School of Life Sciences and Biopharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe, Shenyang, 110016, People's Republic of China.
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe, Shenyang, 110016, People's Republic of China.
Enzyme Microb Technol. 2021 Oct;150:109880. doi: 10.1016/j.enzmictec.2021.109880. Epub 2021 Jul 29.
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.
烯还原酶（ERs）来源于古老的黄色酶（OYEs）家族，具有将活化的烯烃还原生成多达两个手性中心的能力，因此它们作为强大的生物催化剂受到了广泛关注。在这项研究中，通过基因挖掘，从根瘤固氮菌、荧光假单胞菌和恶臭假单胞菌的基因组中鉴定出了四种 ERs。鉴定了这四种 ERs 的生物催化特性，并进一步评估了它们在二氢香芹酮和丙酰衍生物合成过程中的应用。其中，属于经典 OYEs 的三种 ER（EaER2、PvER1 和 PvER2）在 30°C 和 pH 7.0（100mM 磷酸钾缓冲液）下表现出最佳的催化活性，属于嗜热样 OYEs 的 PfER2 在 40°C 和 pH 7.0（100mM 磷酸钾缓冲液）下表现出最佳的催化活性。在探索有机溶剂对催化效率的影响时，发现这四种 ER 对甲苯更敏感，并且对几种其他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% de。EaER2 和 PfER2 可以催化多种丙酰衍生物的合成，立体选择性超过 99%ee。此外，通过同源建模和分子对接，我们初步解释了这四种 ER 催化活性和立体选择性的机制，为今后合理设计其立体偏好提供了坚实的基础。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 的生物催化工具包。我们对这四种 ER 酶学性质的探索，为今后的研究和产业化进程提供了充分的数据基础。
