Purification and biochemical characterization of a chymotrypsin-like serine protease from Euphorbia neriifolia Linn.

Abstract Neriifolin, a chymotrypsin-like serine protease, has been purified from the latex of Euphorbia neriifolia Linn. by ammonium sulfate precipitation, cation exchange chromatography and gel filtration. The molecular mass of the enzyme is 35.24 kDa, with an isoelectric point of pH 5.7. The enzyme consists of 18 tryptophan, 25 tyrosine and 9 cysteine residues with 4 disulfide bridges. The extinction coefficient ( e 280 nm 1 % ) is 38.28. The K m values are 1.39 ± 0.08 mM and 1.94 ± 0.17 mM, with N -succinyl- l -Phe- p -nitroanilide and α-leucine- p -nitroanilide as substrates, respectively. Neriifolin retains proteolytic activity over a wide range of pH and temperature value, with pH optima of 8.5 and an optimal temperature of 55 °C. Inhibition of enzyme activity by chymostatin and amidolytic activity against synthetic substrates specific to chymotrypsin indicates that the enzyme belongs to chymotrypsin-like serine protease class. Polyclonal antibodies specific to neriifolin and immunodiffusion reveal that the enzyme has unique antigenic determinants. The amino terminal sequence of the first 14 residues of neriifolin is D–F–P–P–N–T–H–I–G–I–P–N–G–Y. A high ratio of milk-clotting activity to proteolytic activity as well as stability against variations in pH and temperature, surfactants, oxidizing agents and compatibility with detergent additives make neriifolin an excellent candidate for industrial applications.