Clonging And Prokaryotic Expression Of Cypermethrin Degrading Enzyme From Pseudomonas Aeruginosa GF31

Pyrethroids are a class of highly efficient,broad-spectrum insecticides that have evolved from natural pyrethrin for two generations.Due to its extensive use,It is urgent for us to deal with the problem it bring to ecosystem and people's health.Microbial degradation is currently the most effective methods to degrade pyrethroids.In recent years,study on degradation of pyrethroids at home and abroad is mostly concentrated in the screening of degrading microorganisms,degrading characteristics and degrading pathways.While studies on cloning and expression of pyrethroids degrading enzymes,molecular modification or the degrading mechanism are ralely.This research takes Pseudomonas aeruginosa GF31 preserved in our laboratory which has the ability of degrading pyrethroids as the research object.According to the amino acid sequence obtained from measuring the purified pyrethroids degrading enzyme,we have blast its homologous sequences and consequently determined to use the deduced aminopeptidase from PAO1 as template to clone cypermethrin degrading enzyme from GF31,named it APs.According to the analysis of amino acid sequence of APs from SMART datebase,we cloned four different length of genes according to its structure,namely ORF?SgI?PA?Pep,with the length of 1611?1503?1014?645 by,respectively.Finally,we constructed the APs gene with plasmids pET28a/pET30a/pET32a and transformed the expression plasmids to host cells like BL21(DE3).Rosseta(DE3)and Rosseta-gami,formed the engineering bacteria.We explored the condition for expression cypermethrin degrading enzyme with the engineering bacteria.The enzyme was found to be expressed in inclusion bodies in BL21(DE3)and Rosetta(DE3),expressed in soluble form in Rosseta-gami.We optimized the condition from reducing the concentration of IPTG or adding sorbitol to the medium,while the dilemma did not change.We purified the soluble enzyme with Nickel column and collected the purified enzyme for degrading cypermethrin and Leu-pNA.The result show that the purified enzyme can degrade cypermethrin at 60 ? with a efficiency of 12.7%in 24h.The degrading enzyme belongs to aminopeptidase of protease and the aminopeptidase activity toward Leu-pNA is 1255.2U/mg.In order to study the expression level of the target gene,we constructed standard plasmids of target and reference genes and used them for qRT-PCR,resulted the relative standard curve of each.The curve equation with R~2>0.99 and amplification efficiency between 90%and 100%,which means we have successfully established the relatively qRT-PCR methods.We used it to measure the expression level of APs gene in genetically engineered bacterium in comparion to GF31.