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

► Neriifolin S is a dimeric serine protease with broad substrate specificity. ► Enzyme is active over a wide range of pH (6–10.5) and temperature (20–60°C). ► Enzyme reveals high conformational stability against chaotrops and organic solvents. ► Enzyme exhibit milk clotting activity with higher MCA/PA of 433.25U/OD 660nm. ► Enzyme is resistant to autodigestion at higher concentration. 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 (Km of 0.195mM) and physiological efficiency (Kcat/Km 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 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 27.54kJmol−1, 24.89kJmol−1, −82.34kJmol−1 and 337.20Jmol−1K−1.