Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and v. suchlasporium

Abstract:
Culture filtrates of the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium growing on colloidal chitin showed increasing chitinolytic activity and production of two (32- and 43-kDa) main proteins. Maximum activity was found 18-20 days after inoculation, but V. suchlasporium always displayed higher activity. Zymography of such filtrates on carboxymethyl-chitin-Remazol brilliant violet 5R/acrylamide gels showed five bands of substrate degradation for V. suchlasporium and three for V. chlamydosporium. Filtrates with maximum activity were chromatographed on macroporous cross-linked chitin affinity matrix, showing a peak of main (50-60%) activity, which only contained a 43-kDa protein for both fungi. Zymography and colloidal chitin degradation showed that it was a single endochitinase (CHl43) with optimum pH range of 5.2-5.7. The main isoforms had pis of 7.6 for V. suchlasporium and 7.9 for V. chlamydosporium. Eggs of the nematode Globodera pallida treated with CHl43 and the serine protease P32 from V. suchlasporium alone or in combination showed surface damage in comparison with controls when examined by scanning electron microscopy. (C) 2002 Elsevier Science (USA).