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 phos- phate 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. All the six bands observed in the crude extract were distributed in these three fractions. Bands 1 and 2 in fraction-I, bands 3, 4 and 5 in fraction-II and band 6 in fraction-III were revealed. The fraction-III

Table 1. Purification of carboxylesterase of germinated finger millet. Purification of a carboxylesterase from finger millet 293

Figure 1. Elution pattern and electrophoretic zymograms of finger millet, carboxylic esterases from DEAE-cellulose using stepwise elution. (O), Esterase activity, (●)protein (A280 nm ).

esterase was slightly contaminated with non-esterolytic protein but this was eliminated by repeatedly applying this fraction onto a Sephadex G-200 column. The fraction-III esterase was purified about 46 fold with a recovery of 16%. The enzyme showed no appreciable loss of activity for over 3 months when stored at — 10°C.

Criteria of homogeneity

The homogeneity of fraction-III esterase was tested by carrying out PAGE. The gel patterns obtained following esterase staining and protein staining are shown in figure 2A. A single band in both the patterns was seen. Isoelectric focussing of the purified enzyme resulted in a single esterase band (figure 2B) corresponding to a pI of 5·1. The purified enzyme also showed only one protein band on subjecting it to SDS- PAGE both in the presence and absence of 2-mercaptoethanol as shown in figure 2C. All these results indicate the homogeneity of the fraction-ΙΠ esterase.

Determination of molecular weight

The molecular weight of the fraction-III esterase estimated by gel filtration on Sephadex G-200 using a calibrated curve was found to be about 70,000 (figure 3A). Using SDS- PAGE technique, the molecular weight of the fraction-III esterase was estimated to be 70,800 (figure 3B). The sedimentation constant (S 20,w) was calculated from the measurements made directly from the Schlieren pattern and a value of 5·8 was obtained. The partial specific volume (V) of the carboxylesterase, calculated from the amino acid 294 Aravinda Upadhya et al.

Figure 2. A. Polyacrylamide gel electrophoresis of fraction-III carboxylesterase. The gels were stained for activity (i) and protein (ii). B. Esterase staining of fraction-III carboxylester ase after gel electrofocussing. C. SDS-PAGE of fraction-III carboxylesterase in 10 % gel. The gels were stained with Coomassie brilliant blue-G without 2-mercaptoethanol (i) and with 2- mercaptoethanol (ii). Direction of migration is from top (cathode) to bottom (anode).

composition shown in table 2, was 0·718 ml g-1. The diffusion coefficient of the enzyme determined using the Stokes-Einstein equation was 7·023 × 10-6 cm2/sec. The molecu lar weight of fraction-III esterase using these values was found to be 71,741.

Amino acid composition

The results of the amino acid analysis of the fraction-III esterase expressed as residues/mol of the enzyme are given in table 2. The enzyme is composed of 648 amino acid residues/mol. wt. The enzyme contained less of half-cystine, methionine, tyrosine, Purification of a carboxylesterase from finger millet 295

Figure 3. A. Molecular weight determination of fraction-III carboxylesterase by gel filtration on Sephadex G-200. The marker proteins used were (1), cytochrome-c, (2), α- chymotrypsinogen; (3), ovalbumin; (4), BSA-monomer; (5), IgG; (6), catalase. Arrow cor responds to fraction-III carboxylesterase. B. Molecular weight determination of fraction-III carboxylesterase by SDS-polyacrylamide gel. The marker proteins used were, (1), cytochrome c, (2), lysozyme; (3), α-chymotrypsinogen; (4), ovalbumin; (5), BSA-monomer; (6), IgG; (7). catalase. The arrow corresponds to fraction-III carboxylesterase.

Table 2. Amino acid composition of fraction III carboxylesterase of germinated finger millet.

Tryptophan was estimated spectrophotometri cally from the alkaline spectrum.

296 Aravinda Upadhya et al. tryptophan, lysine and histidine residues but had relatively more neutral and acidic amino acid residues. It did not show the presence of free thiol groups as indicated by a negative test for free thiol groups with DTNB. The sulphur, therefore, must be present in disulphide bonds. It is not a glycoprotein as the protein band obtained by PAGE did not take up the PAS stain.

