Purification and Characterization of a Highly Thermostable Chitinase from the Stomach of the Red Scorpionfish Scorpaena scrofa with Bioinsecticidal Activity toward Cowpea Weevil Callosobruchus maculatus (Coleoptera: Bruchidae)

摘要

This present study is the first attempt to report on the purification and characterization of a chitinase from the stomach of the red scorpionfish Scorpaena scrofa. A 50-kDa chitinase (SsChi50) was purified to homogeneity, and matrix assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) analysis showed that SsChi50 was a monomer with a molecular mass of 50,103 Da. The 25 N-terminal residues of SsChi50 displayed high homology with family-18 chiti-nases. Optimal activity was obtained at pH 5.0 at 80 °C. SsChi50 was stable at pH and temperature ranges of 3.0 to 7.0 and 70 to 90 °C for 48 and 4 h respectively. Among the inhibitors and metals tested, p-chloromercuribenzoic acid, N-ethylmaleimide, Hg~(2+), and Hg~+ completely inhibited enzyme activity. Chitinase activity was high on colloidal chitin, glycol chitin, glycol chitosane, chitotriose, and chitooligosaccharide. Chitinase activity towards synthetic substrates in the order of p-NP-(GlcNAc)_n (n = 2-4) was p-NP-(GlcNAc)2 >p-NP-(GlcNAc)4 >p-NP-(GlcNAc)3. Our results suggest that the SsChi50 enzyme preferentially hydrolyzed the second glycosidic link from the non-reducing end of (GkNAc)_n. This enzyme obeyed Michaelis-Menten kinetics, the K_m and K_(cat) values being 0.412 mg, colloidal chitin mL~(-1) and 5.33 s-1 respectively. An in vivo bioinsecticidal assay was developed for SsChi50 against Callosobruchus maculatus adults. The enzyme showed bioinsecticidal activity toward Callosobruchus maculatus, indicating the possibility of using it in biotechno-logical strategies for insect management for stored cowpea seeds.