Purification and characterization of moderately halophilic alkaline serine protease from marine Bacillus subtilis AP-MSU 6

Purification and characterization of moderately halophilic alkaline protease secreted by marine fish intestinal isolate Bacillus subtilis AP-MSU 6 was studied. The protease was purified from the culture supernatant to homogeneity using three steps viz. ammonium sulfate precipitation, DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-75 with 23.99% recovery and 27.63 fold increase in specific activity. The molecular weight of purified protease was determined as 18.3 kDa. The optimum pH, temperature and NaCl concentration required for maximum protease activity were 9.0, 40 °C and 0.5 M respectively. The activity of the protease was stimulated by Cu2+, Hg2+, Mn2+ and Ba2+ and it was resistant to SDS, Tween 20 and Tween 40. The protease activity was strongly inhibited in the presence of PMSF and thus it was confirmed as serine protease.