| Summary: | 碩士 === 國立交通大學 === 應用化學系所 === 93 === A powerful chitosanase for the preparation of chitooligosaccharide was previously purified from Aspergillus fumigatus. The corresponding gene was also cloned and the enzyme was further classified into glycosyl hydrolase family 75. The recombinant chitosanase was over-expressed in E. coli with a form of inclusion body, which was rescued by treating with 5 M urea and subsequently purified by cation-exchanged chromatography. Alternatively, the recombinant enzymes were also expressed in a pRSET_SP system containing a signal peptide. The recombinant chitosanases were found to produce as soluble protein intracellularly. A time-course 1H-NMR experiment on the enzymatic formation of chitooligosaccharides revealed that the mechanism of the enzyme involved an inversion of an anomeric configuration. Through analysis of the products and their corresponding methylated derivatives with LC/MS/MS, the pattern of enzymatic hydrolysis of the GlcNAc-GlcN and GlcN-GlcN linkages in chitosan were unequivocally determined, whereas the GlcNAc-GlcNAc and GlcN-GlcNAc linkages were not digestible. Site-directed mutagenic studies on the ten conserved carboxylic amino acids of the family were performed. Among them, the mutants of D160N and E169Q lost all activity, whereas the other mutants retained > 40 % activity of the wild-type chitosanase. Measurements of circular dichroism of D160N, E169Q, wild-type enzyme and other active mutants yielded similar spectra, indicating that activity loss of the two mutants was not due to the change of protein structure. Surface plasma resonance (SPR) studies revealed that the binding properties of D160N and the wild type enzyme with either chitotetramer or chitotrimer are comparable. We conclude that Asp160 and Glu169 are the two essential residues of A. fumigatus chitosanase.
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