Construction of System for Formation of Chitin Oligomer Having Highly Physiological Activity by Enzymatic Hydrolysis Reaction

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Chitinase from the extract of pupae of Pieris rapae crucivcra Boisduval was purified through the successive steps of CM-Sephadex C-50 ion exchange chromatography and gel filtration chromatography with Sephadex G-150 from crude enzyme extract. Then the active fractions named Chi-A and Chi-B were obtained. The purity of the enzyme increased up to 12.4- and 2.17-fold and the recovery of the enzyme activity were 42.4 and 4.58%, for the fraction Chi-A and.Chi-B, respectively. The homogeneity and molecular weight of isolated Chi-A were evaluated by SDS PAGE. The homogeneity of Chi-A was confirmed as a single band on SDS-PAGE and the molecular weight was estimated to be 48,000. The purified Chi-A had an optimal pH of 5.0 for the hydrolysis reaction when glycol chitin was used as a substrate. Chi-A was stable in the pH range of 4.0-8.0 and retained its 70% activity at 310K. Chitinase from pupae of Pieris rapae crucivara Boisduval exhibited typical Michaelis-Menten type kinetics. We also found that Chi-A revealed a chitin synfase activity. A large amount of N-acetylchitopentaose was effectively formed by the transglycosylation from N-acetylglucosamine with Chi-A.On the other hand, three kinds of N-acetylglucosamine transferase were purified from the crude extract of pupae of Papilio xuthus Linne through Sephadex G-25 gel filtration chromatography and CM-Sephadex C-50 ion exchange chromatography. Thermal stability of these enzymes was 308-328 K and optimal pH for transferase activity appeared at 6.5-7.0. Each enzyme could react with N-acetylglucosamine, and produced insoluble deposit suggesting highly polymerized chitooligosaccharides. These catalytic properties somewhat differ from those of chitintransferase fron pupae of Pieris rapae crucivora Boisduval belonging to the same order.