title: Purification and characterization of carboxylesterase from the seeds of Jatropha curcas.
abstract: Carboxylesterases are hydrolases which catalyze the hydrolysis of various types of esters. Carboxylesterase from the seeds of Jatropha curcas has been purified to homogeneity using ammonium sulfate fractionation, CM-cellulose chromatography, Sephadex G-100 chromatography and preparative polyacrylamide gel electrophoresis (PAGE). The homogeneity of the purified enzyme was confirmed by PAGE, iso-electrofocusing and SDS-PAGE. The molecular weight of the purified enzyme was determined by both gel-permeation chromatography on Sephadex G-150 and SDS-PAGE. The molecular weight determined by Sephadex G-150 chromatography and SDS-PAGE both in the presence and absence of 2-mercaptoethanol was 31 kDa. The isoelectric point of the purified enzyme was found to be 8.9. JCSE-I (J. curcas seed esterase-I) was classified as carboxylesterase on the basis of substrate and inhibitor specificity. The K(m) of JCSE-I with 1-naphthyl acetate, 1-naphthyl propionate, 1-naphthyl butyrate and 2-naphthyl acetate as substrates were found to be 0.0,794, 0.0,658, 0.0,567 and 0.1 mM, respectively. The enzyme exhibited an optimum temperature of 45 °C and an optimum pH of 6.5. The enzyme was stable up to 15 min at 65 °C. The enzyme was resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB) and inhibited by organophosphates (dichlorvos, parathion and phosphamidon).
authors.affiliation: Department of Biochemistry, Bangalore University, Bangalore, Karnataka, India.
authors.forename: T
authors.initials: T
authors.lastname: Subramani
authors.forename: K S
authors.initials: KS
authors.lastname: Chandrashekharaiah
authors.forename: N Ramachandra
authors.initials: NR
authors.lastname: Swamy
authors.forename: K R Siddalinga
authors.initials: KR
authors.lastname: Murthy
mesh_terms: D043182:Carboxylesterase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D004396:Coloring Agents / Q000737:chemistry / Q000378:metabolism; D004006:Dichlorvos / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005232:Fatty Acids, Volatile / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007306:Insecticides / Q000494:pharmacology; D007526:Isoelectric Point; D031286:Jatropha / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009284:Naphthols / Q000737:chemistry / Q000378:metabolism; D010278:Parathion / Q000494:pharmacology; D010707:Phosphamidon / Q000494:pharmacology; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002800:Cholinesterase Inhibitors; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D005232:Fatty Acids, Volatile; D007306:Insecticides; D009284:Naphthols; D010940:Plant Proteins; D010707:Phosphamidon; D010278:Parathion; D004006:Dichlorvos; D043182:Carboxylesterase