Purification and characterization of a highly stable cysteine protease from the latex of Ervatamia coronaria

A highly stable cysteine protease was purified to homogeneity from the latex of Ervatamia coronaria by a simple purification procedure involving ammonium sulfate precipitation and ion-exchange chromatography. The molecular mass was estimated to be approximately 25,000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(280nm)(1%)) Of the enzyme was 24.6. The enzyme hydrolyzed denatured natural substrates like casein, hemoglobin, azoalbumin, and azocasein with a high specific activity but showed low specific activity towards synthetic substrates. The pH and temperature optima were 7.5-8.0 and 50 degrees C respectively. The activity of the enzyme was strongly inhibited by thiol-specific inhibitors like leupeptin, iodoacetamide, PCMB, NEM, and mercuric chloride. The striking property of this enzyme was its stability over a wide pH range (2-12) and other extreme conditions of temperature, denaturants, and organic solvents. The N-terminal sequence showed marked similarity to known cysteine proteases.