Purification and Properties of a Carboxylesterase from Germinated Finger Millet (Eleusine Coracana Gaertn.)

J. Biosci., Vol. 7, Numbers 3 & 4, June 1985, pp. 289-301. © Printed in India . Purification and properties of a carboxylesterase from germinated finger millet (Eleusine coracana Gaertn.) G. ARAVINDA UPADHYA, L. GOVARDHAN and P. S. VEERABHADRAPPA Department of Chemistry, Central College, Bangalore University, Bangalore 560001, India MS received 5 August 1984; revised 28 January 1985 Abstract. A carboxylesterase (EC 3.1.1.1) was purified from germinated finger millet by ammonium sulphate fractionation, diethylaminoethyl-cellulose chromatography and Sephadex G-200 filtration. The homogeneity of the enzyme was established by Polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme has a single polypeptide chain with a molecular weight of 70,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to, basic amino acid residues. The isoelectric pH of the enzyme was found to be 5·1. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was more sensitive to organophosphate inhibitors than carbamates. The rate constants ki and l50 for different inhibitors were calculated. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear noncompetitive inhibition with 1-naphthol. Keywords. Carboxylesterase; finger millet; Eleusine coracana; purification and properties. Introduction Carboxylic esterases are a group of enzymes which catalyze the hydrolysis of various types of carboxylic esters. They are a class of enzymes with wide substrate specificity, usually for a short chain acid and an alcohol with only one hydroxyl group. They are widespread in nature and are present in animals, plants and microorganisms. In these systems, carboxylic esterases mostly exist in multiple molecular forms. The widespread occurrence of these enzymes has prompted many biochemists to purify and study them. The carboxylesterases (EC 3.1.1.1) from various animals and insects have been purified and extensively studied (Krisch, 1972; Narise and Hubby, 1966; Veerabhadrappa et al., 1980). Unlike in the case of animal and insect carboxylesterases, reports on the purification of these enzymes from plants and microorganisms are scanty. Among the plant carboxylesterases, the carboxylesterases from pea aqueous extract was the first to be separated and purified (Montgomery et al., 1968). Subsequently, carboxylesterases have been purified from other plant sources such as Abbreviations used: SDS, Sodium dodecyl sulphate; PAGE, Polyacrylamide gel electrophoresis; PAS, periodic acid Schiff; BSA, bovine serum albumin; CM, carboxy methyl; DTNB, 5,5 '-dithiobis (2-nitrobenzoic acid); pI, isoelectric pH; PCMB, p-chloromercuribenzoate; ki, bimolecular rate constant; I50, inhibitor concentration to give 50 % inhibition in enzyme activity; IgG, immunoglobulin-G. 289 290 Aravinda Upadhya et al. barley (Berger et al., 1970), green beans (Veerabhadrappa and Montgomery, 1971), sorghum (Sae et al., 1971), leaves of Festuca pratensis (Thomas and Bingham, 1977),and apple (Bartley and Stevans, 1981). None of these enzymes have been reported to be homogeneous. However, carboxylesterase from the latex of S. grantii has been purified to homogeneity recently (Govindappa, T. and Veerabhadrappa, P. S., unpublished data). In view of the importance of finger millet as a food grain and the scarcity of information on the esterase enzymes of this millet, an investigation was initiated to isolate and characterise the esterases of finger millet. Our earlier work (Veerabhadrappa and Aravinda Upadhya, 1979) has indicated that the three-day germinated finger millet possessed highest esterase activity. The present paper describes the isolation, purify- cation and properties of a carboxylesterase from the germinated finger millet. Materials and methods Chemicals All the chemicals used were of analytical grade or purchased from Sigma Chemical Co., St. Louis, Missouri, USA except the ampholyte carrier (pH 4-6) which was obtained from Serva-Fein Biochemica, Heidelberg, Federal Republic of Germany. The inhibit tors, dichlorvos (2,2-dimethyl dichlorovinyl phosphate) and phosphamidon were gifts from Pesticides and Industrial Chemical Repository, MD-8, Research Triangle Park, North Carolina, USA and CIBA-GEIGY Ltd., Basel, Switzerland respectively. Finger millet Finger millet (Purna variety) was obtained from the germ-plasm collection, Main Research Station, University of Agricultural Sciences, Bangalore. Esterase assay Esterase assay technique with naphthyl esters was carried out as reported earlier (Veerabhadrappa and Aravinda Upadhya, 1979). One unit of enzyme activity was defined as the amount of enzyme that released one µmol of product per min at pH 7 and 28°C. Cholinesterase activity was measured photometrically by the method of Ellman et al. (1961) using acetylthiocholine chloride as substrate. Protein was estimated by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as standard. Purification of carboxylesterase