Abstract Ketoreductase ChKRED12 catalyzes the asymmetric bioreduction of ethyl 3-oxo-3-(2-thienyl) propanoate to produce the key chiral intermediate of duloxetine. To improve the robustness of the enzyme, we used the FireProt web server to predict potential thermostabilizing amino acid substitutions, and we experimentally confirmed 4 beneficial substitutions (S79 P, L128 M, V162I and G163A). These substitutions were combined to form a quadruple mutant (M7234), which displayed substantially enhanced thermostability with half-life of inactivation increased to 58 h at 45 °C, approximately 21-fold of that of the wild-type. Moreover, the temperature optimum increased to 45 °C, which is 5 °C higher than that of the wild-type. M7234 was able to catalyze the complete conversion of 100 g/L substrate within 8 h and produce (S)-3-hydroxy-3-(2-thienyl) propanoate at >99% ee, achieving a space-time yield of 289 g/L/d after column chromatography.

中文翻译：

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使用 FireProt Web 服务器增强酮还原酶 ChKRED12 的热稳定性

摘要 酮还原酶 ChKRED12 催化 3-oxo-3-(2-thienyl) 丙酸乙酯的不对称生物还原反应，生成度洛西汀的关键手性中间体。为了提高酶的稳健性，我们使用 FireProt 网络服务器来预测潜在的热稳定氨基酸替代，我们通过实验确认了 4 个有益的替代（S79 P、L128 M、V162I 和 G163A）。这些取代组合形成四重突变体（M7234），其显示出显着增强的热稳定性，灭活半衰期在 45°C 下增加至 58 小时，约为野生型的 21 倍。此外，最适温度增加到 45°C，比野生型高 5°C。