Purification and characterization of an extracellular 24 kDa chitobiosidase from the mycoparasitic fungus Trichoderma saturnisporum.

A Trichoderma saturnisporum Hamill isolate GITX-Panog (C) exhibiting strong chitinolytic and antifungal activity against Fusarium oxysporum f.sp. dianthi, the causal agent of vascular wilt in carnation was used to purify extracellular chitobiosidase using Czapek-Dox broth amended with the fungal mycelium as the carbon source. The protein was purified by precipitation with ammonium sulphate, followed by DEAE-Cellulose anion-exchange and Sephacryl S-200 high resolution gel filtration chromatography. The purity of the enzyme was determined by SDS-PAGE, with an estimated molecular mass of 24 kDa. In native gel assay with 4-methylumbelliferyl -N,N ′ diacetyl-β-D-chitobioside (4-Mu-(GluNAc)2, the purified chitobiosidase was visualized as single fluorescent band. Enzyme activity towards short oligomeric natural substrates indicated that the enzyme has properties that are characteristic to exochitinases. The enzyme was active up to 60 °C and at pH 4.0, and displayed maximum stability at 50 °C. Mn2+ and Zn2+ stimulated the enzyme activity by 63% and 41%, respectively. The Km and Vmax values of the purified enzyme for 4-Mu-(GluNAc)2were 338.9 μM ml–1 and 0.119 μM ml–1 min–1, respectively. This appears to be the first report of characterization of a chitobiosidase from antagonistic Trichoderma saturnisporum. (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)