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

A dimeric serine protease Neriifolin S of molecular mass 94 kDa 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 (Km of 0.195 mM) and physiological efficiency (Kcat/Km of 144.5 mM s). 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 Tm, 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 27.54 kJ mol−1, 24.89 kJ mol−1, −82.34 kJ mol−1 and 337.20 J mol−1 K−1.