Neriifolin S, a dimeric serine protease from Euphorbia neriifolia Linn.: Purification and biochemical...

A dimeric serine protease Neriifolin S of molecular mass 94kDa with milk clotting activity has been purified from the latex of Euphorbia neriifolia by anion exchange and size-exclusion chromatography. It hydrolyses peptidyl substrates l-Ala-pNA with highest affinity (K_m of 0.195mM) and physiological efficiency (K_cat/K_m of 144.5mMs). Enzyme belongs to the class of neutral proteases with pI value of 6.8, optimal proteolytic activity displayed at pH 9.5 and temperature 45°C. Its proteolytic activity is strongly stimulated in the presence of Ca⁺² ions and exclusively inhibited by serine protease inhibitors. Enzyme is fairly stable toward chemical denaturants, pH and temperature. The apparent T_m, was found to be 65°C. Thermal inactivation follow first order kinetics with activation energy (Ea), activation enthalpy (ΔH∗), free energy change (ΔG∗) and entropy (ΔS∗) of 27.54kJmol^-1, 24.89kJmol^-1, -82.34kJmol^-1 and 337.20Jmol^-1K^-1.