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

• Published in: Journal of microbiology and biotechnology Vol. 21; no. 8; pp. 808 - 817
• Main Authors: Colussi, Francieli, Universidade de Sao Paulo, Sao Carlos, SP, Brazil, Serpa, Viviane, Universidade de Sao Paulo, Sao Carlos, SP, Brazil, Delabona, Priscila da Silva, Universidade de Sao Paulo, Sao Carlos, SP, Brazil, Manzine, Livia Regina, Universidade de Sao Paulo, Sao Carlos, SP, Brazil, Voltatodio, Maria Luiza, Universidade de Sao Paulo, Sao Carlos, SP, Brazil, Alves, Renata, Universidade de Sao Paulo, Sao Carlos, SP, Brazil, Mello, Bruno Luan, Universidade de Sao Paulo, Sao Carlos, SP, Brazil, Pereira Jr., Nei, Universidade Federal do Rio de Janeiro, RJ, Brazil, Farinas, Cristiane Sanches, EMBRAPA Instrumentacao Agropecuaria, Sao Carlos, SP, Brazil, Golubev, Alexander M., St. Petersburg Nuclear Physics Institute, Gatchina, Russia, Santos, Maria Auxiliadora Morim, Universidade de Sao Paulo, Sao Carlos, SP, Brazil, Polikarpov, Igor, Universidade de Sao Paulo, Sao Carlos, SP, Brazil
• Format: Journal Article
• Language: English
• Published: Seoul Korean Society for Applied Microbiology 01.08.2011; 한국미생물·생명공학회
• Subjects: Amino Acid Sequence; Biophysical Phenomena; Biophysics; catalytic core domain; Cellobiohydrolase I; Cellulose 1,4-beta-Cellobiosidase - chemistry; Cellulose 1,4-beta-Cellobiosidase - genetics; Cellulose 1,4-beta-Cellobiosidase - isolation & purification; Cellulose 1,4-beta-Cellobiosidase - metabolism; Enzyme Stability; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Fungal Proteins - metabolism; IDENTIFICACION; IDENTIFICATION; Models, Molecular; Molecular Sequence Data; Protein Structure, Tertiary; PURIFICACION; PURIFICATION; Substrate Specificity; Trichoderma - chemistry; Trichoderma - enzymology; Trichoderma - genetics; Trichoderma harzianum 3844; 생물학
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.1010.10037

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 small-angle 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.