PRODUCTION OF AN EXTRACELLULAR TRYPSIN-LIKE PROTEASE BY THE FUNGAL PLANT PATHOGEN VERTICILLIUM-DAHLIAE

The plant pathogenic fungus Verticillium dahliae produced extracellula r alkaline protease activity when grown in liquid medium supplemented with a protein source. A serine protease was purified 80-fold in a sin gle step, using cation-exchange chromatography, from the filtrate of c ultures grown with skim milk as a protein source. N-terminal amino aci d sequence analysis of the 30-kDa protein (VDP30) that copurified with the serine protease activity suggested that VDP30 is a trypsin-like p rotein. The purified enzyme hydrolyzed the synthetic substrate N alpha -benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA), and the act ivity on BAPNA was inhibited by leupeptin, further verifying the tryps in-like nature of the enzyme.