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首页> 外文期刊>Biotechnology Progress >Design of a Cytochrome P450BM3 Reaction System Linked by Two-Step Cofactor Regeneration Catalyzed by a Soluble Transhydrogenase and Glycerol Dehydrogenase
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Design of a Cytochrome P450BM3 Reaction System Linked by Two-Step Cofactor Regeneration Catalyzed by a Soluble Transhydrogenase and Glycerol Dehydrogenase

机译：可溶性转氢酶和甘油脱氢酶催化的两步辅因子再生联动的细胞色素P450BM3反应系统的设计

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A cytochrome P450BM3-catalyzed reaction system linked by a two-step cofactor regeneration was investigated in a cell-free system. The two-step cofactor regeneration of redox cofactors, NADH and NADPH, was constructed by NAD~+-dependent bacterial glycerol dehydrogenase (GLD) and bacterial soluble transhydrogenase (STH) both from Escherichia coli. In the present system, the reduced cofactor (NADH) was regenerated by GLD from the oxidized cofactor (NAD~+) using glycerol as a sacrificial cosubstrate. The reducing equivalents were subsequently transferred to NADP~+ by STH as a cycling catalyst. The resultant regenerated NADPH was used for the substrate oxidation catalyzed by cytochrome P450BM3. The initial rate of the P450BM3-catalyzed reaction linked by the two-step cofactor regeneration showed a slight increase (approximately twice) when increasing the GLD units 10-fold under initial reaction conditions. In contrast, a 10-fold increase in STH units resulted in about a 9-fold increase in the initial reaction rate, implying that transhydrogena-tion catalyzed by STH was the rate-determining step. In the system lacking the two-step cofactor regeneration, 34% conversion of 50 μM of a model substrate (p-nitrophenoxydeca-noic acid) was attained using 50 μM NADPH. In contrast, with the two-step cofactor regeneration, the same amount of substrate was completely converted using 5 μM of oxidized cofactors (NAD~+ and NADP~+) within 1 h. Furthermore, a 10-fold dilution of the oxidized cofactors still led to approximately 20% conversion in 1 h. These results indicate the potential of the combination of GLD and STH for use in redox cofactor recycling with catalytic quantities of NAD~+ and NADP~+.
机译：在无细胞系统中研究了由两步辅因子再生连接的细胞色素P450BM3催化的反应系统。氧化还原辅因子NADH和NADPH的两步辅因子再生是由来自大肠杆菌的NAD〜+依赖性细菌甘油脱氢酶（GLD）和细菌可溶性转氢酶（STH）构建的。在本系统中，还原甘油辅酶（NADH）是由GLD使用甘油作为牺牲性辅助底物从氧化辅因子（NAD〜+）再生的。还原当量随后通过STH作为循环催化剂转移至NADP +。生成的再生NADPH用于细胞色素P450BM3催化的底物氧化。当在初始反应条件下将GLD单元增加10倍时，通过两步辅因子再生连接的P450BM3催化的反应的初始速率显示出轻微的增加（大约两倍）。相反，STH单位增加10倍导致初始反应速率增加约9倍，这意味着由STH催化的转氢反应是决定速率的步骤。在缺少两步辅因子再生的系统中，使用50μMNADPH可实现50μM模型底物（对硝基苯氧基癸酸）的34％转化。相反，通过两步辅助因子再生，在1小时内使用5μM氧化辅助因子（NAD〜+和NADP〜+）完全转化了相同量的底物。此外，氧化辅因子的10倍稀释仍可在1小时内导致约20％的转化率。这些结果表明，GLD和STH的组合具有催化量的NAD〜+和NADP〜+用于氧化还原辅因子回收的潜力。

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