The aldo‐keto reductase (AKR) KdAKR from Kluyvermyces dobzhanskii can reduce t‐butyl 6‐chloro‐(5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) to t‐butyl 6‐chloro‐(3R,5S)‐dihydroxyhexanoate ((3R,5S)‐CDHH), which is the key chiral intermediate of rosuvastatin. Herein, a computer‐aided design that combined the use of PROSS platform and consensus design was employed to improve the stability of a previously constructed mutant KdAKRM6. Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The “best” mutant KdAKRM10 (KdAKRM6‐S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T5015 and Tm values were increased by 10.2 and 10.0°C, respectively, and half‐life (t1/2) at 40°C was increased by 17.6 h. Additionally, KdAKRM10 demonstrated improved resistance to organic solvents compared to that of KdAKRM6. Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of KdAKRM10, thus improving its stability. The results validated the feasibility of the computer‐aided design strategy in improving the stability of AKRs.

中文翻译：

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计算机辅助设计提高醛酮还原酶KdAKR的稳定性

醛酮还原酶 (AKR)钾AKR 来自多布詹氏克鲁维酵母可以减少t‐丁基6‐氯‐(5S)-羟基-3-氧代己酸酯 ((5S)-CHOH) 至t‐丁基 6‐氯‐(3右,5S)-二羟基己酸酯((3右,5S)-CDHH)，是瑞舒伐他汀的关键手性中间体。在此，采用结合 PROSS 平台和共识设计的计算机辅助设计来提高先前构建的突变体的稳定性钾AKRM6。实验验证表明，S196C、T232A、V264I和V45L产生了改善的热稳定性和活性。“最好的”突变体钾AKRM10（钾AKRM6‐S196C/T232A/V264I/V45L）是通过组合四种有益突变构建的，显示出增强的热稳定性。它是时间5015和时间米值分别增加了 10.2 和 10.0°C，半衰期（t1/2）在 40°C 下增加了 17.6 小时。此外，钾AKRM10与有机溶剂相比，表现出更好的耐有机溶剂性钾AKRM6。结构分析表明，氢键数量的增加和稳定的疏水核心有助于提高其刚性钾AKRM10，从而提高其稳定性。结果验证了计算机辅助设计策略在提高 AKR 稳定性方面的可行性。