Carboxylesterases are hydrolases which catalyze the hydrolysis of various types of esters. Carboxylesterase from the seeds of Jatropha curcas has been purified to homogeneity using ammonium sulfate fractionation, CM-cellulose chromatography, Sephadex G-100 chromatography and preparative polyacrylamide gel electrophoresis (PAGE). The homogeneity of the purified enzyme was confirmed by PAGE, iso-electrofocusing and SDS-PAGE. The molecular weight of the purified enzyme was determined by both gel-permeation chromatography on Sephadex G-150 and SDS-PAGE. The molecular weight determined by Sephadex G-150 chromatography and SDS-PAGE both in the presence and absence of 2-mercaptoethanol was 31 kDa. The isoelectric point of the purified enzyme was found to be 8.9. JCSE-I (J. curcas seed esterase-I) was classified as carboxylesterase on the basis of substrate and inhibitor specificity. The K(m) of JCSE-I with 1-naphthyl acetate, 1-naphthyl propionate, 1-naphthyl butyrate and 2-naphthyl acetate as substrates were found to be 0.0,794, 0.0,658, 0.0,567 and 0.1 mM, respectively. The enzyme exhibited an optimum temperature of 45 °C and an optimum pH of 6.5. The enzyme was stable up to 15 min at 65 °C. The enzyme was resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB) and inhibited by organophosphates (dichlorvos, parathion and phosphamidon).
Objective:The main aim of this scientific report was to investigate a series of phytochemicals in silico and the pharmacology of four plants found at higher altitude in the ginger family, Zingiberaceae (incl. Costaceae) from North-East India, particularly Sikkim. First, the goal was to determine the biological activities of the four herbs (used under Zingiberaceae family) using antioxidant assays to identify the best species. Second, previously reported compounds in litero were subsequently screened for their anticancerous activities using in silico methods.Materials and Methods:Using the methanolic extracts of herbs, quantitative detection of phytochemicals such as total phenols and total flavonoids was detected, and the free radical scavenging activity was also studied using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay. Docking process was studied, using Discovery Studio version 3.5, to identify suitable molecules at the protein-binding sites through annealing and genetic simulation algorithms. Grids centered on active sites were obtained with spacing of 54 × 55 × 56, and 0.503 grid spacing was calculated. The methods adopted and used in this study were comparisons of Global and Local Search Methods to determine the parameters such as maximum number of 250,000 energy evaluations as well as generations of 27,000, followed by mutation and crossover rates of 0.02 and 0.80. The number of docking runs was set to 10. Molecular dynamics study was done to check the stability of the complex.Results:Among all the genus of Zingiberaceae family investigated in this study, Curcuma angustifolia and Hedychium sp. exhibited the highest 537 ± 12.45; 292 ± 9.16 mg gallic acid equivalent/g total polyphenols and 38 ± 1.54; 75 ± 6.75 mg quercetin equivalent/g flavonoids, respectively. Depending on the concentration, the Hedychium sp. extract exerted the highest scavenging activity on DPPH radical (IC50 36.4 μg/mL). In silico result demonstrated that the synergetic effects of β-phellandrene with other compounds might be responsible for its anticancerous activity. β-phellandrene and farnesene epoxide showed bonding with Leu298, Ala302, Met336, Leu339, Leu343, Phe356, Ala302, Glu305, Met340, Leu343, Arg346, Phe356, Ile373, Ile376, Leu380, His475, Leu476, and Leu491.Conclusion:Based on the current available literature, this is the first study to understand the interaction of compounds found in the rhizomes of Zingiberaceae family.SUMMARY The aqueous methanolic extract of Zingiberaceae family Curcuma angustifolia and Hedychium sp. has potent antioxidant activity as assessed by 2,2-diphenyl-1-picryl-hydrazyl assaysHedychium sp. is understood to possess more active compounds than other varietiesIn silico studies indicated synergetic effects of β-phellandrene and other compounds for its anticancerous activity. Abbreviations used: CADD: Computer-aided drug designing; ROS: Reactive oxygen species; ADMET: Absorption, distribution, metabolism, and excretion-toxicity; FeCl3: Ferric chloride; DPPH: 2,2-diphenyl-1-picryl-hydrazyl; NaNO2: Sodium ni...
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).
Germinating seeds of Tamarindus indica synthesizes various enzymes which are required for the degradation of seed reserves such as xyloglucans, fatty acid esters and proteins. Among these, esterases, belonging to a group of hydrolytic enzymes catalyze the hydrolysis of various types of esters. They play an important role in cell expansion as well as detoxification of xenobiotics and many agrochemicals and insecticides. The esterases are extracted from the germinating tamarind seeds using 50 mM phosphate buffer, pH 7. The Km with α-naphthyl acetate as the substrate is 19.23 µM and the enzymes are optimally active at pH 7.0 to 7.5 and are stable between pH 5.0 to 9.0. The optimum temperature of esterase activity of tamarind seed is between 37˚C-50˚C and is stable up to 40˚C. The activity declined by 30% at 60˚C and about 90% at 70˚C. Highest esterase activity and specific activity are observed on the 21 st day of germination. The polyacrylamide gel electrophoresis (PAGE) indicated the presence of nine isozymes of esterases. Band numbers 1, 5 and 6 are the major esterolytic bands present throughout the germination period while band numbers 2 & 3 are minor bands present only during the latter period of the germination. Based on substrate and inhibitor specificity in conjunction with electrophoresis, the esterases 1 to 8 have been classified as carboxylesterases sensitive to organophosphate inhibitor (OP) and PCMB (p-chloromercuribenzoate) while esterase 9 is classified as carboxylesterase sensitive to OP. These esterases are unaffected by carbamate inhibitor, eserine sulphate.
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