Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul 04310, Republic of Korea.
Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul 04310, Republic of Korea; Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.
Int J Biol Macromol. 2019 Sep 1;136:1042-1051. doi: 10.1016/j.ijbiomac.2019.06.108. Epub 2019 Jun 21.
Cold-active enzymes with distinctive properties from a psychrophilic Exiguobacterium antarcticum B7 could be excellent biocatalysts in industrial and biotechnological processes. Here, the characterization, immobilization, and site-directed mutagenesis of a novel cold-active acetylesterase (EaAcE) from E. antarcticum B7 is reported. EaAcE does not belong to any currently known lipase/esterase family, although there are some sequence similarities with family III and V members. Biochemical characterization of EaAcE was carried out using activity staining, mass spectrometry analysis, circular dichroism spectra, freeze-thaw experiments, kinetic analysis, acetic acid release assays, and enantioselectivity determination. Furthermore, immobilization of EaAcE using four different approaches was explored to enhance its thermal stability and recyclability. Based on a homology model of EaAcE, four mutations (F45A, S118A, S141A, and T216A) within the substrate-binding pocket were investigated to elucidate their roles in EaAcE catalysis and substrate specificity. This work has provided invaluable information on the properties of EaAcE, which can now be used to understand the acetylesterase enzyme family.
来自嗜冷极端古菌 Antarcticimicrobium antarcticum B7 的具有独特性质的低温活性酶可能是工业和生物技术过程中的优秀生物催化剂。本文报道了一种新型低温活性乙酰酯酶（EaAcE）的特性、固定化和定点突变。EaAcE 不属于任何已知的脂肪酶/酯酶家族，尽管与家族 III 和 V 成员有一些序列相似性。通过活性染色、质谱分析、圆二色光谱、冻融实验、动力学分析、乙酸释放测定和对映选择性测定对 EaAcE 的生化特性进行了研究。此外，还探索了四种不同方法固定 EaAcE 以提高其热稳定性和可重复使用性。基于 EaAcE 的同源模型，研究了底物结合口袋内的四个突变（F45A、S118A、S141A 和 T216A），以阐明它们在 EaAcE 催化和底物特异性中的作用。这项工作为 EaAcE 的特性提供了宝贵的信息，现在可以用来了解乙酰酯酶酶家族。
