Purification and characterization of chitinase from pupae of Pieris rapae crucivora Boisduval

Chitinase from the extract of pupae of Pieris rapae crucivora 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 310 K. The chitinase from pupae of Pieris rapae crucivora Boisduval exhibited typical Michaelis-Menten type kinetics. The kinetic parameters for the hydrolysis reaction with glycol chitin by Chi-A were determined to be 1.43 x 10(-2) kg/(m(3).h) as V-max and 23.9 kg/m(3) as K-m at 310 K. We also found that Chi-A revealed a chitin synthase activity. A large amount of N-acetylchitopentaose was efficiently formed by the transglycosylation from N-acetylglucosamine with Chi-A.