Journal
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
Volume 75, Issue 2, Pages 240-246Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.100529
Keywords
endoglucanase; EG65; carbohydrate-binding module; adsorption isotherm; Ampullaria crossean
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Funding
- National Natural Science Foundation of China [30370336]
- Major State Basic Research Development Program of China [2003CB716006, 2004CB719702]
- Creation Foundation of the Shanghai Institutes for Life Sciences
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Three endoglucanase cDNAs, eg65a, eg65b, and eg65c, were cloned from the mollusk Ampullaria crossean in previous work. To characterize the full-length enzymes as well as their individual functional modules via heterologous expression analysis, the three full-length putative endoglucanases (rEG65a, rEG65b, and rEG65c) and the corresponding catalytic modules (EG65a-CM, EG65b-CM, and EG65c-CM) were expressed in Pichia pastoris GS115, and the three corresponding carbohydrate-binding modules (EG65a-CBM, EG65b-CBM, and EG65c-CBM) were expressed in Escherichia coli BL21 (DE3). The properties of recombinant rEG65b, EG65a-CM, EG65b-CM, and EG65c-CM were characterized. Binding assays of CBMs with insoluble polysaccharides indicated that both EG65b-CBM and EG65c-CBM bound to phosphoric-acid swollen cellulose (PASC), Avicel, and oat-spelt xylan, while EG65a-CBM did not. The relative equilibrium constants (K-r) of EG65b-CBM and EG65c-CBM were determined by absorption isotherm measurements. In this study, the CBMs of animal cellulases were expressed and characterized for the first time.
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