Prokaryotic Expression Of Carboxylesterases CarE001C From Helicoverpa Armigera And Hydrolytic Activity Of Mutant Esterases

Helicoverpa armigera is a major pest of many crops, its have serious resistance to the organophosphates(Ops) and synthetic pyrethroids(SPs). The aim of this study is to understand the connection between metabolic resistance and carboxylesterase from H.armigera, as well as the molecular mechanism of metabolic resistance. We established the carboxylesterase prokaryotic expression system by using three different vectors in prokaryotic expression and optimized purification conditions of recombinant protein. Carboxylesterase gene CarE001 C from GR strain of H.armigera and mutant CarE001CA127 D were successfully expressed in this prokaryotic expression system. The hydrolatic activity of purified proteins against α-NA and real pesticides were analyzed by using a spectrophotometer and HPLC. Furthermore, 7 single-site mutants and 6 multi-site mutants of CarEs were constructed by using the site-directed mutagenesis technique, the effects of amino acid mutant on catalytic activities of esterase toward pesticides were analyzed. Our results will be useful to integrated resistant pest management and bioremediation. The main results of this study were obtained as below:1.Compared of different carboxylesterase prokaryotic expression systemCarboxylesterase gene CarE001 D cloned by H.armigera(WH) from Wuhan was successfully expressed in prokaryotic expression systems using different expression vectors. Firstly,the expression products were purified by different concentrations of the imidazole elution buffer, the result of SDS-PAGE told us concentration of 50 mM and 80 mM imidazole elution buffer was more suitable for purified expression products. Secondly, the results of SDS-PAGE and Western-Blot of three purified protein illustrated that vector pET30a(+) can be obtained purity and high concentration of carboxylesterase. Finally, the hydrolytic activity of CarE001 D from different expression vector toward β-cypermethrin were measured with HPLC. The results showed there was some difference in.hydrolytic activity of the esterases expressed by different expression vectors, and esterase expressed by vector pET30a(+) had higher hydrolytic activity toward β- cypermethrin(0.40 μM·min-1·μM-1).2.Prokaryotic expression and hydrolysis activity assay by carboxylesterase CarE001 C from H.armigeraThe recombinant plasmid pET30a/001 C was successfully expressed in vitro by prokaryotic expression system was established, while the recombinant plasmid pET30a/001CA127 D as the control of expression.The purified CarE001 C and CarE001CA127 D from p ET30a(+) showed only a single band of approximately 67 kDa on the Western-Blot, in good agreement with the theoretical molecular mass of 6×His/S-tag/CarE001 C. Meanwhile, the kinetic assay with α-NA were carried out for CarE001 C and CarE001CA127 D using a microplate reader. The rate constants( kcat/Km value) of CarE001 C was 0.002 μM-1·s-1. Then the hydrolytic activity of CarE001 C and CarE001CA127 D toward insecticide were measured with HPLC assays repectively, the results showed CarE001 C has certain activity against pyrethrum and organophosphorus, and CarE001CA127 D has no activity toward β-cypermethrin, but it has higher hydrolytic activity toward chlopyrifos(1.342 μM·min-1·μM-1). The experimental results indicated the prokaryotic expression system was established are suitable for the carboxylesterase gene from H.armigera. and the preparation of active products.3.Construction of mutants CarE001 C and the hydrolytic activities of mutant esterase against SPs7 single-site mutants of CarEs were maken using the QuikChange site-directed mutagenesis technique. Then its were successfully expressed in vitro by prokaryotic expression system was established. And the results of kinetic assay with α-NA showed that the hydrolytic activity of mutant esterases were lower than CarE001 C. The result of HPLC showed the hydrolytic activity mutant esterases CarE001CF276 L and CarE001CH423 I towards fenvalerate higher than the activity of CarE001 C. It indicated the mutation of amino acids in carboxylesterase gene can enhance the hydrolytic activities of esterases to insecticide.4.Construction of multi-site mutants CarE001 C and the hydrolytic activities of mutant esterase against OPs6 multi-site mutants of CarEs were maken using the QuikChange multi site-directed mutagenesis technique. Then its were also successfully expressed in vitro by prokaryotic expression system was established. And the results of kinetic assay with α-NA showed that mutant esterases had lost the hydrolytic activity toward α-NA. The result of HPLC showed the hydrolytic activity mutant esterases of OP4-1, OP4-2 and OP5 was 1.5~2.5 times than the activity of CarE001 C toward chlopyrifos.In summary, this paper compare three prokaryotic expression system to establish a more stable and efficient preparation for carboxylesterase expression from H.armigera, and it was detacred by gene CarE001 C and CarE001CA127 D. On this basis, 7 single-site mutants and 6 multi-site mutants of CarEs were expressed in vitro by prokaryotic expression system. And the mutant esterases CarE001CF276 L and CarE001CH423 I significantly increased the metabolic activity of carboxylesterase toward fenvalerate. And the mutant esterases OP4-1, OP4-2 and OP5 increased the metabolic activity of carboxylesterase toward chlopyrifos.The results of this study demonstrate the metabolic activity of carboxylesterase from H.armigera was improved by one amino acids mutation. On the one hand, the result indicated may H.armigera enhance its ability against insecticides by mutation of carboxylesterase in the future, on the other hand the result also shows that the carboxylesterase of some insects including H.armigera had the potential to develop detoxifying enzymes in bioremediation of contaminated environment.