Structural and functional analysis of insoluble-polysaccharide-hydrolyzing-enzyme by chimeric enzymes construction.

为了了解纤维素酶和木聚糖酶这两种不溶性多糖水解酶的结构和功能关系，我们计划基于两种酶的立体结构构建刺曲霉纤维素酶和矮芽孢杆菌木聚糖酶之间的嵌合蛋白（酶）的研究。利用这两种酶的基因制备嵌合酶蛋白。采用限制性内切位点法和/或PCR法分别构建了XCX和CXC两个化学基因。构建的基因经核苷酸测序鉴定。这两个基因在大肠杆菌中均有表达。在大肠杆菌中表达的嵌合蛋白用常规色谱技术纯化至均匀性。纯化的两种嵌合蛋白均与原菌株的纤维素酶和木聚糖酶抗血清反应。分析了嵌合蛋白对敏感底物（4-methylumbeiferyl- β - d -cellobiose, cellotriose，木糖）的活性。嵌合蛋白的活性低于可食用水平。

To understand the structure and function relationsip of the insoluble polysaccharide-hydrolyzing-enzymes, such as cellulase and xylanase, we planned the research that construct chimeric protein (enzyme) between cellulase of Aspergillus aculeatus and xylanase of Bacillus pumilus that based on both stereostructure of the enzymes. To produce the chimeric enzyme proteins, the genes of the both enzymes were used. The chemeric genes which named XCX and CXC,were constructed by using restriction site and/or PCR method. The constructed genes were identified by the nucleotide sequencing. Both genes were expressed in Escherichia coli. The chimeric proteins that expressed in E.coli were purified to homogeneity by conventional chromatographic tequnique. Both purified chimeric proteins were reacted to antiserum against cellulase and xylanase from original strains. The activity of the chimeric proteins were analyzed to sensitive substrates, which were 4-methylumbeiferyl-beta-D-cellobiose, cellotriose, and xylose, respectively. The activities of the chimeric proteins were less than ditectable level.