Calcium ion induced thermodynamic stability, bisubstrate specificity, and differential organic solvent tolerance of a predominantly β-sheet serine protease from Bacillus aquimaris VITP4.

Abstract

The present study was aimed to get insights on the role of calcium ions on the thermodynamic stability, substrate specificity, and organic solvent compatibility of the extracellular protease produced by Bacillus aquimaris VITP4. Presence of Ca2+ enhanced the activity of the enzyme in the temperature range of 30-60°C and increased the half-life from 164 to 234Min. Circular dichroism experiments indicated that the temperature of half-denaturation (Tm ) of the protease increased from 76 to 86°C. As judged by fluorescence emission profiles, the overall fold of the enzyme around the tryptophan residues could be similar. Further, thermal inactivation experiments revealed that the enzyme followed first order kinetics, with increase in energy for inactivation (Eai ) by 24.2±1.2kJmol-1 in the presence of Ca2+ . Studies with synthetic peptides as well as with bovine serum albumin signified preferential hydrolysis of the peptide bonds at the C-terminus of alanine residues (with a kcat /KM of 141,400M-1 Sec-1 ) and at the C-terminus of arginine residues with a lower specificity (72,400M-1 Sec-1 ), indicating bisubstrate specificity of the enzyme. The enzyme was found to be compatible with organic solvents (50%, v/v) such as acetonitrile and butanol, indicating possible application under demanding nonaqueous conditions.