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

含有聚乙二醇结合 NAD 和热稳定性脱氢酶的酶反应器的运行和稳定性

基本信息

批准号：
58850198
负责人：
OKADA Hirosuke
金额：
$ 9.09万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1983
资助国家：
日本
起止时间：
1983 至 1985
项目状态：
已结题

项目摘要

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.

脱氢酶活性的连续利用需要辅酶如NAD和NADP的保留和再生。我们已经制备了聚（乙二醇）结合NAD（PEG-NAD）作为酶反应器的大分子NAD衍生物。PEG-NAD对多种酶具有良好的辅因子活性。特别地，嗜热栖热菌苹果酸脱氢酶和嗜热脂肪芽孢杆菌乳酸脱氢酶显示出对PEG-NAD的高活性。因此，我们将这些热稳定酶用于我们的酶反应器。两种酶和PEG-NAD被保留在具有超滤膜的反应器中，并且底物（丙酮酸和苹果酸）被连续进料。在不同温度下，连续偶联反应7 ~ 21 d。在30、40、45和50 ℃下，反应器L-乳酸生产速率的半衰期分别为9.7、6.2、5.4和3.6 d。还测量了连续操作期间反应器中酶和PEG-NAD活性的降低。酶的稳定性优于PEG-NAD，PEG-NAD的稳定性限制了反应器的稳定性。酶和PEG-NAD在反应器中的稳定性与在缓冲液中的不同。由此获得的PEG-NADP对各种酶具有良好的辅因子活性，并且可应用于连续酶反应器。