Isolation and purification of bacteriostatic protein from Bacillus velezensis MCCC 1A15695 and its application
付欢 1李亚婷 1肖贵谦 2彭磊磊 2夏金梅 2邵宗泽 2曹军伟 1赖其良2
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作者信息
1. 上海海洋大学海洋科学学院,上海 201306
2. 自然资源部第三海洋研究所、自然资源部海洋生物遗传资源重点实验室,福建厦门 361005
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摘要
对虾"玻璃苗"病的暴发,会导致对虾幼苗大批死亡,给水产养殖带来巨大损失.利用本实验室最近鉴定的"玻璃苗"致病菌副溶血弧菌(Vibrio parahaemolyticus)vp-HL,从海洋来源芽孢杆菌中筛选拮抗菌,获得一株能够高效抑制vp-HL菌株的贝莱斯芽孢杆菌(Bacillus velezensis)MCCC 1A15695.经分离鉴定与活性测定,其有效抑菌物质为蛋白类,该蛋白抑菌谱较广,对包括多种弧菌病原在内的17种病原微生物有抑制作用.经Q Seharose Fast Flow阴离子柱层析、聚丙烯酰胺凝胶电泳(SDS-PAGE)及液相色谱-质谱联用技术(LC-MS)分离纯化鉴定,确定活性蛋白组分中的有效抑菌物质为枯草杆菌素(subtilisin)、未知蛋白(hypothetical protein)及α-淀粉酶(Alpha-amylase).蛋白类物质具有高生物安全性,本研究为后续益生菌的开发利用提供了理论基础.
Abstract
The outbreak of shrimp"glass post-larval disease"has led to a large number of diseases of shrimp lar-vae,bringing huge losses to aquaculture.Using the recently identified Vibrio parahaemolyticus vp-HL of the"glass post-larval disease"pathogen Vibrio parahaemolyticus,the antagonistic antibacterial screening of Bacillus of marine origin was carried out to obtain a strain of Bacillus velezensis MCCC 1A15695 that can effectively inhibit the strains MCCC 1A15695.After isolation and activity determination,the effective bacteriostatic substances were proteins,with a wide inhibitory spectrum,and had inhibitory effects on 17 pathogenic microorganisms,including a variety of Vibrio pathogens.After Q Seharose Fast Flow anion column chromatography,polyacrylamide gel electrophoresis(SDS-PAGE)and liquid chromatography-mass spectrometry(LC-MS),the effective bacteriostatic substances in the active protein components were subtilisin,hypothetical protein and Alpha-amylase.In view of the high biosafety of proteins,the results provide a theoretical basis for the subsequent development and utilization of probiotics.
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