Purification and Characterization of Novel α-Amylase from Bacillus subtilis KIBGE HAS

Purification of extracellular α-amylase from Bacillus subtilis KIBGE HAS was carried out by ultrafiltration, ammonium sulfate precipitation and gel filtration chromatography. The enzyme was purified to homogeneity with 96.3-fold purification with specific activity of 13011 U/mg. The molecular weight of purified α-amylase was found to be 56,000 Da by SDS-PAGE. Characteristics of extracellular α-amylase showed that the enzyme had a Km and Vmax value of 2.68 mg/ml and 1773 U/ml, respectively. The optimum activity was observed at pH 7.5 in 0.1 M phosphate buffer at 50°C. The amino acid composition of the enzyme showed that the enzyme is rich in neutral/non polar amino acids and less in acidic/polar and basic amino acids. The N-terminal protein sequence of 10 residues was found to be as Ser-Ser-Asn-Lys-Leu-Thr-Thr-Ser-Trp-Gly (S-S-N-K-L-T-T-S-W-G). Furthermore, the protein was not N-terminally blocked. The sequence of α-amylase from B. subtilis KIBGE HAS was a novel sequence and showed no homology to other reported α-amylases from Bacillus strain

Methanol extract of Melastoma malabathricum leaves exerted antioxidant and liver protective activity in rats

Background Melastoma malabathricum L. (Melastomaceae) is a small shrub with various medicinal uses. The present study was carried out to determine the hepatoprotective activity of methanol extract of M. malabathricum leaves (MEMM) against the paracetamol-induced liver toxicity in rats model. Methods The respective chemicals and herbal solutions (10% DMSO, 200 mg/kg silymarin or MEMM (50, 250 and 500 mg/kg)) were administered orally to rats once everyday for 7 days followed by the hepatotoxicity assay. The blood samples and livers were collected and subjected to biochemical and microscopical analysis. Prior to the hepatoprotective study, MEMM was subjected to determination of the total phenolic content (TPC) and the antioxidant properties using several standard assays (e.g. 2, 2-diphenyl-1-picrylhydrazyl- and superoxide anion- radical scavenging assay, and oxygen radical absorbance capacity assay). Results MEMM exerted significant (p < 0.05) and high antioxidant activity in which high TPC was recorded; while in the hepatotoxicity study, the extract exhibited significant hepatoprotective effects against the paracetamol-induced hepatotoxic model. The results observed for serum liver enzymes (ALT, ALP and AST) as well as the microscopic observations and microscopic scoring supported the hepatoprotective potential of MEMM. The phytochemical and HPLC analysis of MEMM demonstrated the presence of flavonoids as its major constituents. Conclusions The MEMM-induced hepatoprotective activity could be allied partly to its antioxidant activity and the presence of flavonoids