Molecular hybridization approach is an emerging tool in drug discovery for designing new pharmacophores with biological activity. A novel, new series of coumarin-benzimidazole hybrids were designed, synthesized and evaluated for their broad spectrum antimicrobial activity. Among all the synthesized molecules, compound (E)-3-(2-1H-benzo[d]imidazol-1-yl)-1-((4-chlorobenzyl)oxy)imino)ethyl)-2H-chromen-2-one showed the most promising broad spectrum antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Proteus vulgaris. In addition, it has showed no cytotoxicity and hemolysis at 10 times the MIC concentration. SAR studies indicate that position of the chlorine atom in the hybrid critically determines the antibacterial activity.
An extracellular cholesterol oxidase (cho) enzyme was isolated from the Streptomyces parvus, a new source and purified 18-fold by ion exchange and gel filtration chromatography. Specific activity of the purified enzyme was found to be 20 U/mg with a 55 kDa molecular mass. The enzyme was stable at pH 7.2 and 50 °C. The enzyme activity was inhibited in the presence of Pb(2+), Ag(2+), Hg(2+), and Zn(2+) and enhanced in the presence of Mn(2+). The enzyme activity was inhibited by the thiol-reducing reagents (DTT, β-mercaptoethanol), suggesting that disulfide linkage is essential for the enzyme activity. The enzyme activity was found to be maximum in the presence of Triton X-100 and X-114 detergents whereas sodium dodecyl sulfate fully inactivated the enzyme. The enzyme showed moderate stability towards all organic solvents except acetone, benzene, chloroform and the activity increased in the presence of isopropanol and ethanol. The K(m) value for the oxidation of cholesterol by this enzyme was 0.02 mM.
The applicability of the statistical tools coupled with artificial intelligence techniques was tested to optimize the critical medium components for the production of extracellular cholesterol oxidase (COD; an enzyme of commercial interest) from Streptomyces rimosus MTCC 10792. The initial medium component screening was performed using Placket-Burman design with yeast extract, dextrose, starch and ammonium carbonate as significant factors. Response surface methodology (RSM) was attempted to develop a statistical model with a significant coefficient of determination (R2 = 0.89847), followed by model optimization using Genetic Algorithm (GA). RSM-GA based optimization approach predicted that the combination of yeast extract, dextrose, starch and ammonium carbonate at concentrations 0.99, 0.8, 0.1, and 0.05 g/100 ml respectively, has resulted in 3.6 folds increase in COD production (5.41 U/ml) in comparison with the un-optimized medium (1.5 U/ml). COD was purified 10.34 folds having specific activity of 12.37 U/mg with molecular mass of 54 kDa. The enzyme was stable at pH 7.0 and 40 °C temperature. The apparent Michaelis constant (Km) and Vmax values of COD were 0.043 mM and 2.21 μmol/min/mg, respectively. This is the first communication reporting RSM-GA based medium optimization, purification and characterization of COD by S. rimosus isolated from the forest soil of eastern India.
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