Purification and Characterization of Chitinase from Pupae of Pieris rapae crucivora Boisduval

JOURNAL OF CHEMICAL ENGINEERING OF JAPAN

Akane Kojo¹  Ryosuke Maeda¹  Michiaki Matsumoto¹  Kazuo Kondo¹ 

[1] Department of Chemical Engineering and Materials Science, Doshisha University

关键词: Chitinase;    Purification;    N-Acetylglucosamine;    Chitooligosaccharide;    Transglycosylation;   

DOI  :  10.1252/jcej.35.241

来源: Maruzen Company Ltd

【 摘 要 】

References(13)Cited-By(3)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 × 10–2 kg/(m3·h) as Vmax and 23.9 kg/m3 as Km 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.

【 授权许可】

Unknown