Directed evolution and secretory expression of a pyrethroid-hydrolyzing esterase with enhanced catalytic activity and thermostability

摘要: Pyrethroids are potentially harmful to human health and ecosystems. It is necessary develop some efficient strategies degrade pyrethroid residues. Biodegradation generally considered as a safe, efficient, inexpensive way eliminate environmental contaminants. To date, although several pyrethroid-hydrolyzing esterases have been cloned, there has no report about hydrolase with high hydrolytic activity, good stability, productivity, indispensable enzymatic properties in practical biodegradation. Almost all hydrolases intracellular enzymes, which require complex extraction protocols present issues terms of easy inactivation low production. In this study, random mutagenesis was performed on one esterase, Sys410, enhance its activity thermostability. Two beneficial mutations, A171V D256N, were obtained by gave rise the mutant M2. The displayed ~1.5-fold improvement kcat/Km value 2.46-fold higher catalytic activity. optimal temperature 10 °C than that wild-type enzyme (55 °C). half-life at 40–65 °C 3.3–310 times longer. surprising M2 12 h 70 °C while Sys410 completely inactivated 70 °C. addition, desired gene extracellularly expressed Pichia pastoris host system. soluble expression level reached up 689.7 mg/L. Remarkably, could efficiently various pyrethroids moderate for 15 min, exceeding hydrolysis rate 98%, highest ever reported. This first secretory esterase high-level productivity purity P. pastoris. Broad substrate specificity, enhanced thermostability make an ideal candidate biodegradation