苦瓜几丁质酶的分离及生物学特征分析  

Isolating and Bio-characterization of Chitinase from Momordica charantia

作　　者：朱新产[1] 王倩文[1] 于群[1] 

机构地区：[1]青岛农业大学生命科学学院,山东省高校植物生物技术重点实验室,山东青岛266109

出　　处：《华北农学报》2015年第6期182-187,共6页Acta Agriculturae Boreali-Sinica

基　　金：山东省自然基金项目(ZR2011CM008)

摘　　要：为了探究苦瓜几丁质酶在生物防治和植物抗真菌病害中的潜势作用,以青皮苦瓜为试验材料,采用0~60%（NH4）2SO4分级分离苦瓜蛋白质,通过几丁质亲和柱层析色谱工作站,分步聚集纯化出几丁质酶。SDS-PAGE凝胶电泳显示单一条蛋白带;经生化分析鉴定,苦瓜几丁质酶的分子量为35.4 k Da,等电点p I=6.2;酶活力为24 U,比活力为32 U/mg。理化性检测表明,苦瓜几丁质酶的最适温度和p H值分别为50℃、6.0;氧化剂、还原剂和金属离子影响酶活力;其清除羟自由基能力和总抗氧化能力均有上升的趋势。抑菌试验显示,苦瓜几丁质酶对霉菌有一定的抑制效力,对植物致病真菌有不同程度的抑制作用,提示偏酸性几丁质酶具有明显的抗菌活性及抗氧化能力,利用区隔性分级提纯苦瓜几丁质酶,将能开发出有发展潜力的新型抗真菌病虫害药物。Chitinases are pathogensis-related proteins,which play an important role in plant defense mechanisms. In order to explore biological control and plant antifungal preparations,the characteristics of chitinase was investigated by using the biochemical technology from Momordica charantia. The chitinase was isolated and purified from the green skin of Momordica charantia by ammonium sulphate（ 0- 60%） fractionation and on affinity chromatography. The results of SDS-PAGE and biochemical analysis showed that the chitinase of Momordica charantia was homogeneous single band with p I 6. 2,the relative molecular weight of 35. 4 k Da was determined by SDS-PAGE,the enzyme activity and the specific activity were 24 U and 32 U / mg respectively. The physicochemical property analysis showed that the optimum temperature and p H of the chitinase were respectively 50 ℃ and 6. 0 from Momordica charantia,metal ion and oxidant / reducing agent affected the activity of the chitinase from Momordica charantia,and its total antioxidantive activity and clearance on·OH free radical were rising. The results of antimicrobial activities analysis showed that the chitinase from Momordica charantia had strong inhibition to testing mould（ Aspergillus niger,Penicillium,Rhizopus） and fungi（ Valsa mali Miyabe et Yamada,Physalospora piricola Nose,Alternaria mali Roberts,Coniothyrium diplodiella,Fusarium oxysporum f. sp. Vasinfectum）. Therefore,the partial acidity chitinase from Momordica charantia had the obviously antimicrobial activity and antioxidant capacity. The chitinase from Momordica charantia could exploit new type antifungal agents with development potential through grading purification by molecular weight interval fractionation way.

关 键 词：苦瓜几丁质酶 分离纯化 SDS-PAGE 抑菌活性 

分 类 号：Q556.2[生物学—生物化学]