Purification and Biochemical Characterization of Chitin Deacetylase 7 from Bombyx mori

doi:10.16409/j.cnki.2095-039x.2019.06.012

Abstract

Abstract: Insect chitin deacetylases (CDAs) are essential chitin modifying enzymes that play important roles in chitin formation, thus being potential targets for developing insecticides. Our recent study revealed that BmCDA7 exhibited the highest activity towards peritrophic membrane chitin among all the three midgut CDAs from Bombyx mori. The expression level of BmCDA7 was dramatically upregulated at feeding stage, suggesting its possible role in the formation of peritrophic membrane. In this study, we further optimized the purification of the recombinant BmCDA7 by a two-step purification procedure, namely a metal chelate chromatography followed by an anion exchange chromatography, which produced BmCDA7 of high purity. The activity of BmCDA7 towards glycol chitin was qualitatively determined using SDS-PAGE coupled with Calcofluor White M2R staining. The catalysis optimal temperature and pH, the stability and the substrate selectivity of BmCDA7 were quantitatively determined based on the amount of released acetic acid during enzymatic catalysis. The optimal temperature and pH were 60℃ and 8.0, respectively. And the stable conditions for BmCDA7 was 30-60℃ and pH range of 7-11. BmCDA7 showed activity of 2.9926 and 0.4270 μmol/(min·μmol) towards glycol chitin and colloidal chitin, respectively, while was inactive towards α-chitin and β-chitin. These results have paved a biochemical basis for future investigation on BmCDA7.

Key words: Lepidopteran insect, peritrophic membrane, chitin deacetylase, pest control

CLC Number:

S432

GUO Xingming, LIU Lin, YANG Jun, YANG Qing. Purification and Biochemical Characterization of Chitin Deacetylase 7 from Bombyx mori[J]. Chinese Journal Of Biological Control, 2020, 36(1): 72-78.

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