Expression And Hydrolytic Activities Of Carboxylesterases From Helicoverpa Armigera

Carboxylesterases（CCEs） are a major class of enzymes involved in the detoxification oforganophosphates （OPs）, synthetic pyrethroids （SPs） and carbamates （CBs） in insects. Theaim of this research is to elucidate the role of CCEs from H. armigera in the development ofmetabolic resistance to these chemical insecticides. Furthermore, identification of pesticidesdegrading enzymes from cotton bollworm will have its application in bioremedation ofcontaminated environment. Nine CCEs genes from GR strain of H. armigera weresuccessfully expressed in the Sf9cells using the Baculovirus Expression Vector System（BEVS）. The hydrolysis activities of eight CCEs against OPs and SPs were measured andanalyzed so further in term with molecular and biochemical methods. In addition, a total18mutants of CCEs were maken using the site-directed mutagenesis technique, the kineticefficiency of the CCEs and the effects of the mutations on the catalytic activities toward OPsand SPs were studied. Lastly, following above the gentic engineering bacteria strain E.coliBL21were constructed for potential hydrolytic degradation of insecticides. The significancesof this research were showed as below:1. Baculovirus expression and esterase activity assay of CCEs in vitroNine CCEs genes were cloned from the cDNA library of the H. armigera GR strain.Alignment of anmino acid sequences showed that they all contain the critical motifs in theactive centre: the catalytic triad （S-E-H）, the conservative pentapeptide （GxSxG） and the highconservative oxyanion hole （G-G-A）. It has shown they should have hydrolytic activitiestoward some insecticides. Baculovirus expression system was used to produce native folded（active form） protein of CCEs. The expressed CCEs were analyzed by native-PAGE andstained with artificial substrate α-naphthyl acetate （α-NA）, the resulting stained bands （closelytogether） indicated that the relative mobility （Rm） of all CCEs fell in two zones （0.27～0.36and0.37～0.48）, which thus has shown most CCEs are probabaly associated with metabolicresistance. The kinetic assay with α-NA and p-NA were carried out for all CCEs using amicroplate reader. Eight of nine CCEs showed hihger affinity to α-NA and their Km valuewere between3.3μmol/L and671μmol/L. The rate constants （kcat/Km value） varied from0.3μmol^-1·S-1·L to95μmol^-1·S-1·L. The eight CCEs showed relatively lower hydrolyticactivities against para-nitrophyl acetate （p-NA） compared with those of α-NA. 2. Hydrolysis of organophosphrous insecticides by CCEs expression in vitroThe molar concentration of esterase protein in the Sf9cell extracts and the OP hydrolysiswere determined fluorometrically by using diethyl-and dimethyl-phosphates （dECP anddMUP） in term with the enzymatic reaction. The results showed the kinetic constants （kcatvalue） varied from1.3×10-3min^-1to15.8×10-3min^-1and resembled those of the E3controlfrom Lucilia cuprina. Although the eight CCEs had hydrolytic activities toward OPs, noclosed correlations between the OPs hydrolysis and the carboxylesterase activity were found.3. Hydrolysis of individual isomers of fenvalerate and cypermethrin insecticides byCCEsThe catalytic activities of the CCEs toward individual SP insecticides and analogs weremeasured with fluorometric and HPLC assays repectively. The reuslts showed four of eightCCEs （001B,001C,001D and001J） had relatively higher hydrolytic activities toward twoisomers2（R）-α（R） and2（R）-α（S） of fenvalerate analogs, and001G had some activities againstesfenvalerate2（S）-α（S）(13.19μmol·min^-1·μmol^-1). In contrast with001G others hadnegligible activities. The same four CCEs also were observed higher activities toward thelight insecticidal1S trans cypermethrin isomers (17μmol·min^-1·μmol^-143μmol·min^-1·μmol^-1)as well as the corresponding analogs. But their hydrolytic activities for the most insecticidalisomer1（R）cis-α（S） of cypermethrin and its analog were relatively lower. Other four CCEsshowed even lower or undetectable activities toward the isomers of cypermethrin.4. Construction of mutant CCEs and their kinetic properties for model substratesOne or two amino acids were introduced to mutate in oxyanion hole （A/G to D） and acylbinding pocket （F to L） in the CCEs using the site-directed mutagenesis technique to make18mutant enzymes. The results of kinetic assay with a-NA showed that the single point mutationin oxyanion hole had dramatically reduced the esterase activities. The constants Km values ofthese mutants were generally higher than that of the wild type enzymes and the rate constantskcat/Km were between20-and437-fold lower than that of the wild type enzymes. By contrast,the single point mutations in acyl binding pocket had relatively less effects on the kinceticefficiencies of CCEs than that of the A to D mutants. The kcat/Km values were between1.5-and5-fold lower than that of the wild type enzymes. The double point