桥联黄素再生NAD^(+)耦联L-谷氨酸脱氢酶高效产α-酮戊二酸

Enhancement of α-ketoglutaric acid production via L-glutamate dehydrogenase with NAD^(+) regenerated by coupled system of bridged flavinium catalyst

在L-谷氨酸脱氢酶(EC 1.4.1.3,L-GluDH)催化L-谷氨酸氧化脱氨过程中涉及昂贵的氧化型烟酰胺辅因子NAD^(+),因此需要构建其再生体系以降低成本。通过构建桥联黄素(F4)耦联L-谷氨酸脱氢酶催化L-谷氨酸生产α-酮戊二酸(α-KG)的新型均相体系,桥联黄素能高效氧化天然还原型烟酰胺辅因子NADH至其氧化态NAD^(+),从而减缓NADH对L-谷氨酸脱氢酶的抑制,提高α-KG产率的同时降低NAD^(+)用量。通过单因素考察,筛选得到来自艰难梭菌的L-谷氨酸脱氢酶作为酶源,在Tris-HCl缓冲液环境下进行氧化脱氨反应,结合响应面法(RSM)确立了最佳反应条件:NAD^(+)浓度1 mmol/L,F4浓度1 mmol/L,pH 8。在底物L-谷氨酸15 mmol/L条件下放大反应,24 hα-KG的收率达到92%。

In the process of the oxidative deamination of L-glutamic acid catalyzed by L-glutamate dehydrogenase(EC 1.4.1.3,L-GluDH),the oxidized nicotinamide cofactor NAD^(+)need to be regenerated in virtue of its high price.We developed a novel approach for producingα-ketoglutaric acid(α-KG)from sodium L-glutamate monohydrate by a fully homogeneous coupled system of L-glutamate dehydrogenase and bridged flavinium catalyst(F4).With the aid of highly active bridged flavinium catalyst,the dosage of NAD^(+)was reduced and the product inhibition caused by NADH was partly released.Through single factor study,L-glutamate dehydrogenase from Clostrdium difficile was screened as the source of enzyme,and oxidative deamination reaction was carried out in Tris-HCl buffer solution.The yield ofα-KG was optimized by response surface method,the concentration of NAD^(+),F4,and pH value were 1 mmol/L,1 mmol/L,pH 8,respectively.At an L-glutamate concentration of 15 mmol/L,the scale-up reaction reached a maximum yield of 92%in 24 h.