Purification and characterization of Bombyx mori chitinases

Two isozymes of chitinase (EC 3.2.1.14) were purified from the fifth-instar larvae of Bombyx mori by chromatography on DEAE-Cellulofine A-500, hydroxylapatite, Butyl-Toyopearl 650M, and Fractogel EMD DEAE 650 (M). These two isozymes were glycoproteins with different apparent molecular masses of 65 and 88 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pHs of the 65 and 88 kDa chitinases were 5.5 and 6.5, respectively, towards a short substrate, N-acetylchitopentaose (GlcNAc5), whereas their high activities were observed in a wide pH range between 4 and 10 towards a longer substrate, glycolchitin. Steady-state kinetic analysis of these chitinases was performed using a series of N-acetylchitooligosaccharides (GlcNAcn, n = 2-6) and glycolchitin as the substrates. Kinetic parameters for both chitinases could be obtained in the hydrolysis of glycolchitin, but not in that of N-acetylchitooligosaccharides because of strong substrate inhibition. Both chitinases similarly hydrolysed N-acetylchitooligosaccharides except for GlcNAc2 as follows: GlcNAc3 to GlcNAc plus GlcNAc2, GlcNAc4 to two molecules of GlcNAc2, GlcNAc5 to GlcNAc2 plus GlcNAc3, and GlcNAc6 to GlcNAc2 plus GlcNAc4 as well as two molecules of GlcNAc3. These results suggest that these chitinases are endo-type hydrolases, and preferred the longer-chain N-acetylchitooligosaccharides. With respect to activity, the 65 kDa chitinase was 1.7-fold more active than the 88 kDa chitinase with regard to the initial velocity in the reaction of 0.1 mM N-acetylchitooligosaccharides (GlcNAcn, n = 3-6), whereas in the overall reaction of glycolchitin (kcat/K(m)), the 88 kDa chitinase was four times more active than the 65 kDa chitinase. Regarding the affinity (1/K(m)) to glycolchitin, the affinity of the 88 kDa chitinase was 5.8-fold higher than that of the 65 kDa chitinase. The protein amino acid and gene nucleotide sequences were partly determined. Both N-terminal amino acid sequences of the 65 and 88 kDa chitinases were identical as ADSRARIVXYFSNWAVYRPG. The partial amino acid (113 amino acids) and nucleotide sequences (278 nucleotides) analysed from a mixture of 65 and 88 kDa chitinases included the two conserved regions of the family of 18 glycosyl hydrolases. All these results suggest that the B. mori chitinases are similar to Manduca sexta chitinase in primary structure and kinetic behaviour, and may be involved in the initial and intermediate stages of chitin degradation.