Yin Qiuyu, Teng Yigang, Li Yanhong, Ding Ming, Zhao Fukun
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Biosci Biotechnol Biochem. 2011;75(2):240-6. doi: 10.1271/bbb.100529. Epub 2011 Feb 7.
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.
在之前的研究中，从软体动物福寿螺中克隆出了三个内切葡聚糖酶cDNA，即eg65a、eg65b和eg65c。为了通过异源表达分析来表征全长酶及其各个功能模块，将三个全长推定内切葡聚糖酶（rEG65a、rEG65b和rEG65c）以及相应的催化模块（EG65a-CM、EG65b-CM和EG65c-CM）在毕赤酵母GS115中进行表达，将三个相应的碳水化合物结合模块（EG65a-CBM、EG65b-CBM和EG65c-CBM）在大肠杆菌BL21(DE3)中进行表达。对重组rEG65b、EG65a-CM、EG65b-CM和EG65c-CM的性质进行了表征。CBM与不溶性多糖的结合试验表明，EG65b-CBM和EG65c-CBM均能与磷酸膨胀纤维素（PASC）、微晶纤维素和燕麦麸木聚糖结合，而EG65a-CBM则不能。通过吸收等温线测量确定了EG65b-CBM和EG65c-CBM的相对平衡常数（K(r)）。在本研究中，首次对动物纤维素酶的CBM进行了表达和表征。
