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The 54 kDa protein that was suggested to be processed from the 65 kDa and 88 kDa chitinases of Bombyx mori [Koga et al., Insect Biochem. Mol. Biol. 27, 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 ofB. mori by chromatography on DEAE-Cellulofine A‐500, hydroxylapatite, Butyl-Toyopearl 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 short substrate, N-acetylchitopentaose (GlcNAc5), while in its reaction with a longer substrate, glycolchitin, the enzyme showed a wide pH-range between 4.0 and 10. Kinetic parameters for the chitinase could be obtained in the hydrolysis of glycolchitin but not in that of N-acetylchitooligosaccharides (GlcNAc n, n=2‐6) because of substrate inhibition. The chitinase hydrolyzed 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 hydrolase and preferred the longer-chain N-acetylchitooligosaccharides. Moreover, the anomeric forms of N-acetylchitooligosaccharides were analyzed in the reaction with the 54-kDa chitinase. It was revealed that this enzyme cleaves the substrate to produce the b anomeric product. With respect to inhibition of the 54 kDa chitinase, it was specifically inhibited by allosamidin in a competitive way with Ki values depending on the pH of the reaction mixture (Ki=0.01320.746 μM). Comparing the properties and kinetic behavior 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 chitinase was 2.15 and 2.8 times more active than the 88 and 54-kDa chitinases, respectively. However, in the overall reaction of glycolchitin ( kcat/Km), the 88-kDa enzyme was 4 and 40 times more active than the 65-kDa and the 54-kDa enzymes, respectively. Concerning the affinity (1/Km) to glycolchitin, the 88 kDa chitinase affinity (at pH 6.5) was 5.8 times higher than that of the 65 kDa chitinase (at pH 5.5) and 4.0 times higher than that of the 54 kDa chitinase (at pH 6.0). These kinetic results suggest that B. mori chitinases are processed during ecdysis from the larger chitinase to smaller ones that leads to changes in their kinetic properties such as Km, kcat and kcat/Km successively. © 1999 Elsevier Science Ltd. All rights reserved. |
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