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

Two 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 CM-cellulose, 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). © 2011 Elsevier Ltd. All rights reserved

CHARACTERIZATION OF ALPHA-AMYLASE FROM THE SEEDS OF Mucuna pruriens

Amylases are hydrolytic enzymes which are widely distributed in nature, animals, plants and microorganisms. Amylases are of great significance in present-day biotechnology. In present study, amylases are isolated from the soaked seeds of Mucuna pruriens under extreme acidic conditions. Conventional protein purification techniques such as salt fractionation, ion exchange chromatography on CM-cellulose and sephadex G-75 was employed for the purification of amylase from the seeds of Mucuna pruriens. The amylase activity was eluted in one peak. The specific activity and yield of the purified amylase was 6.25 and 29.99, respectively. Native PAGE, SDS-PAGE and gel electrofocussing were employed to establish homogeneity of the purified amylase. SDS-PAGE and gel-filtration chromatography on sephadex G-75 was used to determine the molecular weight of the purified amylase. The purified amylase was nearly homogenous and its molecular weight was found to be 78.4 kDa. The optimum pH and temperature of the purified amylase were 7.0 and 50oC, respectively. The isolectric pH of the purified amylase was 7.2 and the activity was linear up to 60 minutes

Mechanistic investigations of the oxidation of phenethyl alcohols by sodium-N-bromo-benzenesulphonamide in acid medium

The kinetics of oxidation of six substituted phenethyl alcohols (X-C6H4-CH2-CH2-OH where X = -H, -Cl, -Br, -CH3, -OCH3 and -NO2) by sodium-N-bromo-benzenesulphonamide or bromamine-B(BAB) in the presence of HCl at 35 degrees C showed that the rate has first-order dependence on BAB](0) and H+] and fractional order on PEA](0) and Cl-]. Tonic strength variations, addition of reaction product of benzenesulphonamide and variation of dielectric constant of the medium have no effect on the rate. The solvent isotope effect k(1)(H2O)/k(1)(D2O) similar or equal to 0.82. Proton inventory studies have been made in H2O-D2O mixtures. The Hammett plot is biphasic and the reaction constant rho was -3.2 for electron-releasing substituents and -0.34 for electron-withdrawing groups. The activation parameters Delta H#, Delta S# follow an isokinetic relationship

Kinetics and mechanism of oxidation of phenethyl alcohols by bromamine-T in acid medium

The kinetics of the oxidation of six substituted phenethyl alcohols (X - C6H4 - CH2 - CH2 - OH where X = -H, -Cl, -Br, -CH3, -OCH3 and -NO2) by sodium N-bromo-p-toluenesulphonamide or bromamine-T (BAT) in the presence bf HCl has been studied at 35 degrees C. The reaction rate shows a first order dependence both on the BAT](0) and the H+] and is fractional order with respect to PEA](0) and Cl-]. The change in the ionic strength and dielectric constant of the medium and the addition of reaction product, p-toluenesulphonamide have no effect on the reaction rate. The solvent isotope effect k(1(H2O))/k(1(D2O)) similar or equal to 0.86. Proton inventory studies have been made in H2O-D2O mixtures. The rates satisfactorily correlate with Hammett's LFER. The reaction constant rho has been found to be -3.1 for electron releasing substituents and -0.26 for electron withdrawing groups at 35 degrees C. Activation parameters Delta H-not equal, Delta S-not equal, Delta G(not equal) and logA are calculated by studying the reaction at different temperatures (308-318K). It is seen that Delta H-not equal and Delta S-not equal are linearly related and an isokinetic relationship is observed with beta = 330K, indicating enthalpy to be a controlling factor

Osmium(VIII) catalyzed kinetics and mechanism of indoles oxidation with aryl-N-haloamines in alkaline medium

The kinetics of oxidation of the title substrates by sodium N-haloarylsulfonamides (or aryl-N-haloamines), chloramine-T (CAT), bromamine-T (BAT), chloramine-B (CAB), and bromamine-B (BAB), catalyzed by osmium(VIII) in alkaline medium has been studied ae 30 degrees C. The corresponding oxindoles and arylsulfonamides have been characterized as reaction products The reaction rate shows a first-order dependence each on indole](0) and oxidant](0), a fractional-order on Os(VIII)], and an inverse first-order on OH-]. Addition of arylsulfonamide, chloride and bromide, and variation of ionic strength of the medium have no effect on the reaction rate. There is a negative effect of dielectric constant of the solvent. Activation parameters have been calculated from the Arrhenius and Eyring plots. Hammett correlation of substituent effects indicates an LFE relationship with rho = -1.0, showing the formation of an electron deficient transition state. From enthalpy-entropy relationships and Exner correlations, the isokinetic temperatures (333 K and 326 K) have been determined for the reactions of CAT and BAT, respectively. Proton inventory studies in H2O-D2O mixtures have shown the involvement of a single exchangeable proton of OH- ion in the transition state. A mechanism consistent with the observed kinetics has been proposed. (C) 1996 John Wiley & Sons, Inc

Status and future transition of rapid urbanizing landscape in central Western Ghats - CA based approach

The land use changes in forested landscape are highly complex and dynamic, affected by the natural, socio-economic, cultural, political and other factors. The remote sensing (RS) and geographical information system (GIS) techniques coupled with multi-criteria evaluation functions such as Markov-cellular automata (CA–Markov) model helps in analysing intensity, extent and future forecasting of human activities affecting the terrestrial biosphere. Karwar taluk of Central Western Ghats in Karnataka state, India has seen rapid transitions in its forest cover due to various anthropogenic activities, primarily driven by major industrial activities. A study based on Landsat and IRS derived data along with CA–Markov method has helped in characterizing the patterns and trends of land use changes over a period of 2004–2013, expected transitions was predicted for a set of scenarios through 2013-2022. The analysis reveals the loss of pristine forest cover from 75.51% to 67.36% (1973 to 2013) and increase in agriculture land as well as built-up area of 8.65% (2013), causing impact on local flora and fauna. The other factors driving these changes are the aggregated level of demand for land, local and regional effects of land use activities such as deforestation, improper practices in expansion of agriculture and infrastructure development, deteriorating natural resources availability. The spatio temporal models helped in visualizing on-going changes apart from prediction of likely changes. The CA-Markov based analysis provides us insights into the localized changes impacting these regions and can be useful in developing appropriate mitigation management approaches based on the modelled future impacts. This necessitates immediate measures for minimizing the future impacts

Oxidation of substituted phenethyl alcohols by sodium N-chlorobenzenesulphonamide: A kinetic study

The kinetics of the oxidation of six substituted phenethyl alcohols by sodium N-chlorobenzenesulphonamide or chloramine-B (CAB) in the presence of HCl was studied at 35 degrees C, The rate shows a first order dependence on CAB](0) and H+] and is of fractional order in PEA](0) and Cl-]. Ionic strength variations, addition of the reaction product of benzenesulphonamide and variation of the dielectric constant of the medium have no effect on the rate. The solvent isotope effect k(H2O)(1)/k(D2O)(1) approximate to 0.78. Proton inventory studies were made in H2O-D2O mixtures. The rates correlate satisfactorily with Hammett's LFER. The reaction constant rho was -3.5 for electron-releasing substituents and -0.30 for electron withdrawing groups at 35 degrees C. Activation parameters Delta H-double dagger, Delta S-double dagger, Delta G(double dagger) and log A were computed for the reaction, An isokinetic relationship is observed with beta=338 K, indicating enthalpy as a controlling factor