A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration
A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration
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DOI：
10.1007/s00253-005-0289-y
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发表时间：
2006-09-01
影响因子：
5
通讯作者：
Goto, Masahiro
中科院分区：
文献类型：
--
作者：
Mouri, Tsuyoshi;Michizoe, Junji;Goto, Masahiro
来自土壤细菌恶臭假单胞菌的细胞色素 P450cam 单加氧酶 (P450cam) 系统需要三种不同蛋白质和辅因子烟酰胺腺嘌呤二核苷酸 (NADH) 之间的电子转移，以对其天然底物樟脑进行氧化。在此，我们报告了一种简便的方法，通过将 P450cam 系统的天然电子转移系统与大肠杆菌全细胞生物催化剂中甘油脱氢酶 (GLD) 催化的酶促 NADH 再生相结合，显着提高 P450cam 系统的催化效率。含有 P450cam 系统但缺乏 GLD 的重组大肠杆菌表现出很少的樟脑羟基化活性。相比之下，GLD 与 P450cam 的蛋白质电子转移成分共表达导致底物转化率提高约十倍，这意味着全细胞生物催化剂利用细胞中的分子氧、内源性 NADH 和甘油进行催化。向反应介质中添加甘油进一步促进了樟脑羟基化，表明外源甘油也可用于宿主细胞中的 GLD 并积极参与催化循环。这些结果清楚地表明了 GLD 在原生 P450cam 系统功能重建方面的实用性。本方法也可用于具有其他 NADH 依赖性加氧酶和氧化还原酶的大肠杆菌全细胞生物催化剂。
A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase (GLD) in Escherichia coli whole cell biocatalysts. Recombinant E. coli harboring the P450cam system, but lacking GLD, exhibited little activity for camphor hydroxylation. In contrast, coexpression of GLD with the proteinaceous electron transfer components of P450cam resulted in about tenfold improvement in the substrate conversion, implying that the whole cell biocatalyst utilized molecular oxygen, endogenous NADH, and glycerol in the cell for catalysis. The addition of glycerol to the reaction media further promoted camphor hydroxylation, suggesting that exogenous glycerol is also available for GLD in the host cell and actively participates in the catalytic cycle. These results clearly show the utility of GLD towards functional reconstruction of the native P450cam system. The present approach may also be useful for E. coli whole cell biocatalysts with the other NADH-dependent oxygenases and oxidoreductases.