Purification, characterization and properties of carboxylesterase from the midgut of the silkworm, Bombyx mori L.

A carboxylesterase has been purified from the midgut of the silkworm Bombyx mon L. by a combination of ammonium sulphate fractionation, DEAE-cellulose ion-exchange chromatography, Sephacryl S-200 gel-filtration and preparative polyacrylamide gel electrophoresis (PAGE). The homogeneity of the enzyme was established by PAGE, isoelectricfocusing (IEF) and SDS-PAGE. The enzyme consists of two identical subunits with a subunit molecular weight of 72,000. The two subunits are held by non-covalent bonds. Amino acid analysis of the purified enzyme revealed a high content of hydrophobic amino acid residues. It lacks proline and tryptophan residues and free thiol groups. The data from substrate specificity study in conjunction with kinetic parameters indicate the hydrophobic nature of the active site. Copyright © 1996 Elsevier Science Ltd. All rights reserved

Purification and characterisation of a carboxylesterase from the latex ofSynadenium grantii Hook, ‘f’

The latex ofSynadenium grantii was found to contain esterolytic activity. Polyacrylamide gel electrophoretic study coupled with substrate and inhibitor specificity studies revealed the presence of multiple forms of carboxylesterases and cholinesterases in the latex. One of the carboxylesterases of the latex was purified by acetone fractionation, carboxymethyl-Sephadex chromatography and Sepharose-6B gel filtration. The homogeneity of the enzyme was established by polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme consists of a single polypeptide chain with a molecular weight of 14,000. 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 4.0. The enzyme was a glycoprotein as revealed by periodic acid Schiff-staining technique. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was sensitive to organophosphates. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear non-competitive inhibition with 1-naphthol

Purification and properties of carboxylesterases from the mid-gut of the termite Odentotermes horni. W.

Two carboxylesterases (TE-I and TE-II) from the mid-gut of the termite Odentotermes horni. W., have been purified to apparent homogeneity by means of ammonium sulfate fractionation, gel-permeation on Sephadex G-75 and Ultragel AcA-34 and ion-exchange chromatography on DEAE-Sephacel. The homogeneity of the preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and Ouchterlony double immunodiffusion. The apparent molecular weights determined by gel-permeation on Sephadex G-200 was 78,350 and by SDS-PAGE 78,500. In the presence of 2-mercaptoethanol, the proteins were split into two subunits of equal size with subunit molecular weight of about 40,000. The enzymes were found to have a Stokes radius of 3.35 nm. The amino acid analysis of the purified enzymes revealed the presence of a greater number of acidic and neutral amino acids than in other insect carboxylesterases. The isoelectric points of the enzymes, TE-I and TE-II, were 5.4 and 5.6, respectively. The two enzymes were inhibited by organophosphates. The substrate preference and inhibition patterns classify these enzymes as carboxylesterases (EC 3.1.1.1), but the physiological function is unknown. The apparent Km, Vmax, ki and I50 values are listed. The product inhibition studies with the enzymes revealed the linear competitive inhibition with acetate and linear non-competitive inhibition with 1-naphthol. In addition, optimum temperature and pH, thermal stability, effect of temperature and pH on Km and Vmax of the two enzymes were determined. © 1991

Isolation and properties of carboxylesterases of the termite gut-associated fungus, Xylaria nigripes. k., and their identity from the host termite, Odentotermes horni. w., mid-gut carboxylesterases

The termite, Odentoiermes horni. W., houses three fungal species, viz. Xylaria nigripes, Termitomyces microcorpus, and Trichoderma (species not identified), in its gut. X. nigripes was found to possess higher esterase activity levels than the other two. 2. 2. Four esterase enzymes, viz. FE-I, -II, -III and -IV, with pI values 5.1, 5.25, 5.4 and 5.6, respectively, were identified, isolated and purified to apparent homogeneity from the fungus X. nigripes, their biochemical and enzymological properties were determined, and compared with those of the previously characterized host termite mid-gut enzymes, TE-I and -II. 3. 3. The Mr, ofFE-I and -II was 85.1 kDa and those of FE-III and -IV was 87.5 kDa. However, TE-I and -II were relatively smaller (Mr ~ 78.5 kDa). Each of the fungal enzymes, viz. FE-I to -IV, was a homodimer with subunits associated non-covalently. The subunit Mr, were 42.6 kDa for FE-I and -II, and 43.7 kDa for FE-III and -IV. On the other hand, the termite mid-gut enzymes, TE-I and -II, were also homodimeric, but the subunits were associated covalently (subunit M, = 40 kDa). Immunologically the fungal esterase enzymes, viz. FE-I to -IV, were different from those of the host termite mid-gut esterases, viz. TE-I and -II. 4. 4. The substrate specificity and inhibitor sensitivity studies classify these enzymes, i.e. FE-I to -IV, as carboxylesterases (EC 3.1.1.1). Steady-state product inhibition kinetics suggested; an ordered release of products, i.e. alcohol followed by acid, and a Uni-Bi kinetic reaction mechanism. 5. 5. The two preliminary studies, i.e. the confinement of most esterase activity to the gut-tissue free from microorganisms and starvation of termites not leading to complete loss of esterase activity in the gut of the termites, suggested that there may not be any symbiotic relationship between termite, O. horni, and its gut associated microorganisms with regard to ester metabolism. Though the enzymes from the two sources were carboxylesterases, several of their properties were different and hence, they are different entities. © 1993

Changes in soluble esterases during germination of ragi (Eleusine coracana Gaertn.)

