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http://dx.doi.org/10.4014/jmb.1010.10037

Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844  

Colussi, Francieli (Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo)
Serpa, Viviane (Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo)
Da Silva Delabona, Priscila (Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo)
Manzine, Livia Regina (Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo)
Voltatodio, Maria Luiza (Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo)
Alves, Renata (Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo)
Mello, Bruno Luan (Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo)
Nei, Pereira Jr. (Centro de Tecnologia, Escola de Quimica, Laboratorio de Desenvolvimento de Bioprocessos (LaDeBio), Universidade Federal do Rio de Janeiro)
Farinas, Cristiane Sanches (EMBRAPA Instrumentacao Agropecuaria)
Golubev, Alexander M. (Department of Molecular and Radiation Biophysics, St. Petersburg Nuclear Physics Institute)
Santos, Maria Auxiliadora Morim (Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo)
Polikarpov, Igor (Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo)
Publication Information
Journal of Microbiology and Biotechnology / v.21, no.8, 2011 , pp. 808-817 More about this Journal
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 $50^{\circ}C$ with specific activities against Avicel and p-nitrophenyl-${\beta}$-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.
Keywords
Cellobiohydrolase I; catalytic core domain; Trichoderma harzianum 3844; purification; identification;
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