Isolation,Identification And Degrading Characteristics Of A Haloxyfop-R-Methyl-Degrading Bacterium,and Cloning,Expression Of A Haloxyfop-R-Methyl-Hydrolyzing Gene

The wide applications of haloxyfop-R-methyl has led to environmental pollution and ecological toxicity,so the isolation of haloxyfop-R-methyl degrading bacteria and investigation of their degradation mechanisms have very important theoretical values and application potentials.In this study,a haloxyfop-R-methyl-degrading strain,designated as strain ZS-15,was isolated from a rice field soil which has been long-term polluted by haloxyfop-R-methyl.Based on its physiological and biochemical characteristics and the result from the 16S rRNA gene sequence analysis,the haloxyfop-R-methyl-degrading strain was preliminarily identified as Rhodococcus sp.(strain ZS-15).Then we study its growth and degrading characteristics.Strain ZS-15 is a Gram-positive,without flagella,short rod shaped aerobe.Colonies grown on LB agar for 2-3 days are orange,circular,convex and opaque with entire margins.Nitrate reduction,starch hydrolysis,indole test are positive.Strain ZS-15 can not use citric acid.The optimum growth is observed at 30?,pH 7.0 with 1-3%NaCl.The optimal carbon source for the growth of strain ZS-15 is glucose and the optimal nitrogen source is yeast extract(organic nitrogen)and potassium nitrate(inorganic nitrogen).Strain ZS-15 could efficiently degrade haloxyfop-R-methyl when haloxyfop-R-methyl was used as the sole carbon source.However,it could not utilize haloxyfop-R-methyl as the sole carbon source for growth.Strain ZS-15 degraded 94.5%of 50 mg/L haloxyfop-R-methyl in 36 h.The optimum temperature for degradation of haloxyfop-R-methyl by strain ZS-15 was 30? and the optimal pH value was observed to be approximately at 7.0.Strain ZS-15 cells and cell-free extracts produced visible transparent halos due to haloxyfop-R-methyl transformation on agar plates supplemented with 200 mg/L haloxyfop-R-methyl.The metabolite from haloxyfop-R-methyl degradation by strain ZS-15 was identified as haloxyfop by mass spectrometry analysis.An esterase gene,harmH,encoding the haloxyfop-R-methyl-hydrolyzing carboxylesterase was cloned from Rhodococcus sp.ZS-15 by short-gun method.Sequence analysis indicated that harmH gene consists of 840 bp and encods a protein of 280 amino acids.The molecular mass of the denatured enzyme was approximately 31 kDa.The deduced protein was compared with other known enzymes available in the Protein Data Bank(NCBI database).HarmH showed the highest identities with some hypothesis or putative esterases or hydrolases,e.g.,an alpha/beta hydrolase from Pseudonocardia sp.P1(54%identity).The gene harmH was expressed in Escherichia coli BL21(DE3)and the HarmH was purified using Ni-nitrilotriacetic acid affinity chromatography.The catalytic efficiency of HarmH toward different AOPP herbicides followed the order of haloxyfop-R-methyl ?diclofop-methyl>quizalofop-p-ethyl? fenoxaprop-P-ethyl>cyhalofop-butyl;the results indicated that the chain length of the alcohol moiety strongly affected the biodegradability of the aryloxyphenoxypropionate herbicides,while the substitutions in the aromatic ring only had slight influence.