Abstract：
As an industrial enzyme that catalyzes the formation and cleavage of ester bonds, carboxylesterase has attracted attention in fine chemistry, pharmaceutical, biological energy and bioremediation fields. However, the weak thermostability limits their further developments in industrial applications. In this work, a novel carboxylesterase (EstF) from Streptomyces lividans TK24, belonging to family XVII, was acquired by successfully heterologous expressed and biochemically identified. The EstF exhibited optimal activity at 55 °C, pH 9.0 and excellent catalytic performances (Km = 0.263 mM, kcat/Km = 562.3 s-1 mM-1 for p-nitrophenyl acetate (pNPA2) hydrolysis). Besides, the EstF presented exceptionally high thermostability with a half-life of 387.23 h at 55 °C and 2.86 h at 100 °C. Furthermore, the EstF was modified to obtain EstFP144G using the site-directed mutation technique to investigate the effect of single glycine on thermostability. Remarkably, the mutant EstFP144G displayed a 5.10-fold increase of half-life at 100 °C versus wild-type without affecting catalytic performance. Structural analysis implied that the glycine introduction could release a steric strain and induce cooperative effects between distal residues to increase the thermostability. Therefore, the thermostable EstF and EstFP144G with prominently catalytic characteristics have potential industrial applications and the introduction of a single glycine strategy opens up alternative avenues for the thermostability engineering of other enzymes.
摘要：
作为催化酯键形成和裂解的工业酶，羧酸酯酶在精细化学中引起了人们的关注，Pharmaceutical,生物能源和生物修复领域。然而,弱的热稳定性限制了它们在工业应用中的进一步发展。在这项工作中,一种新的羧酸酯酶（EstF），来自链霉菌TK24，属于家族XVII，通过成功的异源表达和生化鉴定获得。EstF在55°C时表现出最佳活性，pH9.0和出色的催化性能（Km=0.263mM，kcat/Km=562.3s-1mM-1，对硝基苯基乙酸酯（pNPA2）水解）。此外,EstF具有极高的热稳定性，在55°C下的半衰期为387.23h，在100°C下的半衰期为2.86h。此外，使用定点突变技术对EstF进行修饰以获得EstFP144G，以研究单一甘氨酸对热稳定性的影响。值得注意的是,与野生型相比，突变体EstFP144G在100°C下的半衰期增加了5.10倍，而不影响催化性能。结构分析表明，引入甘氨酸可以释放空间应变并诱导远端残基之间的协同作用以增加热稳定性。因此,具有显著催化特性的热稳定性EstF和EstFP144G具有潜在的工业应用,并且单一甘氨酸策略的引入为其他酶的热稳定性工程开辟了替代途径。
