Purification and Characterization of A Novel Chitinase Isozyme from Yam Tuber

概要

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A new chitinase isozyme (Chitinase A), which had only one optimum pH toward a long substrate, glycolchitin, was purified from the peel of yam tuber by CTAB (hexadecyl trimethyl ammonium bromide) treatment and ammonium sulfate fractionation, followed by column chromatography on DEAE-Cellulofine A-500,chromatofocusing, and gel filtration on Sephacryl S-100. The molecular weight was 28,000 by SDS-PAGE. The isoelectric point was 3.6. The optimum pH was 4.0 toward both a polymer substrate, glycolchitin, and an oligosaccharide substrate, GlcNAc_5. The optimum temperature was 60℃. Chitinase A was stable between pH 6 and 11 and below 45℃. Kinetic analysis was done using a series of N-acetylchitooligosaccharides (GlcNAc_n, n=2 to 6) and glycolchitin as the substrates. Chitinase A hydrolyzed N-acetylchitooligosaccharides in an endo/random fashion except the disaccharide, and released the monosaccharide from all hydrolyzed oligosaccharides. This enzyme preferred oligosaccharides with the longer chain lengths. Chitinase A was inhibited 76% by 55 μM allosamidin, which is known to be a specific inhibitor insect chitinases.
社団法人日本農芸化学会の論文
1999-11-23