title: Purification, and biochemical and biophysical characterization of cellobiohydrolase I from Trichoderma harzianum IOC 3844.
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.
authors.affiliation: Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo Av. Trabalhador Saocarlense 400, Sao Carlos 13560-970, SP, Brazil.
authors.forename: Francieli
authors.initials: F
authors.lastname: Colussi
authors.forename: Viviane
authors.initials: V
authors.lastname: Serpa
authors.forename: Priscila da Silva
authors.initials: Pda S
authors.lastname: Delabona
authors.forename: Livia Regina
authors.initials: LR
authors.lastname: Manzine
authors.forename: Maria Luiza
authors.initials: ML
authors.lastname: Voltatodio
authors.forename: Renata
authors.initials: R
authors.lastname: Alves
authors.forename: Bruno Luan
authors.initials: BL
authors.lastname: Mello
authors.forename: Nei
authors.initials: N
authors.lastname: Pereira
authors.forename: Cristiane Sanches
authors.initials: CS
authors.lastname: Farinas
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Golubev
authors.forename: Maria Auxiliadora Morim
authors.initials: MA
authors.lastname: Santos
authors.forename: Igor
authors.initials: I
authors.lastname: Polikarpov
mesh_terms: D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014242:Trichoderma / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase