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

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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.