Purification and characterization of a novel isozyme of chitinase from Bombyx mori

Abstract

75-kDa chitinase, which showed potential as a biocontrol agent against Japanese pine sawyer, was characterized after purification from the integument of the fifth instar larvae of Bombyx mori by chromatography on diethylaminoethyl (DEAE)-Toyoperal 650 (M), hydroxylapatite, and Fractogel EMD DEAE 650 (M) columns. The optimum pH was 6.0 toward N-acetylchitopentaose (GlcNAc 5) and 10 toward glycolchitin. The optimum temperature was 60°C toward GlcNAc5 and 25°C toward glycolchitn. The enzyme was stable at pH 7-10 and below 40°C. Kinetic analysis and reaction-pattern analysis using glycolchitin and N-acetylchitooligosacchraides as substrates indicated that 75-kDa chitinase is an endo- or random-type hydrolytic enzyme to produce the β anomeric product and that it prefers the longer N- acetylchitooligosaccharides, suggesting, together with the N-terminal amino acid sequence, that the 75-kDa chitinase belongs to family 18 of glycosyl hydrolases.