RESUMO
The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.
Assuntos
Carboxilesterase/genética , Carboxilesterase/metabolismo , Herbicidas/metabolismo , Oxazóis/metabolismo , Propionatos/metabolismo , Rhodococcus/isolamento & purificação , Rhodococcus/metabolismo , Biotransformação , Clonagem Molecular , Análise por Conglomerados , Carboxilesterase/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Dados de Sequência Molecular , Peso Molecular , Filogenia , /genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodococcus/enzimologia , Rhodococcus/genética , Análise de Sequência de DNA , Microbiologia do Solo , Especificidade por Substrato , Triticum/crescimento & desenvolvimentoRESUMO
Carboxylesterase was purified from Fasciola gigantica through ammonium sulfate precipitation, chromatography on DEAE-Sepharose and gel filtration on a sephacryl S300. Three enzymes [El, EII and EIII] were separated. EII and EIII were purified to homogeneity. The molecular weight of EII and EIII enzyme were 66 and 50 KDa, respectively as detected by gel filtration and SDS-polyacrylamide gel electrophoresis. EII and EIII had Km 1.3 and 1.7 mM of p-nitrophenyl acetate. Affinity of esterase EII and EIII decreased as increasing carbon atom number of the substrates. Esterase EII and EIII had optimum temperature at 40 °C. Esterase EII and EIII had pH optima at pH 7.5 in phosphate buffer and pH 8.0 in Tris-HCl buffer, respectively. Studying effect of metal ions on esterase EII and EIII indicated that Li[+], Mn[++], Ba[++] and Mg[++] had activation effect on each isoenzyme. An activation effects could be detected with N- ethylmalimaide on EII and EIII]. The order of inhibition on EII was beta- mercaptoethanol > PMSF > DTNB > PCMB > iodoacetate. While the order of inhibition on EIII was beta-mercaptoethanol > iodoacetate > DTNB> PCM > PMSF