from germinated finger millet All operations were carried out at 4-6°C unless otherwise stated. The finger millet seeds were germinated for 72 h under sterile conditions and acetone powder was prepared from these germinated seeds. Ammonium sulphate fractionation The acetone defatted flour (20 g) was stirred with 200 ml of 0·1 Μ sodium phosphate buffer pH 7 for 3 h at 4°C. The extract was centrifuged at 1000 g for 30 min. To the Purification of a carboxylesterase from finger millet 291 supernatant obtained, solid ammonium sulphate was added gradually with constant stirring at 0°C to obtain 60 % saturation. The mixture was kept stirred for 1 h thereafter and the precipitated protein was collected by centrifugation at 1000 g for 30 min. The pellet was dissolved in 50 mM sodium phosphate-citrate buffer pH 7 and dialysed. This was once again centrifuged to remove insoluble residues. Diethylaminoethyl (DEAE)-cellulose chromatography The supernatant obtained above was lyophilized and dissolved in 5 ml of starting buffer (50 mM sodium phosphate-citrate buffer, pH 7) and applied onto a DEAE-cellulose column (1·8 × 25 cm). The column was washed with 100 ml of the starting buffer. The enzymes were eluted by stepwise elution using 150 ml each of 0·2 Μ and 0·3 Μ sodium chloride in starting buffer at a flow rate of 30 ml/h. Fractions (10 ml) were collected. Three peaks of esterase activity were eluted from the column. Fraction-I was not adsorbed onto the column and hence eluted out first. Fraction-II was eluted by 0·2 Μ NaCl and fraction-III by 0·3 Μ NaCl in the starting buffer. The enzyme eluted with 0·3 Μ NaCl was pooled, dialysed against distilled water and concentrated. Sephadex G-200 chromatography The concentrated enzyme solution from fraction III (approx. 1 ml) was then passed through Sephadex G-200 column (1 × 75 cm) equilibrated with 0·1 Μ sodium phosphate buffer pH 7 and the enzyme was eluted with the same buffer at a flow rate of 15 ml/h. The fractions (2 ml) having esterase activity were pooled, dialysed against distilled water and concentrated. Electrophoresis Polyacrylamide gel electrophoresis (PAGE) was carried out at 40C with 7·5 % gels using 0·03 Μ boric acid-sodium hydroxide buffer pH 8·7 at a current of 2 mA/gel (Ornstein, 1964; Davis, 1964). Esterase activity was detected by placing the gels for 30 min at 37o C in 100 ml of 0·1 Μ phosphate buffer pH 7·0 containing 40 mg Fast blue RR and 20 mg of the substrate (1-naphthyl propionate) in 2 ml of acetone (Hunter and Markert, 1957). Protein bands in the gels were stained with 1 % amido black in 6 % acetic acid, and for glycoproteins the periodic acid Schiff (PAS) staining technique was followed (Rennert, 1967). The PAS staining technique was standardised using ovomucoid and ovalbumin. Sodium dodecyl sulphate (SDS)-PAGE was done in 10 % gels according to the method of Weber and Osborn (1969). The marker proteins used to determine the molecular weight were cytochrome-c, lysozyme, chymotrypsinogen, ovalbumin, BSA-monomer, immunoglobulin-G (IgG) and catalase. Gel electrofocussing was performed by the method of Wringley (1969) in 7·5 % Polyacrylamide gel (pH 4–6). Determination of molecular weight by gel filtration The molecular weight of the purified enzyme was determined by the gel filtration method of Andrews (1964) using a Sephadex G-200 column (1 × 75 cm) equilibrated with 0·1 Μ sodium phosphate buffer pH 7 and operated at a flow rate of 15 ml/h. The 292 Aravinda Upadhya et al. elution profiles of the marker proteins were detected by measuring the absorbance at 280 nm and that of esterase by detecting the activity with 1-naphthyl acetate. Ultracentrifugation The sedimentation velocity (S 20,w) of the purified enzyme was determined in a Beckman Model Ε Analytical Centrifuge equipped with autoscanner and Schlieren optics at 60,000 rpm and 20°C by dissolving the protein in 0·1 Μ KCl. Amino acid analysis The amino acid analysis of the purified enzyme was carried out according to the method of Moore and Stein (1963). The protein sample was hydrolysed at 110°C in 6 Μ HCl in an evacuated and sealed tube for 24 h. The amino acid analysis was done in a Beckman 121 MB automatic amino acid analyser. Tryptophan content in the intact protein was determined spectrophotometrically from the alkaline spectrum in 0·1 Ν NaOH by the method of Bencze and Schmid (1957). The free thiol groups in the protein were estimated by reaction with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) (Ellman, 1959; Habeeb, 1972). Results Purification of the enzyme Table 1 summarises the results of a typical purification for the isolation of the carboxylesterases from germinated finger millet. The elution pattern of the carboxylic esterases from DEAE-cellulose column, reveals three peaks of esterase activity (fraction-I, fraction-II and fraction-III) as presented in figure 1 which also shows the gel electrophoretic analysis performed for each fraction.

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