家蚕几丁质脱乙酰基酶BmCDA7的纯化与生化性质

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

中国生物防治学报 ›› 2020, Vol. 36 ›› Issue (1): 72-78.DOI: 10.16409/j.cnki.2095-039x.2019.06.012

家蚕几丁质脱乙酰基酶BmCDA7的纯化与生化性质

郭兴明, 刘霖, 杨君, 杨青

大连理工大学生物工程学院, 大连 116024

收稿日期:2019-05-30 出版日期:2020-02-08 发布日期:2020-02-26
通讯作者: 杨青,教授,E-mail:qingyang@dlut.edu.cn
作者简介:郭兴明,硕士研究生,E-mail:guoxingming@mail.dlut.edu.cn
基金资助:
国家重点研发计划（2017YFD0200501）；国家自然科学基金（31772193）

Purification and Biochemical Characterization of Chitin Deacetylase 7 from Bombyx mori

GUO Xingming, LIU Lin, YANG Jun, YANG Qing

School of Bioengineering, Dalian University of Technology, Dalian 116024, China

Received:2019-05-30 Online:2020-02-08 Published:2020-02-26

摘要/Abstract

摘要： 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0.4270 μmol/（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。

关键词: 鳞翅目昆虫, 围食膜, 几丁质脱乙酰基酶, 害虫防治

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

中图分类号:

S432

郭兴明, 刘霖, 杨君, 杨青. 家蚕几丁质脱乙酰基酶BmCDA7的纯化与生化性质[J]. 中国生物防治学报, 2020, 36(1): 72-78.

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.

参考文献

[1] Merzendorfer H, Zimoch L. Chitin metabolism in insects:structure, function and regulation of chitin synthases and chitinase[J]. The Journal of Experimental Biology, 2003, 206(24):4393-4412.
[2] Yong Z, Park R D, Muzzarelli R A. Chitin deacetylases:properties and applications[J]. Marine Drugs, 2010, 8(1):24-46.
[3] Xi Y, Pan P L, Ye Y X, et al. Chitin deacetylase family genes in the brown planthopper, Nilaparvata lugens (Hemiptera:Delphacidae)[J]. Insect Molecular Biology, 2014, 23(6):695-705.
[4] Luschnig S, Batz T, Armbruster K, et al. serpentine and vermiform encode matrix proteins with chitin binding and deacetylation domains that limit tracheal tube length in Drosophila[J]. Current Biology, 2006, 16(2):186-194.
[5] Wang S, Jayaram S A, Hemphala J, et al. Septate-junction-dependent luminal deposition of chitin deacetylases restricts tube elongation in the Drosophila trachea[J]. Current Biology, 2006, 16(2):180-185.
[6] Muthukrishnan S, Merzendorfer H, Arakane Y, et al. 7-Chitin metabolism in insects[J]. Insect Molecular Biology and Biochemistry, 2012, 4:193-235.
[7] Yu R, Liu W, Li D, et al. Helicoidal organization of chitin in the cuticle of the migratory locust requires the function of the chitin deacetylase2 enzyme (LmCDA2)[J]. The Journal of Biological Chemistry, 2016, 291(47):24352-24363.
[8] Liu S H, Li H F, Yang Y, et al. Genome-wide identification of chitinase and chitin deacetylase gene families in the oriental fruit fly, Bactrocera dorsalis (Hendel)[J]. Comparative Biochemistry and Physiology Part D:Genomics & Proteomics, 2018, 27:13-22.
[9] Arakane Y, Dixit R, Begum K, et al. Analysis of functions of the chitin deacetylase gene family in Tribolium castaneum[J]. Insect Biochemistry and Molecular Biology, 2009, 39(5-6):355-365.
[10] Zhang Z, Yan J, Liu Q, et al. Genome-wide analysis and hormone regulation of chitin deacetylases in silkworm[J]. International Journal of Molecular Sciences, 2019, 20(7):1679.
[11] Jakubowska A K, Caccia S, Gordon K H, et al. Downregulation of a chitin deacetylase-like protein in response to baculovirus infection and its application for improving baculovirus infectivity[J]. Journal of Virology, 2010, 84(5):2547-2555.
[12] Toprak U, Baldwin D, Erlandson M, et al. A chitin deacetylase and putative insect intestinal lipases are components of the Mamestra configurata (Lepidoptera:Noctuidae) peritrophic matrix[J]. Insect Molecular Biology, 2008, 17(5):573-585.
[13] Zhong X, Wang X, Tan X, et al. Identification and molecular characterization of a chitin deacetylase from Bombyx mori peritrophic membrane[J]. International Journal of Molecular Sciences, 2014, 15(2):1946-1961.
[14] Liu L, Qu M, Liu T, et al. Biochemical characterization of three midgut chitin deacetylases of the Lepidopteran insect Bombyx mori[J]. Journal of Insect Physiology, 2019, 113:42-48.
[15] Liu L, Zhou Y, Qu M, et al. Structural and biochemical insights into the catalytic mechanisms of two insect chitin deacetylases of the carbohydrate esterase 4 family[J]. The Journal of Biological Chemistry, 2019, 119:7597.
[16] Trudel J, Asselin A. Detection of chitin deacetylase activity after polyacrylamide pel electrophoresis[J]. Analytical Biochemistry, 1990, 189(2):249-253.
[17] 张永勤, 薛长湖, 汤浩源, 等. 还原糖的可见分光光度法研究进展[J]. 食品与发酵工业, 2007, 5:97-99, 104.
[18] 顾本贤, 徐玥, 方继康. 荧光胺的合成和应用[J]. 生物化学与生物物理进展, 1979, 6:60-63.
[19] Tsigos I, Bouriotis V. Purification and characterization of chitin deacetylase from Colletotrichum lindemuthianum[J]. The Journal of Biological Chemistry, 1995, 270(44):26286-26291.
[20] Tellam R L, Wijffels G, Willadsen P. Peritrophic matrix proteins. Insect Biochemistry and Molecular Biology[J]. 1999, 29(2):87-101.
[21] Toprak U, Erlandson M, Hegedus D D. Peritrophic Matrix Proteins[J]. Trends in Entomology, 2010, 6:23-51.
[22] Guo W, Li G X, Pang Y, et al. A novel chitin-binding protein identified from the peritrophic membrane of the cabbage looper, Trichoplusia ni[J]. Insect Biochemistry and Molecular Biology, 2005, 35(11):1224-1234.
[23] 赵盼, 张学尧, 刘晓健, 等. 飞蝗几丁质脱乙酰基酶的真核表达、亲和纯化及酶活性[J]. 中国农业科学, 2017, 50(6):1057-1066.
[24] 闫晓平, 赵丹, 郭巍, 等. 美国白蛾几丁质脱乙酰酶的克隆、表达及酶学性质[J]. 中国农业科学, 2017, 50(5):849-858.

