Extracellular alkaline protease from Bacillus licheniformis NCIM-2042: Improving enzyme activity assay and characterization

Optimization of enzyme assay conditions for alkaline protease from Bacillus licheniformis NCIM-2042 was carried out by a statistical approach. Four key determinants such as pH, temperature, buffer concentration, and incubation time were optimized by response surface methodology using rotatable central composite design. Maximum enzyme activity was found to be at pH 9.0, temperature 75 degrees C in phosphate buffer (50mM) when incubated for 10 min. Protease was stable over a broad range of pH 6.0-12.0 and it was stable at 50 degrees C for 1 h. The protease was completely inhibited by PMSF (5 mM), suggesting that the enzyme is a serine alkaline protease. This enzyme had good stability in the presence of H2O2, SDS, Triton X-100, and retained more than 88% of its initial activity after preincubation for 30 min at room temperature in the presence of 25% v/v DMSO, methanol, ethanol, ACN, and 2-propanol.