Enhancement of thermostability and resistance against autolysis in a zinc metalloprotease

Neutral proteases are inactivated at higher temperatures because of autolysis. It appears that autolysis involves some specific solvent-exposed regions that become prone to local unfolding as temperature increases. Accordingly, we designed surface-located mutations at the N-terminal loops (A56P and T73F) of the neutral protease from Salinivibrio proteolyticus and compared the thermostability and autolysis as well as structural properties of wild-type (WT) and mutant proteins. Circular dichroism in far-UV region and intrinsic fluorescence data indicated that compactness of protein increases upon mutation. It was revealed that the catalytic efficiency (kcat/Km) of the enzyme is improved in mutants and optimum temperature of mutants increases relative to WT enzyme. It was also shown that the mutant enzymes are more resistant against autolysis and their thermostability and kinetic parameters are also changed compared to WT protein. This study shows that the stability of enzyme against autolysis and temperature may be increased even by changing only a single amino acid, which in turn is important from application point of view.