Purification and properties of a carboxylesterase from germinated finger millet (Eleusine coracana Gaertn.)

Upadhya, Gundumi A.; Govardhan, L.; Veerabhadrappa, Patnagere S.

doi:10.1007/bf02716791

Cited by 8 publications

(17 citation statements)

References 23 publications

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“…They have been purified from various plant sources including finger millet (Eleusine coracana) (Upadhya et al, 1985), Cucurbita maxima fruit tissue (Nourse et al, 1989), Jatropa curcas L. seeds (Staubmann et al, 1999), Avena fatua (Mohamed et al, 2000), tomato (Stuhlfelder et al, 2002) and Cucurbita pepo (Afaf S Fahmy et al, 2008) by employing different purification processes including ammonium sulphate fractionation, ion exchange chromatography and gel filtration chromatography. However, not all preparations have been shown to be homogeneous.…”

Section: Purificationmentioning

confidence: 99%

“…Carboxylesterases have been purified to homogeneity and purity was established by both gel electrophoresis and isoelectric focussing (IEF) (Upadhya et al, 1985). The homogeneity of the carboxylesterase isolated from the latex of S. grantii was established by PAGE, IEF and SDS-PAGE (Govindappa et al, 1987).…”

Section: Criteria Of Puritymentioning

confidence: 99%

“…However, most of the carboxylesterases studied so far in both animals and plants have low isoelectric pH values and contain large proportions of acidic amino acids. The purification of carboxylesterases in most of the cases involved the use of anion exchangers (Upadhya et al, 1985;Govindappa et al, 1987;Sreerama et al, 1991). The low isoelectric pH values, the presence of large proportions of acidic amino acids and binding affinity to the anion exchanger clearly indicate the acidic nature of carboxylesterases in many cases.…”

Section: Isoelectric Phmentioning

confidence: 99%

“…It is reported that most of the plant carboxylesterases have low molecular weights and contain a single polypeptide chain. The carboxylesterases from finger millet (Upadhya et al, 1985) and sorghum (Sae et al, 1971) were reported to have molecular weights of 60 to 70 kDa and found to contain a single polypeptide chain. Bartley and Stevens (1981) characterized and reported four carboxylesterases, each containing a single polypeptide chain, with molecular weights in the range of 35-50 kDa from apple.…”

Section: Molecular Weightmentioning

confidence: 99%

“…The physiological substrate of these carboxylesterases is not known and only naphthyl esters were used in the present investigation. Substrate specificity studies of purified fractions of bean, pea and finger millet carboxylesterases with phenyl esters, glyceryl esters and naphthyl esters (Montgomery et al, 1968;Veerabhadrappa and Montgomery, 1971;Upadhya et al, 1985) revealed that in all these cases, esterases hydrolysed short chain esters exhibiting preferential action towards propionyl esters. On the other hand, the rate of ester hydrolysis catalyzed by partially purified apple esterases increased with increase in the carbon number of the substrate in the order C 2 -C 6 , but activity declined with higher molecular weight esters.…”

Section: Substrate Specificitymentioning

confidence: 99%

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Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization

2011

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a b s t r a c tTwo 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 CMcellulose, 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).

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Section: Purificationmentioning

confidence: 99%

Section: Criteria Of Puritymentioning

confidence: 99%

Section: Isoelectric Phmentioning

confidence: 99%

Section: Molecular Weightmentioning

confidence: 99%

Section: Substrate Specificitymentioning

confidence: 99%

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Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization

2011

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Identification and characterization of a novel carboxylesterase from Phaseolus vulgaris for detection of organophosphate and carbamates pesticides

et al. 2018

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

et al. 1987

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Abstract. The latex of Synadenium 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.

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Purification and properties of a carboxylesterase from germinated finger millet (Eleusine coracana Gaertn.)

Cited by 8 publications

References 23 publications

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization

Identification and characterization of a novel carboxylesterase from Phaseolus vulgaris for detection of organophosphate and carbamates pesticides

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

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