the 6-amino group of NADP. PEG-NADP thus obtained has good cofactor activity for various dehydrogenases and is applicable in a continuous enzyme reactor.