Purification, catalytic, kinetic and thermodynamic characteristics of a novel ficin from Ficus johannis

Ficin from Ficus johannis was purified to homogeneity by (NH4)2SO4 precipitation, CM-Sepharose and SP-Sepharose chromatography, and gel filtration Sephadex G-25 chromatography. The enzyme, that is characterized by a molecular weight of 25 kDa measured by SDS-PAGE, showed higher affinity towards casein and the highest catalytic efficiency at pH 7.0 and 60 °C. Catalytic hydrolysis rate follows the Michaelis-Menten equation and values of 32.9 s−1 and 13.7 µM for kcat and Km respectively, by Michaelis-Menten, were found. The pKa1 and pKa2 values of the active site ionizable groups controlling Vmax were 4.2 and 8.7, respectively. Thermodynamic parameters for soluble casein hydrolysis and for irreversible inactivation of ficin at different temperatures in the range from 45 to 60 °C were also determined. This is the first report on the thermodynamic parameters of ficin produced by Ficus johannis. These results indicate that Ficin from Ficus johannis may have potential applications in the industrial biotechnology.