Purification and properties of extracellular proteases produced by the nematophagous fungus Verticillium suchlasporium

Abstract

The fungal parasite of eggs of cyst nematodes, Verticillium suchlasporium, produced extracellular proteases when grown in semiliquid culture with gelatin as the only source of nitrogen and carbon. The proteolytic activity of culture filtrates was maximum 12–14 days after inoculation. Gel filtration chromatography in Sephadex G-100 resolved two peaks of proteolytic activity. The peak accounting for most of the activity was further purified by ion-exchange chromatography in SP-Sephadex C-25 as a single peak. This protease had a molecular mass of 32 kDa calculated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The enzyme was an endopeptidase that degraded fibrinogen in zymograms and had an optimum pH of 8.5 using fluorescein isothiocyanate – casein as the substrate. It was inhibited by phenylmethylsulfonyl fluoride, indicating that it was a serine protease. The purified protease was able to degrade certain cyst nematode proteins suggesting the involvement and specificity of the 32-kDa protease during nematode egg penetration by Verticillium suchlasporium. Key words: nematophagous fungi, Verticillium suchlasporium, extracellular enzymes, serine proteases.