title: Expression and characterization of full-length Ampullaria crossean endoglucanase EG65s and their two functional modules.
abstract: 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.
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Qiuyu
authors.initials: Q
authors.lastname: Yin
authors.forename: Yigang
authors.initials: Y
authors.lastname: Teng
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Li
authors.forename: Ming
authors.initials: M
authors.lastname: Ding
authors.forename: Fukun
authors.initials: F
authors.lastname: Zhao
mesh_terms: D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D050260:Carbohydrate Metabolism; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D049851:Gastropoda / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D002480:Cellulase