In this work, we have identified a significantly improved variant (S131Y/Q252I) of the natural ϵ-keto ester reductase CpAR2 from Candida parapsilosis for efficiently manufacturing (R)-8-chloro-6-hydroxyoctanoic acid [(R)-ECHO] through co-evolution of activity and thermostability. The activity of the variant CpAR2S131Y/Q252I towards the ϵ-keto ester ethyl 8-chloro-6-oxooctanoate was improved to 214 U mg-1 -from 120 U mg-1 in the case of the wild-type enzyme (CpAR2WT )-and the half-deactivating temperature (T50 , for 15 min incubation) was simultaneously increased by 2.3 °C in relation to that of CpAR2WT . Consequently, only 2 g L-1 of lyophilized E. coli cells harboring CpAR2S131Y/Q252I and a glucose dehydrogenase (GDH) were required in order to achieve productivity similar to that obtained in our previous work, under optimized reaction conditions (530 g L-1  d-1 ). This result demonstrated a more economical and efficient process for the production of the key (R)-α-lipoic acid intermediate ethyl 8-chloro-6-oxooctanoate.

中文翻译：

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Better-酮酯还原酶的活性和热稳定性共同进化，可以更好地合成（R）-α-硫辛酸前体。

在这项工作中，我们确定了来自准假丝酵母的天然β-酮酯还原酶CpAR2的显着改良变体（S131Y / Q252I），可有效生产（R）-8-氯-6-羟基辛酸[（R）-ECHO]通过活性和热稳定性的共同进化。对于野生型酶（CpAR2WT），变体CpAR2S131Y / Q252I对8-氯-6-氧代辛酸β-酮酯的活性从120 U mg-1提高到214 U mg-1--与CpAR2WT相比，半灭活温度（T50，孵育15分钟）同时提高了2.3°C。因此，仅需2 g L-1的冻干大肠杆菌细胞（带有CpAR2S131Y / Q252I）和葡萄糖脱氢酶（GDH），即可达到与我们以前的工作相似的生产率，在优化的反应条件下（530 g L-1 d-1）。该结果证明了生产关键的（R）-α-硫辛酸中间体8-氯-6-氧辛酸乙酯的经济和有效的方法。