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( Colussi Francieli ),( Viviane Serpa ),( Priscila Da Silva Delabona ),( Livia Regina Manzine ),( Maria Luiza Voltatodio ),( Renata Alves ),( Bruno Luan Mello ),( Nei Pereira Jr. ),( Cristiane Sanches 한국미생물 · 생명공학회 2011 Journal of microbiology and biotechnology Vol.21 No.8
Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed α- helices and β-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50℃ with specific activities against Avicel and p-nitrophenyl-β-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.