半理性设计提高甲酸脱氢酶(Cb FDH)活力及热稳定性

Enhanced Activity and Thermal Stability of Formate Dehydrogenase(CbFDH)via Semi-Rational Design

甲酸脱氢酶(formate dehydrogenase,FDH)是NADH循环再生的最佳酶之一,广泛应用于食品、医药和化工等行业。但是野生型甲酸脱氢酶普遍存在酶活低、催化效率差等缺点,导致产品转化率较低,影响产品的工业化生产。为了获得具有更佳催化性能的甲酸脱氢酶,作者以博伊丁假丝酵母(Candida boidinii)来源的甲酸脱氢酶为模板,利用HOTSPOT WIZARD v3.1进行三维结构模拟预测,构建了P68G、Q197K两个突变体,比酶活较野生型分别提高了11%和33%。这是由于P68G氨基酸残基侧链的苯环被氢取代,减少了甲酸盐底物进入口袋的空间位阻;而Q197K侧链酰胺基突变为胺丁基增强了酶的柔性。然而这两个突变点对甲酸脱氢酶的热稳定性产生了负面影响,因此在I239位引入半胱氨酸突变与C262构成二硫键以提高其热稳定性,最终获得一株热稳定性显著提高,比酶活较野生型提高31%、较I239C提高45%的突变株Cb FDH Q197K/I239C。通过半理性预测蛋白质结构提高了甲酸脱氢酶的活力和热稳定性,为高效构建性能稳定、还原力强的甲酸脱氢酶提供了理论基础。

Formate dehydrogenase(FDH)is one of the most effective enzymes for NADH regeneration and is extensively utilized in the food,pharmaceutical and chemical industries.However,wild-type FDH often suffers from low enzyme activity and poor catalytic efficiency,resulting in lower product conversion and hindering industrial production.In order to obtain FDH with improved catalytic performance,FDH from Candida boidinii was used as a template and HOTSPOT WIZARD v3.1 was employed for three-dimensional structure simulation and prediction.And 2 mutants,P68G and Q197K,were constructed.In comparison to the wild type,they exhibited 11%and 33%higher enzyme activity,respectively.The improved activity was attributed to the replacement of the phenyl ring on the side chain of residue P68G by hydrogen,reducing steric hindrance for formate substrate entry,while the mutation of the side chain amide group in Q197K to aminobutyl enhanced the flexibility of the enzyme.However,these 2 mutations negatively affected the thermal stability of FDH.Hence,a cysteine mutation was introduced at position I239 to form a disulfide bond with C262,thereby enhancing the thermal stability.Ultimately,the mutant strain CbFDH Q197K/1239C was obtained,which exhibited remarkable improvement in thermal stability,displaying a 31%increase in enzyme activity compared to the wild-type and a 45%increase compared to I239C.This study demonstrates that the activity and thermal stability of FDH could be enhanced through semi-rational protein structure prediction,providing a reliable theoretical foundation for the efficient construction of FDH with stable performance and strong reducing power.