Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
Institute of Biotechnology, RWTH Aachen University, Aachen, Germany.
Protein Sci. 2024 Apr;33(4):e4933. doi: 10.1002/pro.4933.
Alcohol dehydrogenases (ADHs) are synthetically important biocatalysts for the asymmetric synthesis of chiral alcohols. The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of KpADH from Kluyveromyces polyspora was semi-rationally evolved. Using tolerant variants obtained, meticulous experiments and computational studies were conducted to explore properties including stability, activity and kinetics in the presence of various organic solvents. Compared with WT, variant V231D exhibited 1.9-fold improvement in ethanol tolerance, while S237G showed a 6-fold increase in catalytic efficiency, a higher , as well as 15% higher tolerance in 7.5% (v/v) ethanol. Based on 3 × 100 ns MD simulations, the increased tolerance of V231D and S237G against ethanol may be ascribed to their enhanced ability in retaining water molecules and repelling ethanol molecules. Moreover, 6.3-fold decreased K value of V231D toward hydrophilic ketone substrate confirmed its capability of retaining hydration shell. Our results suggest that retaining hydration shell surrounding KpADH is critical for its tolerance to organic solvents, as well as catalytic performance. This study provides useful guidance for engineering organic solvent tolerance of KpADH and other ADHs.
醇脱氢酶（ADHs）是手性醇不对称合成的重要合成生物催化剂。由于大多数前手性酮高度疏水，因此 ADHs 在有机溶剂中的催化性能通常很重要。在这里，我们对半理性地进化了来自聚多孢曲霉的 KpADH 的有机溶剂耐受性。利用获得的耐受变体，进行了细致的实验和计算研究，以探索包括稳定性、活性和在各种有机溶剂存在下的动力学在内的特性。与 WT 相比，变体 V231D 在乙醇耐受性方面提高了 1.9 倍，而 S237G 的催化效率提高了 6 倍，Kcat/Km 值提高了 2.6 倍，在 7.5%（v/v）乙醇中的耐受性提高了 15%。基于 3×100ns MD 模拟，V231D 和 S237G 对乙醇的耐受性增加可能归因于它们增强了保留水分子和排斥乙醇分子的能力。此外，V231D 对亲水性酮底物的 K 值降低了 6.3 倍，证实了其保留水合壳的能力。我们的结果表明，保留围绕 KpADH 的水合壳对于其对有机溶剂的耐受性以及催化性能至关重要。本研究为工程化 KpADH 和其他 ADHs 的有机溶剂耐受性提供了有用的指导。