Catalytic properties of the enzyme

The optimum pH and temperature for the enzyme were 7·5 and 37°C respectively. The energy of activation of the enzyme was 6·15 kcal/mol for hydrolysis of 1-naphthyl acetate. The enzyme was assayed at varying concentrations of naphthyl esters and from the Lineweaver-Burk plots the Km and Vmax values were determined (1-naphthyl acetate-Km = 1·176 mM, Vmax = 2·105 nmol/mg protein; 1-naphthyl propionate-Km = 0·86 mM, Vmax = 3·225 nmol/mg protein).

Product inhibition

Product inhibition studies were carried out by incubating the enzyme with a constant concentration of the product (either acetic acid or l-naphthol) for 30 min. The amount of l-naphthol released with 1-naphthyl acetate as substrate was then measured colorimetrically. Figures 4 and 5 show the types of inhibitions obtained with acetic acid

Figure 4. Lineweaver-Burk plots of the inhibition of hydrolysis of 1-naphthyl acetate by the fraction-III carboxylesterase in presence of acetic acid. (●) No acetic acid, (Ο), 0·0125 Μ, (Δ), 0·015 Μ, (□) 0·0175 M acetic acid. Inset: Replot of slopes versus acetic acid concentration. Purification of a carboxylesterase from finger millet 297

Figure 5. Lineweaver-Burk plots of the inhibition of hydrolysis of 1-naphthyl acetate by the fraction-III carboxylesterase in the presence of 1-naphthol. (●), No. 1-naphthol, (O), 4 μΜ, (Δ), 6 μΜ, (□) 8 μΜ-naphthol. Inset: Replot of slopes versus 1-naphthol concentrations.

and 1-naphthol respectively. A linear competitive type of inhibition was obtained with acetic acid and a linear non-competitive inhibition with 1-naphthol.

Stability of the enzyme

The effects of heat and 8 Μ urea on the fraction-III esterase were tested. The enzyme lost about 50% of its activity in 2 min and was rendered inactive after 12 min of incubation at 65°C. On the other hand, fraction-III esterase was quite stable in the presence of 8 Μ urea.

Determination of ki and I50 values

A kinetic study of the fraction-III esterase with different inhibitors was made. The inhibitor rate constant ki was determined according to the method of Aldridge (1950). The enzyme was incubated for different time intervals with inhibitors prior to the addition of substrate. The organophosphates-dichlorvos and phosphamidon and a carbamate like eserine sulphate were used as inhibitors. The results obtained were plotted as logarithm per cent activity versus time (figure 6). The bimolecular rate constants for the reaction Ε +I → EI were calculated from the relationship, slope = Iki ][ and are shown in table 3. The I values for different inhibitors were calculated 303.2 50 from the inhibition curves (figure 7) and are also shown in table 3. 298 Aravinda Upadhya et al.

Figure 6. The effect of time on the inhibition of fraction-III carboxylesterase by or- ganophosphate and carbamate inhibitors. (□)10-5 Μ dichlorvos, (O), 10-6 Μ phos- phamidon, (Δ), 10 -3 Μ eserine sulphate.

Table 3. I50 and ki values obtained with different inhibitors for fraction-III carboxylesterase of germi- nated finger millet.

Discussion

The finger millet fraction-III carboxylesterase is the first plant carboxylesterase to be isolated in the homogeneous state. Though many attempts have been made to purify carboxylic esterases from other plant sources, homogeneity of the isolated enzymes has not been confirmed. However, a carboxylesterase isolated from sorghum grain (Sae et al. 1971) was reported to exhibit only one band on gel electrofocussing and two closely running bands on agarose gel electrophoresis. This has been interpreted as due to the inactivation of one of the esterase isozymes on the ampholyte carrier. The single sigmoidal inhibition patterns obtained for fraction-III esterase with organophosphate and carbamate inhibitors reveal the presence of only one enzyme. Purification of a carboxylesterase from finger millet 299

Figure 7. Plots of per cent inhibition versus log 10 [I] for the hydrolysis of 1-naphthyl esters by fraction-III carboxylesterase with inhibitors. (A), Dichlorvos; (B), Phosphamidon; (C), eserine sulphate. (●)1-Naphthyl acetate, (O), 1-naphthyl propionate.

