Enhancing Catalytic Efficiency of an Actinoplanes utahensis Echinocandin B Deacylase through Random Mutagenesis and Site-Directed Mutagenesis
2020
Cheng, Ying-Nan | Qiu, Shuai | Zheng, Feng | Weng, Chun-Yue | Wang, Ya-Jun | Zheng, Yu-Guo
Echinocandin B deacylase (EBDA), from Actinoplanes utahensis ZJB-08196, is capable of cleaving the linoleoyl group from echinocandin B (ECB), forming the echinocandin B nucleus (ECBN), which is a key precursor of semisynthetic antifungal antibiotics. In the present study, molecular evolution of AuEBDA by random mutagenesis combined with site-directed mutagenesis (SDM) and screening was performed. Random mutagenesis on the wild-type (WT) AuEBDA generated two beneficial substitutions of G287Q, R527V. The “best” variant AuEBDA-G287Q/R527V was obtained by combining G287Q with R527V through SDM, which was most active at 35 °C, pH 7.5, with Kₘ and vₘₐₓ values of 0.68 mM and 395.26 U/mg, respectively. Mutation of G287Q/R527V markedly increased the catalytic efficiency kcₐₜ/Kₘ by 290% compared with the WT-AuEBDA.
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