Purification and Characterization of A Novel Chitinase Isozyme from Yam Tuber.

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, GlcNAc5. The optimum temperature was 60°C. Chitinase A was stable between pH 6 and 11 and below 45°C. Kinetic analysis was done using a series of N-acetylchitooligosaccharides (GlcNAcn, 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 of insect chitinases.