Generally, those carboxylic esterases which exhibit complete inhibition with or- ganophosphates and active towards short chain carboxylic esters are classified as carboxylesterases (Holmes and Masters, 1967). Based on the above criteria the finger millet fraction-III esterase has been characterised as carboxylesterase (EC 3.1.1.1). Although it exhibited some inhibition with higher concentrations of carbamate (eserine sulphate, 0·01 M), this enzyme cannot be considered as a Cholinesterase as it did not hydrolyse any of the choline esters tested. Similar observations were noticed in the case of insect carboxylesterases (Veerabhadrappa et al., 1980; Mehrotra and Singh, 1976).

Although I50 (inhibitor concentration to give 50% inhibition in esterase activity) values have been widely used and recommended (O'Brien, 1960) the inhibitor constant ki is probably the most reliable criterion to measure the inhibitory potency of organophosphate inhibitors (Krysan and Chadwick, 1962). The rate constants obtained for the irreversible inhibition of organophosphate inhibitors were similar to those obtained for rat liver and human brain carboxylesterases (Main and Dauterman, 1967;

Hojring and Svensmark, 1977). The low value of ki obtained for finger millet carboxylesterase indicates that this enzyme is more resistant to the organophosphate inhibitors. This is also in agreement with the higher values of I50 obtained with different inhibitors (table 3). In the case of carboxylesterases from E. lunata (Mehrotra and Singh,

1976) and H. caerulea (Veerabhadrappa et al., 1980) very low values of I50 were obtained. The enzymes in these cases were very sensitive to these inhibitors. Since the physiological substrate of carboxylesterases is not known and only a few naphthyl esters are used in the present investigation, the substrate specificity results are not of much use as far as their physiological role is concerned. However, the hydrolysis of naphthyl esters catalysed by finger millet carboxylesterase follows typical Michaelis- Menten kinetics. The enzyme showed more affinity towards the short chain naphthyl esters and, among these, a preferred one was the propionate ester (low Km and high Vmax). Purified fractions of bean and pea carboxylesterases also showed similar type of 300 Aravinda Upadhya et al. substrate specificities exhibiting preferential action towards propionyl esters (Montgomery et al., 1968; Veerabhadrappa and Montgomery, 1971). The kinetics of ester hydrolysis by carboxylesterase can be described by a mechanism involving three distinct steps namely, adsorption of the substrate on the enzyme, acylation of the enzyme and concomitant liberation of alcohol and finally liberation of acid and reactivation of the enzyme (Hofstee, 1960). The kinetic results obtained with the products of ester hydrolysis in the present investigation (figures 4 and 5) indicated that the alcoholic product 1-naphthol gave linear non-competitive inhibition while acetic acid gave linear competitive inhibition. This is consistent with the ordered release of products by the enzyme, with the alcohol released first and acid second. The random release of products is ruled out. These product inhibition patterns also agreed with the Uni-Bi kinetic scheme with alcohol as the leading product. The amino acid composition (table 2) of fraction-III carboxylesterase is characterized by a relatively high content of acidic and neutral amino acids. Similar was the case with most of the animal carboxylesterases isolated so far (Scott and Zerner, 1975; Krisch, 1972). Further, the low value of isoelectric pH (5·1) for the finger millet carboxylesterase suggest that the enzyme is rich in acidic amino acid residues. Unlike other carboxyleste rases (Scot and Zerner, 1975) the amount of half-cystine residues was found to be more for finger millet carboxylesterase. This may explain its relative stability at room temperature for several days. In addition, the tryptophan content of the enzyme was very low compared to other mammalian carboxylesterases. The enzyme does not contain any carbohydrate and hence is not a glycoprotein. Most of the carboxylesterases isolated and purified from animal sources were reported to be oligomeric having two or three subunits (Krisch, 1972). Unlike these enzymes, the fraction-III carboxylesterase consists of a single polypeptide chain having a molecular weight of about 70,000.

Acknowledgements

The authors wish to acknowledge the encouragement from Prof. G. K. N. Reddy, and the help from Dr. S. Gurusiddaiah, Associate Director, Bioanalytical Centre, Washington State University, Pullman, Washington, USA.

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