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KMID : 0545120110210080808
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Journal of Microbiology and Biotechnology
2011 Volume.21 No. 8 p.808 ~ p.817
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Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844
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Francieli Colussi
Viviane Serpa
Priscila da Silva Delabona
Livia Regina Manzine
Maria Luiza Voltatodio
Renata Alves
Bruno Luan Mello
Nei Pereira Jr.
Cristiane Sanches Farinas
Alexander M. Golubev
Maria Auxiliadora Morim Santos
Igor Polikarpov
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Abstract
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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 50oC 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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KEYWORD
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Cellobiohydrolase I, catalytic core domain, Trichoderma harzianum 3844, purification, identification
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