mutations at both sitesnearly nullified the carboxylesterase activities. In line with the pattern of kinetic assay witha-NA, the mutations also markedly reduced the activities toward p-NA.5. The effects on hydrolysis of OPs by two point mutations of CCEsThe amino acid mutations had marked effects on the hydrolytic activities of CCEstoward dimethyl-and diethyl-phosphates. Some mutants increased hydrolytic activites, but others reduced the hydrolysis. Among the CCEs modified in oxyanion hole, some obviouslyenhanced the hydrolysis of OPs, in particular, the kcat value for dECP of001CA127Dwas32.8×10-3min^-1. By contrast, the F to L mutations in acyl binding pocket showed enhancedhydrolytic activites for relatively smaller substrate dimethylphosphates, the kcat values wereat least1.5-fold higher than that of the wild type enzyme, which were also higher comparedwith the relatively larger substrate diethylphosphates. It’s shown the dimethylphosphates wasas the preferred substrate in the hydrlolysis. The A to D mutations combined with F to Lmutations almost all enhanced the hydrolysis of OPs to some extent.6. The effects of mutations on the hydrolytic activities of CCEs against individual SPsinsecticidesThe catalytic activities toward individual SP insecticides and analogs of the mutant CCEswere also measured with fluorometric and HPLC assays. Among all the mutants, just threemutants (001B^F236L,001D^F235Land001G^F238L) showed some hydrolytic activities against the4isomers of envalerate analogs, but the hydrolysis were lower compared to those of the wildtype CCEs with the exception of the001BF236L.001BF236Lshowed an enhancement ofhydrolysis of2（S）-α（S） isomer analog compared to its wild type. In line with that thismutation increased the activity about4-fold higher for the most insecticidal2（S）-α（S） isomer.Others reduced the hydrolytic activities toward this most insecticidal isomer.The CCEs modified in oxyanion hole had undetectable activities toward any of thecypermethrin analogs, and also showed lowest activities toward most of the isomers ofcypermethrin insecticides. By contrast, three of five CCEs modified in the acyl binding pocket(001B^F236L,001D^F235Land001J^F236L) showed enhancement of hydrolytic activities toward thelight insecticidal1（S） trans isomers of cypermethrin insecticide than that of the wild typeCCEs. The turnover for the1（S） trans-α（R） isomer were20μmol·min^-1·μmol^-1,43μmol·min^-1·μmol^-1and51μmol·min^-1·μmol^-1respectiviely. In particular, the turnover for themost insecticidal1（R） cis-α（S） isomer by001D^F236Land001J^F238Lwere9.3μmol·min^-1·μmol^-1and15.6μmol·min^-1·μmol^-1respectively. All three double point mutations in CCEs had verylower or nearly nullifiled the activities toward both the cypermethrin analogs and the isomers.7. Construction of genetic engineering bacteria with degradation CCEs and itsexpressionThe N-terminal signal peptide sequence were removed from three CCEs and their codingregions were amplification with new designed primers. They were cloned into an expressionvector PETMCS III and transferred into the E.coli BL21cells. The CCEs genes wereexpressed in E.coli BL21at18,28and37degree respectively to yield the CCEs protein. The CCEs expressing BL21（DE3） cells were lysed and the soluble and insoluble proteins wereseparated. Both supernatants and precipitates of cell harvests from the cell cultures wereanalyzed by SDS-PAGE. The results showed that the higher expression level was found whenthe cells were grown at37degree. However, nearly all the over-expressed CCEs wereexpressed as insoluble protein （in precipitates） and very little enzyme were in solubale andactive. Future work will focus on improving correct folding of CCEs in E.coli BL21cellsbefore they can be applied for removing pesticide contamination in the environment. Currentresults provided useful data for further devolopment of degradation enzymes using genticlymicrobial engineering.In summary, CCEs from H. armigera GR strain contain the active sites of typicalhydrolase. All CCEs were successfully expressed in Baculovirus Expression Vector System.Their hydrolytic activities were assayed and the results showed eight CCEs had somehydrolysis activities to some extent toward OPs and SPs insecticides. The kinetic results ofthe substitution of amino acids in oxyanion hole or acyl binding pocket had significant effectson the the enzymatic activities, in which some really showed enhancement of the hydrolysisof OP and the type II SP insecticides. The results demonstrated that the CCEs hydrolaseactivity toward insecticides could be enhanced through mutations at around enzyme activesites. It’s thus shown the mutation in active site of CCEs will play important role for thedeveolepment of OP and SP insecticides resistance in H. armigera in future. The results alsoindicated there are great potentials for using the CCEs of H. armigera to develop geneticengineering bacterial for detoxificaiton in contaminated enviroment.