Affiliation: Marine Biotechnology Laboratory, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam, 629 502 Kanyakumari District, Tamilnadu, India.
Protease enzyme from estuarine sediment bacterium Bacillus flexus was purified by using two steps such as ammonium sulphate precipitation (37.25% enzyme yield and 2.75 fold purification) and sephadex G-75 gel filtration chromatography (9.38% yield and 10.62 fold purification). Molecular weight of the purified protease detected was 44.3 kDa. The protease activity was found to be maximum at pH 8.0 and retained its activity in the pH range of 8-9 after 1.5 h of incubation. Also at 40°C the protease activity was maximum and remained stable between a temperature range of 40 to 50°C after 1.5 h of incubation. This enzyme is slightly halophilic and the maximum activity was observed in 0.5 M NaCl concentration. Further, the tested surfactants were found to enhance the protease activity; also this enzyme maintained its activity in the presence of SDS (5mM). Among the metal ions tested, mercuric chloride and zinc chloride completely inhibited the protease activity and optimum activity was registered in medium added with barium chloride and magnesium chloride. The serine protease inhibitor was found to inhibit 90% activity and hence it was further confirmed as a serine protease type. This enzyme effectively hydrolyzed casein when compared to BSA and gelatin.