Multiparameter Optimization in Directed Evolution: Engineering Thermostability, Enantioselectivity, and Activity of an Epoxide Hydrolase

摘要: The challenge of optimizing several parameters in the directed evolution enzymes remains a central issue. In this study we address thermostability, enantioselectivity, and activity limonene epoxide hydrolase (LEH) as catalyst hydrolytic desymmetrization cyclohexene oxide with formation (R,R)- (S,S)-cyclohexane-1,2-diol. Wild type LEH shows thermostability T5030 = 41 °C an enanioselectivity 2% ee (S,S). Two approaches are described herein. one strategy, mutations generated previously by Janssen, Baker, co-workers for notably increased combined evolved earlier enhanced enantioselectivity. Although highly enantioselective R,R S,S variants (92–93% ee) increases 10–11 were obtained, relative to wild tradeoff was significant. second strategy based on simultaneous optimization both using iterative saturation mutagenesis (ISM) minimized i...