Purification and characterization of a small size protease from Bacillus sp. APR-4.
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.
Kumar
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Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171 005, India.
Bhalla
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TC
D000473:Alkanes / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002364:Caseins / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D000431:Ethanol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature
D000473:Alkanes; D002364:Caseins; D003902:Detergents; D011506:Proteins; D012997:Solvents; D000431:Ethanol; D010450:Endopeptidases; D008274:Magnesium; C004544:naphtha; D002118:Calcium; D000432:Methanol