ABSTRACT
Nocardia globerula NHB-2 exhibited an intracellular acetonitrile hydrolysing activity (AHA) when cultivated in nutrient broth supplemented with glucose (10.0 g/l) and yeast extract (1.0 g/l), at pH 8.0, 30 degrees C for 21 hr. Maximum AHA was recorded in the culture containing 0.1 M of sodium phosphate buffer, (pH 8.8) at 45 degrees C for 15 min with 600 micromol of acetonitrile and resting cells of N. globerula NHB-2 equivalent to 1.0 ml culture broth. This activity was stable up to 40 degrees C and was completely inactivated at or above 60 degrees C. About five-fold increase in AHA was observed after optimization of culture and reaction conditions. Under the optimized conditions, this organism hydrolyzed various nitriles and amides such as propionitrile, benzonitrile. acetamide, and acrylamide to corresponding acids. This nitrile/amide hydrolysing activity of N. globerula NHB-2 has potential applications in enzymatic synthesis of organic acids and bioremediation of nitriles and amides contaminated soil and water system.
Subject(s)
Acetonitriles/metabolism , Catalysis , Culture Media , Hydrogen-Ion Concentration , Hydrolysis , Ions/chemistry , Metals/chemistry , Nocardia/drug effects , Substrate Specificity , Temperature , Time FactorsABSTRACT
A thermostable extracellular protease of Bacillus sp. APR-4 was purified by size-exclusion and ion-exchange chromatographic methods and its properties were studied. The purified enzyme had a specific activity of 21,000 U/mg of protein and gave single band on SDS/PAGE with a molecular mass of 16.9 KDa. This protease had an optimal pH of 9 and exhibited its highest activity at 60 degrees C. The enzyme activity was inhibited by EDTA, suggesting the presence of metal residue at the active site. Ca2+ (5 mM) had stabilising effect on the activity of protease, but Cu2+ (5 mM) had inhibitory effect. The enzyme exhibited highest specificity towards casein (1%) and had a Km of 26.3 mg/ml and a Vmax of 47.6 U/mg with casein as a substrate. The stability of this enzyme was evaluated in the presence of some organic solvents and the enzyme was stable in methanol, petroleum ether and ethanol. Detergents (Wheel, Farishta) had stimulatory effect on the activity of this enzyme.
Subject(s)
Alkanes/chemistry , Bacillus/enzymology , Binding Sites , Calcium/chemistry , Caseins/chemistry , Chromatography, Gel , Chromatography, Ion Exchange , Detergents/pharmacology , Electrophoresis, Polyacrylamide Gel , Endopeptidases/chemistry , Ethanol/chemistry , Hydrogen-Ion Concentration , Kinetics , Magnesium/metabolism , Methanol/chemistry , Proteins/chemistry , Solvents/chemistry , Substrate Specificity , TemperatureABSTRACT
The inhibitory effects of aflatoxin B1 were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0·05 % w/v) or Tween-80 (0·05 % v/v) addition accentuated the aflatoxin B1 growth inhibition in Salmonella typhi and Escherichia coli at different pH values. The inhibition of lipase production was only 5–20 % in Pseudomonas fluorescence ca. 25–48% in Staphylococcus aureus and Bacillus cereus at different aflatoxin B1 concentrations (4–16 μg/ml).However, inhibition of α-amylase induction was complete in Bacillus megaterium whereas the inhibition was partial in Pseudomonas fluorescence (27–40%) at 32 μg aflatoxin B1 concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B1 concentration.