家蚕几丁质脱乙酰基酶BmCDA7的纯化与生化性质

Purification and Biochemical Characterization of Chitin Deacetylase 7 from Bombyx mori

几丁质脱乙酰基酶是几丁质修饰关键酶,在昆虫几丁质组装过程中发挥重要作用,是潜在的绿色农药靶标。课题组近期研究发现家蚕中肠3个几丁质脱乙酰基酶中,BmCDA7对围食膜几丁质具有最高活性,在进食期高表达,可能参与该时期围食膜形成。本研究通过进一步优化分离纯化条件,建立了金属螯合层析和阴离子交换层析两步分离纯化方法,获得了高纯度的BmCDA7重组蛋白。采用SDS-PAGE结合Calcofluor White M2R显色定性确定了BmCDA7对底物乙二醇几丁质的催化活性。通过测定催化反应过程中释放的乙酸量,确定了BmCDA7的最适反应条件、稳定性及底物选择性。BmCDA7催化反应最适温度为60℃,最适pH为8.0,在温度低于60℃以及中性、偏碱性条件下较为稳定。此外,BmCDA7对乙二醇几丁质、胶体几丁质的酶活力分别为2.9926和0.4270μmol/(min·μmol),而对高结晶度的α-几丁质和β-几丁质无活力。这些结果丰富了昆虫几丁质脱乙酰基酶的生物化学基础研究。

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 M2 R 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.