Muhammad Nadeem, Javed Iqbal Qazi, Quratulain Syed, and Muhammad Gulsher

pp. 187 - 195

Abstract

Alkaline protease produced by mutant strain B. licheniformis UV-9 was purified and characterized for its exploitation
in detergent formulation. The enzyme was purified to homogeneity by employing ammonium sulphate precipitation and
sephadex G-100 gel filtration chromatography with a 36.83 fold increase in specific activity and 11% recovery. The molecular
weight of the protease was found to be 36.12 kDa by SDS-PAGE. The Km and Vmax values exhibited by purified protease
were 5 mg/ml and 61.58ìM/ml/min, respectively, using casein as substrate. The enzyme exhibited highest activity at pH 11 and
temperature 60°C. Stability studies showed that the enzyme retained higher than 80% residual activity in the pH and temperature ranges of 8 to 11 and 30 to 50°C, respectively. However, in the presence of 10 mM Ca²⁺ ions the enzyme tained more
than 90% of its residual activity at pH 11 and temperature 60°C. Phenyl methyl sulphonyl fluoride (PMSF) completely
inhibited the enzyme activity suggesting that it was serine protease. Among metal ions, the Mg²⁺ and Ca²⁺ ions enhanced
activity up to 128% and 145%, respectively. The purified enzyme showed extreme stability towards various surfactants
such as Tween-20, Tween- 45, Tween-65 and Triton X-45. In addition, the enzyme also exhibited more than 100% residual
activity in the presence of oxidizing agents, H₂O₂
 and sodium perborate. These biochemical properties indicate the potential
use of B. licheniformis UV-9 enzyme in laundry detergents.