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Purification, and biochemical and biophysical characterization of cellobiohydrolase I from Trichoderma harzianum IOC 3844
Authors:Colussi Francieli  Serpa Viviane  Delabona Priscila da Silva  Manzine Livia Regina  Voltatodio Maria Luiza  Alves Renata  Mello Bruno Luan  Pereira Nei  Farinas Cristiane Sanches  Golubev Alexander M  Santos Maria Auxiliadora Morim  Polikarpov Igor
Institution:Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo Av. Trabalhador Saocarlense 400, Sao Carlos 13560-970, SP, Brazil.
Abstract: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 alpha- helices and beta-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 degrees C with specific activities against Avicel and p-nitrophenyl-β-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.
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