Changes in esterase activity, isozyme patterns, and sol. proteins of shoot and endosperm of ragi seeds were studied during germination. Significant differences in both esterase activity and isozyme patterns between shoot and endosperm were obsd. In endosperm, max. esterase activity was obsd. on the 3rd day of germination, showing a total of 5 bands. In addn., a new band of esterase activity appeared on the 4th day of germination in spite of the subsequent gradual decrease in esterase activity. Compared to endosperm, shoots contained only 3 bands exhibiting greater esterase activity in the earlier stages of germination. Activities of these enzymes decreased in both the tissues at later stages of germination with the disappearance of some bands. Variations obsd. in the esterase activity of the 2 tissues indicate that these enzymes have important roles in ragi seed germination

Purification and characterization of acetylcholinesterase isozymes from the latex of Synadenium grantii Hook, 'f'

Three acetylcholinesterase isoenzymes were purified from S. grantii latex by a combination of acetone fractionation, CM-​Sephadex C-​50 chromatog., Sephadex G-​200 gel filtration, and PE-​Cellulose chromatog. The homogeneity of the isoenzymes was established by PAGE and isoelectrofocusing. The pI values were 5.0, 5.2, and 5.4. The mol. wt. of each enzyme was estd. to be 70,​000 by a gel-​filtration method. SDS-​PAGE indicated that each enzyme consisted of 2 subunits of mol. wt. ∼35,​000. These enzymes were glycoproteins and were more sensitive towards carbamate inhibitors compared to organophosphates. They exhibited substrate inhibition and showed identical substrate specificity and inhibitor sensitivity. The rate consts. for different inhibitors were calcd. These 3 enzymes were considered to be charge isoenzymic forms of the latex acetylcholinesterase

A novel approach for the identification of isozymes using inhibitors.

Carboxylesterase isozymes of a termite and a fungus, have been identified by exploiting their differential sensitivity towards organophosphate inhibitors. The inhibition curves obtained by plotting pI (negative logarithm of inhibitor concentration) versus per cent inhibition are used to distinguish the isozymes. The termite and its associated fungus possess 2 and 4 isozymes respectively. Each of the purified isozyme gave a single sigmoidal inhibition curve with its characteristic I50 value. The pattern of the inhibition curve of the crude extract is found to mimic that of the mixture of the purified isozymes in both termite and fungus. This method provides not only identification of isozymes but also serves as a criterion of homogeneity

Proteinase Inhibitors of Finger Millet (Eleusine coracana gaertn.)

Proteinase inhibitory activities of a number of finger millet and five representative varieties of other millets were determined. Inhibitory activities in the buffer extracts of the millets varied widely. Trypsin inhibitory activity (TIA) was more pronounced than the chymotrypsin inhibitory activity (CIA) in all the finger millet varieties tested. On germination, TIA and CIA were markedly reduced in the endosperm, but in the axis considerable TIA remained while CIA disappeared. Proteinase inhibitors of finger millet were found to be heat-labile under neutral conditions and comparatively heat-stable under acidic conditions. These studies suggest the presence of multiple forms of protein proteinase inhibitors in finger millet

Tissue esterases of Exoristina sorbillans (Uzi fly)

Polyacrylamide gel disc electrophoretic technique was used to examine the esterase pattern of nead, haemolymph, alimentary canal., ovary and testis of the Uzi fly. The zymograms revealed the varied pattern of esterases both in number and type. This varied pattern suggested several roles for these enzymes present in different tissues © 1984 Indian Academy of Sciences

Esterases as Genetic Markers in Finger Millet

Different varieties of finger millet (Eleusine coracana Gaertn.) were screened for esterase activity colorimetrically and electrophoretically using 1-naphthyl acetate and acetylthiocholine chloride as substrates. The Indian brown seed coat variety (Purna), the Indian white seed coat variety (Hamsa), hybrids of these designated as HPB (brown) and HPW (white), African varieties (brown) and Indian-African hybrid varieties (brown) all exhibited 1-naphthyl acetate hydrolysing activity and showed 6,5,6,5,8 and 8 esterolytic bands respectively on gel electrophoresis. The white seed coat varieties, both parental (Hamsa) and hybrid (HPW), did not possess any acetylthiocholine chloride hydrolysing activity while all the brown seed coat varieties did, the African varieties having greater activities than Indian brown seed coat varieties. Thus, the demonstrable variation in esterase isozymic pattern and cholinester hydrolysing activity with the varieties tested provides a useful genetic marker for identifying different varieties of finger millet