Purification and characterization of a small size protease from <i>Bacillus </i>sp. APR-4

Abstract

A thermostable extracellular protease of <i>Bacillus </i>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 <i>p</i>H of 9 and

exhibited its highest activity at 60°C. The enzyme activity was inhibited by EDTA, suggesting the presence of metal residue at the active site. Ca²⁺ (5 m<i>M</i>)<i> </i>had stabilising effect on the activity of protease, but Cu²⁺(5 m<i>M</i>)<i> </i>had inhibitory effect. The enzyme exhibited highest specificity towards casein (1%) and had a K_m of 26.3 mg/ml and a V_max of 47.6 U/mg<i> </i>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 the activity of this enzyme.