Enhancement degradation efficiency of pyrethroid-degrading esterase (Est816) through rational design and its application in bioremediation

The pervasive use of pyrethroids is seriously hazardous to the environment and even human health. Enzymatic bioremediation is potentially a rapid and environmentally friendly technology to combat the pollution of pyrethroid pesticides. The hydrolysis of ester linkages is the initial and critical enzymatic step in microbial degradation pathways. Here, the versatile and thermostable esterase Est816 was cloned and its new function, pyrethroid-hydrolysis activity, was expanded. To further improve its pyrethroid-hydrolysis ability, Est816 was modified by rational design. After two rounds of mutation, the best-performing mutant, Est816A216V/K238N/M97V, was obtained, which could completely degrade 1 mg/L λ-cyhalothrin, cypermethrin, and deltamethrin within 20 min, and efficiently degrade fenvalerate, reaching over 80% conversion. Degradation activity analyses showed that three substitutions (A216V, K238 N and M97V) were beneficial for enhancing the activity of Est816. Enzymatic characterization showed that Est816A216V/K238N/M97V inherited broad substrate specificity and possessed excellent stability and adaptability over wide ranges of temperature and pH, which is essential for bioremediation in frequently changing conditions. Furthermore, Est816A216V/K238N/M97V had the best degradation effect on all four pyrethroid residues in Panax notoginseng root, with more than 87% conversion after 24 h. Pyrethroid residues in tea, cucumber, and soil were reduced by more than 76%, 80%, and 76%, respectively. Taken together, these findings highlight the great potential of Est816A216V/K238N/M97V in the bioremediation of pyrethroid-contaminated soil and agricultural products.