Isolation,Identification And Degrading Genes Cloning Of Picoxystrobin-Degrading Bacteria

Picoxystrobin is a typical fungicide of synthetic methoxyacrylate with the merits of high efficiency,bread spectrum and wide applicability.The problem of picoxystrobin residue in ecosystem was increasing concerned for its residue rapid increasing.Microbial degradation is an potential method for cleaning the fungicides residue such as methoxyacrylate in ecosystem.The picoxystrobin-degrading bacterial strain PY3 was isolated by selective medium of picoxystrobin as sole carbon source.Strain PY3 was identified as Rhodopseudomonas palustris based on physiological and biochemical characteristics and phylogenetic analysis.The optimal picoxystrobin degradation conditions of strain PY3 were both 35 °C and pH 6.0,respectively.Strain PY3 degraded upto 72.0 % of 50 mg/L picoxystrobin in optimal degradation conditions.The metabolic products of picoxystrobin degraded by strain PY3 was scanned and identified by GC/MS for deducing the degradation pathway of picoxystrobin.The picoxystrobin was degraded through braking and esterificating of the oxo-bridging band between benzene ring and N heterocyclic ring,and/or opening the oxo-bridging ring and benzene ring.The degrading proteins of strain PY3 degrading picoxystrobin wwere screened by esterase activation staining plus time of flight mass spectrometer.There were 5 candidate proteins screened.All five candidate genes were cloned using PCR based on genome sequence of Rhodopseudomonas.The recombinant condidate degrading genes(GST tag)was prokaryotic expression,the recombinant proteins were purified by glutathione agarose affinity chromatography.There were 4 candidate proteins were successfully purified including P-RPA1879,P-RPA1844,P-RPA1745 and P-gloB,molecular weight was 65.7 kDa,77.4 kDa,71.3 k Da,and 54.7 kDa,respectively.The capacity of degrading picoxystrobin of 4 candidate proteins were 0.62,0.66,0.59 and 0.72 mg/(mg·min)(picoxystrobin/protein·min).This study was scientifically contributed to bio-remediation of picoxystrobin residues,and also provided novel degrading enzymes for picoxstrobin degradation.