Analysis of Hydrolytic Activity of a 65-kDa Chitinase from the Silkworm,Bombyx mori

The hydrolytic reactions of Bombyx mori 65-kDa chitinase with the short substrates, N-acetyl-chitooligosaccharides, were analyzed by HPLC. Analysis of the hydrolyzed products showed that the newly produced oligosaccharides are all beta anomers, suggesting that, similar to other family 18 glycosyl hydrolases, the 65-kDa chitinase acts in the retaining mechanism. Furthermore, the enzyme cleaves the N-acetylchitooligosaccharides mainly at the linkage between the second and the third GlcNAc moieties from the non-reducing end, while the other sites were cleaved in smaller proportions. Moreover, the initial reaction rates of the enzyme with the longer N-acetylchitooligosaccharides were higher than those with shorter ones. These results suggest that the enzyme is an endo-cleaving type and more efficient on the longer substrates.