PURIFICATION AND CHARACTERIZATION OF HEAVY METAL INDUCED PROTEASE FROM Pseudomonas fluorescens ATCC 948

The microorganisms that live in association with plant or animal cells rely extensively on the production of extracellular proteases, secondary metabolites and siderophores for their survival. In the gram-negative bacteria, the expression of extracellular products is controlled by the conserved two-component regulatory system consisting sensor kinase GacS and cognate response regulator GacA that also influences the stress tolerance in these bacteria. Oxidative stress is one of the major reasons for the formation of oxidized proteins in aerobic bacteria. During stress, bacteria adapt to the presence of reactive oxygen species (ROS) and oxidation of proteins, by increasing the expression of detoxifying enzymes, protein and DNA repair molecules, which helps in keeping the concentration of these species to sub toxic level by removing the unwanted proteins. This role is attributed to proteases, as they are known to scavenge these oxidized proteins. In response to heavy metal stress (lead, copper and cobalt) in Pseudomonas fluorescens ATCC 948, protease increases in culture supernatant. The highest protease activity was found with exposure of culture to lead. The lead-induced protease was purified to homogeneity and was identified as 33kDa metalloprotease. The purified protease was maximally active at 200C and pH 6.0. The nature of the protease was elucidated using different protease inhibitors, which indicated the protease as metalloprotease. The enzyme is characterized and its role in mechanism of adaptation to heavy metal stress has been discussed.