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Protein J (2012) 31:120128 DOI 10.1007/s10930-011-9380-7
Purication and Characterization of Carboxylesterase from the Seeds of Jatropha curcas
T. Subramani K. S. Chandrashekharaiah
N. Ramachandra Swamy K. R. Siddalinga Murthy
Published online: 16 December 2011 Springer Science+Business Media, LLC 2011
Abstract Carboxylesterases are hydrolases which catalyze the hydrolysis of various types of esters. Carboxylesterase from the seeds of Jatropha curcas has been puried to homogeneity using ammonium sulfate fractionation, CM-cellulose chromatography, Sephadex G-100 chromatography and preparative polyacrylamide gel electrophoresis (PAGE). The homogeneity of the puried enzyme was conrmed by PAGE, iso-electrofocusing and SDS-PAGE. The molecular weight of the puried 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 puried enzyme was found to be 8.9. JCSE-I (J. curcas seed esterase-I) was classied as carboxylesterase on the basis of substrate and inhibitor specicity. The Km 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 carba-mates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB) and inhibited by organophosphates (dichlorvos, parathion and phosphamidon).
Keywords Jatropha curcas Carboxylesterase
Purication Characterization Kinetics
AbbreviationsJCSE Jatropha curcas seed esterasePAGE Polyacrylamide gel electrophoresis SDS-PAGE Sodiumdodecylsulphatepolyacrylamide gel electrophoresisCM-cellulose Carboxymethyl-celluloserpm Revolution per minuteDBLS Diazo blue B lauryl sulphatekDa KilodaltonOps OrganophosphatesIEF Iso-electrofocusing
1 Introduction
Carboxylesterases (EC.3.1.1.1, carboxyl ester hydrolases) are enzymes belonging to the group of hydrolases which catalyze the hydrolysis of various types of esters. These are widely distributed in nature and occur in multiple molecular forms. They exhibit a number of unique enzyme characteristics such as substrate specicity, regiospecicity and chiral specicity [21]. These carboxylesterases preferably catalyze the hydrolysis of esters composed of short chain fatty acids [5], particularly; the potential application of these enzymes for the synthesis of short chain esters has attracted the interest of a broad range of industrial elds like foods, pharmaceuticals and cosmetics. These enzymes are also involved in various functions in plants...