Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO2. The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum (CtFDH) with enhanced reduction activity in the conversion of CO2 into formic acid. Four libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous CO2(g) and HCO3−. The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (kcat/KM) compared to that of the wild-type for HCO3− reduction. The improved biocatalysts were also applied as a coupled cofactor recycling system in the enantioselective oxidation of 4-phenyl-2-propanol catalysed by the alcohol dehydrogenase from Streptomyces coelicolor A3 (ScADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-type CtFDH. Two variants presenting up to five-fold increase in catalytic efficiency and kcat were obtained and characterised. They constitute a promising enzymatic alternative for CO2 utilization and will serve as scaffolds to be further developed in order to meet industrial requirements.

中文翻译：

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来自嗜热毛壳菌的工程甲酸脱氢酶，一种用于生物技术 CO2 固定的有前途的酶解

甲酸脱氢酶 (FDH) 是 NAD(P)H 依赖性酶，可催化甲酸可逆氧化为 CO2。主要目标是使用定向进化从嗜热毛壳菌 (CtFDH) 中获得 FDH 的变体，在将 CO2 转化为甲酸时具有增强的还原活性。针对活性位点中的五个残基构建了四个文库。我们鉴定了两种具有增强还原活性的变体（G93H/I94Y 和 R259C），其特征在于同时存在 CO2(g) 和 HCO3-。与野生型相比，A1 变体 (G93H/I94Y) 的催化效率 (kcat/KM) 增加了 5.4 倍，以减少 HCO3-。改进的生物催化剂还被用作耦合辅因子再循环系统，用于由天蓝色链霉菌 A3 (ScADH) 的醇脱氢酶催化的 4-苯基-2-丙醇的对映选择性氧化。当使用 A1 变体代替野生型 CtFDH 时，这些反应中的转化率从 56% 增加到 91%。获得并表征了催化效率和 kcat 增加多达五倍的两种变体。它们构成了二氧化碳利用的有前途的酶替代品，并将作为进一步开发的支架以满足工业要求。获得并表征了催化效率和 kcat 增加多达五倍的两种变体。它们构成了二氧化碳利用的有前途的酶替代品，并将作为进一步开发的支架以满足工业要求。获得并表征了催化效率和 kcat 增加多达五倍的两种变体。它们构成了二氧化碳利用的有前途的酶替代品，并将作为进一步开发的支架以满足工业要求。