小球藻锌结合类金属硫蛋白的结构表征及抗菌活性  

Studies on the structural characterization and antimicrobial activities of Zn-MT-like proteins induced from Chlorella vulgaris

作　　者：李连平[1] 张小东[2] 黄志勇[1] 鄢庆枇[2] 梁英[2] 徐同玲[2] 

机构地区：[1]集美大学生物工程学院,福建厦门361021 [2]集美大学水产学院,福建厦门361021

出　　处：《水产学报》2010年第2期278-284,共7页Journal of Fisheries of China

基　　金：国家自然科学基金项目(40771185);福建省科技计划重点项目(2007Y0028);福建省基金项目(2009J01034);厦门市科技项目(3502Z20055025);集美大学创新团队基金资助项目(2006A003)

摘　　要：小球藻经60μmol/L的锌胁迫培养5d后,离心收集藻泥,经超声波细胞破碎后,离心得到上清液即蛋白质粗提液,再经葡聚糖凝胶层析柱G-75及脱盐柱G-25分离、纯化后,收集的蛋白组分经真空冷冻干燥后获得小球藻锌结合蛋白。通过Tricine-SDS-PAGE电泳、氨基酸组成及紫外光谱等方法进行表征,测得该蛋白的分子量约为8.2ku,半胱氨酸含量为15.4%,具有金属硫蛋白典型的紫外光谱特征,称为类金属硫蛋白(Zn-MT-like proteins)。试验采用琼脂糖孔穴扩散法,研究了小球藻Zn-MT-like蛋白对3种细菌(大肠杆菌、金黄色葡萄球菌和枯草芽胞杆菌)和3种真菌(青霉菌、曲霉菌、酵母菌)的抗菌活性,并与兔肝Zn-MTs的抗菌能力进行比较。结果表明,小球藻Zn-MT-like蛋白对革兰氏阳性菌(金黄色葡萄球菌、枯草芽胞杆菌)和霉菌(青霉、曲霉)具有显著的抗菌活性,1mg/mL的Zn-MT-like蛋白对金黄色葡萄球菌、枯草芽胞杆菌、青霉和曲霉的抑菌圈直径分别为13.2、17.4、7.2和9.8mm;而对大肠杆菌、酵母菌则无抗菌活性。同样条件下,未加锌胁迫的小球藻蛋白质本身和兔肝Zn-MTs并不具有这些抗菌能力。小球藻Zn-MT-like蛋白抗菌活性的发现为进一步综合开发利用小球藻提供了实验基础。Chlorella vulgaris was cultivated for five days under the stress of 60 μmol/L of Zn^2＋ （ ZnCl2 ）. After harvest by centrifugation, the algal cells were washed with EDTA and water, and were homogenized by a supersonic cell disintegrator. After centrifugation, the supernatant, a crude protein-containing extract, was separated and purified with a gel filtration column （ Sephadex G-75, 3.5 i.d. ×80 cm） and a desalting gel filtration column （ Sephadex G-25, 1. 5 i.d.×30 cm）. The obtained Zn-binding proteins were characterized with several analytical methods. Results showed that the molecular weight of the proteins measured with tricine-SDS-PAGE was about 8.2 ku, and the cysteine content of the proteins was 15.4%. In addition, the characteristic ultraviolet spectrum of the proteins was found to be similar to that of the standard MTs from rabbit liver. Therefore, the Zn-binding proteins were referred as metallothionein-like （Zn-MT-like） proteins. Based on the agar hole pervasion method, the activities of anti-bacterial and antifungal of Zn-MT-like proteins were tested. The results showed that Zn-MT-like proteins strongly exhibited the antimicrobial activities against gram-positive bacteria （including Staphylococcus aureus and Bacillus subtilus ） , Penicillium chrysogenum and Aspergillus niger, but was insensitive to Escherichia coli and brewer＇ s yeast. The diameters of inhibition zone for 1 mg/mL of Zn-MT-like protein were 13.2, 17.4, 7.2 and 9.8 mm for S. aureus, B. subtilus, P. chysogenum and A. niger, respectively. However, no anti- microbial activity was found for the standard Zn-MTs from rabbit liver. The antimicrobial activities of Zn- MT-like proteins from C. vulgaris may be helpful for the further comprehensive development and utilization of the alga.

关 键 词：小球藻 类金属硫蛋白 结构表征 抗菌活性 

分 类 号：Q946.1[生物学—植物学] S917[农业科学—水产科学]