title: Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization.
abstract: Two carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CM-cellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS-PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS-PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K(m) of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K(m) was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45°C (ME-III) and 37°C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35°C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB).
authors.affiliation: Department of Biotechnology, PES Institute of Technology, BSK III Stage, Hosakerahalli, Bangalore, Karnataka, India. kschandraks@gmail.com
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: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D031299:Mucuna / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D013379:Substrate Specificity
chemical_list: D010940:Plant Proteins; D002265:Carboxylic Ester Hydrolases