The Research Of Fusing A Thermostable Cellulose Binding Domain To The Xylanase XynⅢ From Aspergillus Niger

Xylanase is the enzyme family that dehydrates xylan into xylose oroligoxylose.Xlyanase has extensive applications in the industries of feed, food, fabric and papermaking, and has become one of the hot spots of modern enzymology.But it doesn’t work well in the extreme environments such as highly acidic or highly alkalic.So it’s all-important to recontrust the enzyme to fulfill the industrial needs.This study chose the gene, xynⅢwhich encoding the mature protein of an endo-β-1,4-xylanase obtained from Aspergillus niger A-25 and the gene cbd which encoding the cellulose binding domain of the XynA obtained from Thermotoga maritime as materials to reconstrust the XYN-Ⅲ, and then overexpressed in Escherichia coli to investigate the characterizations of the recombinant proteins.There were three parts in this study:(1) Construction of the chimeric gene:the gene of CBD (1107bp) from Thermotoga maritime presented from JiangNan University was fused at the carboxyl-termminus of xynⅢ(552bp) by the Staggered extention PCR.The DNA sequencing result indicates that the two genes were fused together accurately.(2) The XynⅢand the XynⅢ-CBD were expressed in Escherichia coli:The xynⅢand xynⅢ-cbd were inserted into the expression vector of pET20b(+) separately and transformed into E.coli BL21(DE3) which were expressed successfully in Escherichia coli by inducing with IPTG. But wasn’t secreted. The recombinant proteins was purified by Co-NTA sepharose 6BFF.The purified protein XynⅢwas detected to reach the enzyme activity of 41.2 IU/ml, while the XynⅢ-CBD was 5.25 IU/ml.(3) The properties of the chimeric enzyme were determined:The optimal temperature of the chimeric enzyme was 2℃higher than the parental enzyme;and the optinal pH was improved 0.2 units.A notable feaure on substrate specificity was that the chimeric enzyme had a larger capacity to hydrolases oat spelt xylan.