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 8

编辑推荐

Metrics

本文评价

联系我们

[1]	靳婷婷, 戈峰, 吴杰. 亚洲玉米螟幼虫膜结合海藻糖酶基因RNAi效应[J]. 中国生物防治学报, 2020, 36(3): 452-457.
[2]	刘臣, 陈琳, 王冰洁, 赵曼, 梁革梅, 郭予元. 四种Bt蛋白对六种重要鳞翅目害虫的杀虫活性评价[J]. 中国生物防治学报, 2017, 33(6): 774-779.
[3]	申昭灿, 陈龙, 邬家栋, 李元喜, 王甦. 稻田寄生蜂和鳞翅目昆虫的多样性及变化动态[J]. 中国生物防治学报, 2017, 33(5): 590-596.
[4]	吴文丹1,2 , 尹姣1 , 曹雅忠1 , 肖春2 , 李克斌1 . 我国昆虫病原线虫的研究与应用现状[J]. , 2014, 30(6): 817-822.
[5]	蒋月丽1 , 武予清1 , 段云1 , 高新国2 . 释放东亚小花蝽对大棚辣椒上几种害虫的防治效果[J]. , 2011, 27(3): 414-417.
[6]	刘琴, 马谈斌, 祁建杭, 施建德, 李传明, 徐健. 苏云金芽胞杆菌毒素蛋白和粘虫颗粒体病毒对甜菜夜蛾中肠围食膜的破坏作用[J]. , 2011, 27(2): 182-187.
[7]	张严峻1 ;谭军2 ;林玉清2 . 低温和几丁质酶处理对棉铃虫围食膜的影响 [J]. , 2000, 16(4): 152-155.
[8]	廖玲洁;彭建新;洪华珠. 昆虫病毒增强蛋白的研究新进展 [J]. , 1999, 15(1): 41-44.