Decolorization of Crude Latex by Activated Charcoal, Purification and Physico-Chemical Characterization of Religiosin, a Milk-Clotting Serine Protease from the Latex of Ficus religiosa

The crude latex of Ficus religiose is decolorized by activated charcoal. Decolorization follows the Freundlich and Langmuir equations. A serine protease, named religiosin, has been purified to homogeneity from the decolorized latex using anion exchange chromatography. Religiosin is a glycoprotein with a molecular mass of 43.4 kDa by MALDI-TOF. Religiosin is an acidic protein with a pI value of 3.8 and acts optimally at pH 8.0-8.5 and temperature 50 degrees C. The proteolytic activity of religiosin is strongly inhibited by PMSF and chymostatin indicating that the enzyme is a serine protease. The extinction coefficient (epsilon(1%)(280)) of religiosin is 29.47 M-1 cm(-1) with 16 tryptophan, 26 tyrosine, and 11 cysteine residues per molecule. The enzyme shows broad substrate specificity against natural as well as synthetic substrates with an apparent K-m of 0.066 mM and 6.25 mM using casein and Leu-pNA, respectively. MS/MS analysis confirms the novelty of the enzyme. Religiosin is highly stable against denaturants, metal ions, and detergents as well as over a wide range of pH and temperature. In addition, the enzyme exhibits milk-clotting as well as detergent activity.