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

被引：22

作者：

Colussi, Francieli ^{[1
]}

Serpa, Viviane ^{[1
]}

Delabona, Priscila da Silva ^{[1
,2
]}

Manzine, Livia Regina ^{[1
]}

Voltatodio, Maria Luiza ^{[1
]}

Alves, Renata ^{[1
]}

Mello, Bruno Luan ^{[1
]}

Pereira, Nei, Jr. ^{[3
]}

Farinas, Cristiane Sanches ^{[2
]}

Golubev, Alexander M. ^{[4
]}

Morim Santos, Maria Auxiliadora ^{[1
]}

Polikarpov, Igor ^{[1
]}

机构：

[1] Univ Sao Paulo, Inst Fis Sao Carlos, BR-13560970 Sao Carlos, SP, Brazil

[2] EMBRAPA Instrumentacao Agropecuaria, BR-13560970 Sao Carlos, SP, Brazil

[3] Univ Fed Rio de Janeiro, Escola Quim, Ctr Tecnol, Lab Desenvolvimento Bioproc LaDeBio, BR-21949900 Rio De Janeiro, Brazil

[4] St Petersburg Nucl Phys Inst, Dept Mol & Radiat Biophys, Gatchina 188300, Russia

来源：

JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY | 2011年 / 21卷 / 08期

基金：

巴西圣保罗研究基金会;

关键词：

Cellobiohydrolase I; catalytic core domain; Trichoderma harzianum 3844; purification; identification; PROTEIN SECONDARY STRUCTURE; PHANEROCHAETE-CHRYSOSPORIUM; REESEI CELLOBIOHYDROLASE; ENZYMATIC-HYDROLYSIS; CRYSTAL-STRUCTURES; CELLULOSE; CELLULASES; ENZYMES; CLASSIFICATION; GLYCOSYLATION;

D O I：

10.4014/jmb.1010.10037

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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 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-beta-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

引用

页码：808 / 817

页数：10

共 59 条

[1]

ABOULENEIN FA, 2010, INT J ACAD RES, V2, P81

[2]

Aden A., 2002, National Renewable Energy Laboratory: Lignocellulosic biomass to ethanol process design and economics utilizing cocurrent dilute acid prehydrolysis and enzymatic hydrolysis for corn stover

[3] Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis [J].

Arantes, Valdeir ;

Saddler, Jack N. .

BIOTECHNOLOGY FOR BIOFUELS, 2010, 3

[4] THE CCP4 SUITE - PROGRAMS FOR PROTEIN CRYSTALLOGRAPHY [J].

BAILEY, S .

ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 1994, 50 :760-763

[5]

Bakare MK, 2005, AFR J BIOTECHNOL, V4, P898

[6]

*BIOCH IUO, 1961, REP COMM ENZ

[7]

*BIOCH IUO, 1965, ENZ NOM REC 1964 INT

[8]

Boer H, 2000, BIOTECHNOL BIOENG, V69, P486, DOI 10.1002/1097-0290(20000905)69:5<486::AID-BIT3>3.0.CO

[9]

2-N

[10]

BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3

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