The ene reductases (ERs) from the old yellow enzymes (OYEs) family have the ability to reduce activated alkenes to generate up to two stereocenters, therefore they have been received extensive attention as powerful biocatalysts. In this study, through gene mining, four ERs were identified from the genomes of Ensifer adhaerens, Pseudomonas fluorescens, and Pseudomonas veronil. The biocatalytic properties of these four ERs were identified, and their applications in the synthesis process of dihydrocarvone and profen derivatives were further evaluated. Among them, three ERs (EaER2, PvER1, and PvER2) belonging to the classic OYEs showed the best catalytic activity at 30 °C and pH 7.0 (100 mM potassium phosphate buffer) and the PfER2, which belongs to the thermophilic-like OYEs exhibited the best catalytic at 40 °C and pH 7.0 (100 mM potassium phosphate buffer). When exploring the influence of organic solvents on the catalytic efficiency, it was found that the four ERs were more sensitive to toluene and had tolerance to several other selected organic solvents. In addition, EaER2, PfER2, PvER1 and PvER2 showed excellent catalytic activity toward carvone, and the stereoselectivity of PvER2 toward carvone could reach up to 88.7 % de. EaER2 and PfER2 can catalyze the synthesis of a variety of profen derivatives with a stereoselectivity over 99 % ee. Moreover, through homology modeling and molecular docking, we preliminarily explained the mechanism of catalytic activity and stereoselectivity of the four ERs, which provided a solid base on the rational design of their stereo-preference in the future. The discovery of EaER2, PfER2, PvER1, and PvER2 provides four new enzyme sources for the study of the OYEs family and enriches the biocatalytic toolbox of ERs. Our exploration of the enzymatic properties of these four ERs will provide the sufficient data basis for future research and industrialization progress.

来自老黄酶 (OYE) 家族的烯还原酶 (ER) 能够还原活化的烯烃以产生多达两个立体中心，因此它们作为强大的生物催化剂受到了广泛关注。本研究通过基因挖掘，从Ensifer adhaerens、Pseudomonas fluorescens和Pseudomonas veronil的基因组中鉴定出4个ERs. 确定了这四种 ER 的生物催化性能，并进一步评估了它们在二氢香芹酮和苯丙芬衍生物合成过程中的应用。其中，属于经典 OYE 的三种 ER（EaER2、PvER1 和 PvER2）在 30°C 和 pH 7.0（100 mM 磷酸钾缓冲液）下表现出最佳催化活性，而属于嗜热类 OYE 的 PfER2 表现出最好的催化活性。在 40 °C 和 pH 7.0（100 mM 磷酸钾缓冲液）下具有最佳催化效果。在探索有机溶剂对催化效率的影响时，发现四种 ER 对甲苯更敏感，并且对其他几种选定的有机溶剂具有耐受性。此外，EaER2、PfER2、PvER1和PvER2对香芹酮表现出优异的催化活性，PvER2对香芹酮的立体选择性可达88。7% 德。EaER2 和 PfER2 可以催化合成多种 profen 衍生物，立体选择性超过 99% ee。此外，通过同源建模和分子对接，我们初步解释了四种ERs的催化活性和立体选择性的机理，为今后对其立体偏好的合理设计提供了坚实的基础。EaER2、PfER2、PvER1和PvER2的发现为OYEs家族的研究提供了四种新的酶源，丰富了ERs的生物催化工具箱。我们对这四种ER的酶学性质的探索将为未来的研究和产业化进程提供充分的数据基础。通过同源建模和分子对接，我们初步解释了四种ERs的催化活性和立体选择性的机理，为今后对其立体偏好的合理设计提供了坚实的基础。EaER2、PfER2、PvER1和PvER2的发现为OYEs家族的研究提供了四种新的酶源，丰富了ERs的生物催化工具箱。我们对这四种ER的酶学性质的探索将为未来的研究和产业化进程提供充分的数据基础。通过同源建模和分子对接，我们初步解释了四种ERs的催化活性和立体选择性的机理，为今后对其立体偏好的合理设计提供了坚实的基础。EaER2、PfER2、PvER1和PvER2的发现为OYEs家族的研究提供了四种新的酶源，丰富了ERs的生物催化工具箱。我们对这四种ER的酶学性质的探索将为未来的研究和产业化进程提供充分的数据基础。PvER2 为 OYEs 家族的研究提供了四种新的酶源，丰富了 ERs 的生物催化工具箱。我们对这四种ER的酶学性质的探索将为未来的研究和产业化进程提供充分的数据基础。PvER2 为 OYEs 家族的研究提供了四种新的酶源，丰富了 ERs 的生物催化工具箱。我们对这四种ER的酶学性质的探索将为未来的研究和产业化进程提供充分的数据基础。