昆虫来源的几丁质酶的分离纯化及酶学性质.PDF

生 物 工 程 学 报 Chin J Biotech 2010, March 25; 26(3): 404–409 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ ©2010 CJB, All rights reserved. 生物技术与方法 昆虫来源的几丁质酶的分离纯化及酶学性质 刘明艳，张洪斌，胡雪芹，魏青丽 合肥工业大学化工学院制药工程系，合肥 230009 摘 要: 几丁质酶在真菌和昆虫的生理和发育过程中起着关键作用，该酶本身及其酶抑制剂是获取生物农药的重要途 径。本研究从蚕蛹体内提取几丁质粗酶，经硫酸铵分级沉淀和 Sephadex G-150 分离得到几丁质酶。用 SDS测得 该酶的分子量为88 kDa。水解胶体几丁质的Km 值为22.3 μmol/L 。酶反应的最适温度为45 ℃，最适pH 值为 6.0 ，金属 离子和有机试剂对几丁质酶活性都有影响，其中高浓度的Mn2+对酶有较强的激活作用，而Cu2+ 、SDS 则有较强的抑制 作用。研究结果为基于几丁质酶的生物农药筛选研究奠定了基础。 关键词: 几丁质酶，分离纯化，酶学性质 Purification and characterization of a chitinase from Bombyx mori Mingyan Liu, Hongbin Zhang, Xueqin Hu, and Qingli Wei Department of Pharmaceutical Engineering , Hefei University of Technology, Hefei 230009, China Abstract: The importance of chitinases in the physiological and developmental processes of fungi and insects makes themselves and their inhibitors important targets for biological pesticides. A chitinase was isolated from Bombyx mori and purified to electrophoretic homogeneity by ammonium sulfate precipitation and Sephadex G-150 column chromatography. The molecular mass was estimated to be about 88 kDa by SDS, while the Km was calculated to be 22.3 μmol/L. Moveover, the optimal reaction temperature was 45°C, and the optimum pH was 6.0. The effect of metal ions and organic reagents on chitinase activity was investigated. The activity was enhanced by high concentration of Mn2+, while was strongly inhibited by Cu2+ and SDS. These results provide a basis for screening the chitinase-based biological pesticide. Keywords: chitinase, isolation and purification, enzyme properties 几丁质是由N- 乙酰-D-氨基葡萄糖以β-1,4 糖苷 表皮、昆虫的外骨骼和围食膜、甲壳动物的外壳和 键连接而成的高分子聚合物，是构成大多数真菌细