Purification and characterization of a 54 kDa chitinase from Bombyx mori

The 54 kDa protein that was suggested to be processed from the 65 kDa and 8 8 kDa chitinases of Bombyx. mori [Koga et al., Insect Biochem. Mel. Biol. 2 7, 757-767 (1997)] was purified and proved to be a third chitinase (EC 3.2. 1.14). This chitinase was purified from the fifth larval instar of B. mori by chromatography on DEAE-Cellulofine A-500, hydroxylapatite, Butyl-Toyopea rl 650M, and Fractogel EMD DEAE 650(M) columns. The apparent molecular mass was confirmed to be 54 kDa by SDS-PAGE. Its optimum pH was 6.0 toward a sh ort substrate, N-acetylchitopentaose (GlcNAc(5)), while in its reaction wit h a longer substrate, glycolchitin, the enzyme showed a wide pH-range betwe en 4.0 and 10. Kinetic parameters for the chitinase could be obtained in th e hydrolysis of glycolchitin but not in that of N-acetylchitooligosaccharid es (GlcNAc(n), n = 2-6) because of substrate inhibition. The chitinase hydr olyzed N-acetylchitooligosaccharides except for dimer as follows: trimer to monomer plus dimer, tetramer to two molecules of dimer, pentamer to dimer plus trimer, and hexamer to dimer plus tetramer as well as two molecules of trimer. These results suggest that the 54 kDa chitinase is an endo-type hy drolase and preferred the longer-chain N-acetylchitooligosaccharides. Moreo ver, the anomeric forms of N-acetylchitooligosaccharides were analyzed in t he reaction with the 54-kDa chitinase. It was revealed that this enzyme cle aves the substrate to produce the beta anomeric product. With respect to in hibition of the 54 kDa chitinase, it was specifically inhibited by allosami din in a competitive way with K-i values depending on the pH of the reactio n mixture (K-i = 0.013-0.746 mu M). Comparing the properties and kinetic be havior of this chitinase with those of the 88 and 65 kDa chitinases from B. mori, regarding the specific activity of the three enzymes, the 65-kDa chi tinase was 2.15 and 2.8 times more active than the 88 and 54-kDa chitinases , respectively. However, in the overall reaction of glycolchitin (k(cat)/K- m), the 88-kDa enzyme was 4 and 40 times more active than the 65-kDa and th e 54-kDa enzymes, respectively. Concerning the affinity (1/K-m) to glycolch itin, the 88 kDa chitinase affinity (at pH 6.5) was 5.8 times higher than t hat of the 65 kDa chitinase (at pH 5.5) and 4.0 times higher than that of t he 54 kDa chitinase (at pH 6.0). These kinetic results suggest that B. mori chitinases are processed during ecdysis from the larger chitinase to small er ones that leads to changes in their kinetic properties such as K-m, k(ca t) and k(cat)/K-m successively. (C) 1999 Elsevier Science Ltd. All rights r eserved.