Biochemical Characterization And Molecular Modification Of A Novel Marine Mud-derived Pyrethroid Hydrolase

Pyrethroids were widely used in our daily life, accounting for more than 20% of the world pesticide market because of its bio-efficiency and safety to human beings. As pyrethroids were not completely safe for the environment, it took more and more attention to deal with the problem of residual amount. So far, the methods used to eliminate the pyrethroids were physical, chemical and biological processes. Among these methods, the biological processes, especially pyrethroids degradated by the biological enzyme, showed potential industrial application due to its environmental safety and no secondary pollution. Over the past two decades, most of the pyrethroid hydrolases were isolated from the cultural microorganisms.In this study, a novel pyrethroids hydrolase gene (est825) originating from a marine mud metagenomic library was ligated into pET-28a(+) and transformed into Escherichia coli BL21(DE3). The expression product reached to 200 mg/L after induced for 8 h at 30°C with 1.0 mM IPTG. SDS-PAGE analysis revealed that the molecular mass of recombinant Est825 was 30.1 kDa. The optimum pH and temperature of Est825 were 6.5 and 40℃, respectively. It kept more than 50% of its activity after incubated for 2 h at 70℃. The enzyme activity was increased by the presence of 1mM Al³⁺, 1% Triton X-100 and 1% Tween 80. Fortunately, Est825 could degradate cyhalothrin, cypermethrin, sumicidin and deltamethrin at 37℃for 1h with the degradation rates of 80.66%,84.95%,81.90% and 76.75%, respectively.In order to improve the ability of Est825 for pyrethroids degradation, est825 was motified by directed evolution. Random mutations were introduced through error-prone PCR (epPCR). A mutant enzyme EstM46 was obtained and its characterization was researched. Compared with Est825, the activity of EstM46 was increased by 1.5 times and its optimum temperature was increased to 45℃. EstM46 showed more thermal stability by remaining 80% of its activity after incubated at 70℃for 2 h. About 92.21% cyhalothrin, 99.75% cypermethrin, 93.21% sumicidin and 89.48% deltamethrin were hydrolyzed by EstM46 at 37℃for 1 h.The results showed that a new marine mud-oriented pyrethroid-hydrolyzing esterase Est825 degraded a variety of pyrethroids with broader substrate specificities and higher activity made it an ideal candidate for situ bioremediation where pyrethroids caused environmental contamination problems. Moreover, the degradation activity was further enhanced by epPCR, which promote its potential applications in dealing with residual pyrethroid pesticides.