生防芽孢杆菌T2胞外蛋白酶的纯化及其抗真菌作用  

Purification,characterization and antagonism of an extracellular protease from Bacillus subtilis strain T2

作　　者：邢介帅[1] 李然[1] 赵蕾[1] 梁元存[2] 竺晓平[2] 

机构地区：[1]山东师范大学生命科学学院,济南250014 [2]山东农业大学植物保护学院,泰安271018

出　　处：《植物病理学报》2008年第4期377-381,共5页Acta Phytopathologica Sinica

基　　金：山东省自然科学基金项目(Y2005D06);“十一五”国家科技支撑计划重点项目(2006BAD17B07)

摘　　要：从小麦根际土壤中分离到一株高产蛋白酶并对棉花枯萎病菌（Fusarium oxysporum f sp．vasinfectum）具有拮抗作用的枯草芽孢杆菌（Bacillus subtilis）T2。采用硫酸铵分级沉淀、DEAE Sepharose Fast Flow、SP Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶过滤层析，从T2发酵液中分离纯化出一种分子量约为29．0kD的蛋白酶。该酶作用的最适温度为65℃，最适pH为8．0，在低于50℃、pH5．0—7．5范围内较稳定。Na^＋、K^＋Pb^2＋、EDTA对酶有激活作用，Ca^2＋、Ba^2＋、Al^3＋、Zn^2＋、Mn^2＋、Hg^2＋、SDS对酶有部分抑制作用，而PMSF可完全抑制酶活，推测该酶是一种丝氨酸蛋白酶。抗菌活性显示，该酶对棉花枯萎病菌的孢子萌发、菌丝生长有明显抑制作用。Bacillus subtilis strain T2, which could produce large amount of extracellular proteases, was isolated from wheat rhizosphere and showed antagonism against Fusarium oxysporum f. sp. vasinfectum in vitro. One of the protease was purified by using ammonium sulfate precipitation following by DEAE Sepharose Fast Flow chromatography, SP Sepharose Fast Flow chromatography and Sephadex G-75. The molecular weight of the protease was estimated to be 29.0 kD by SDS-PAGE analysis. The optimum temperature and pH for the enzyme activity were determined to be 65℃ and 8.0, the protease was more stable when temperature was lower than 50℃ and pH ranged from 5.0 to 7.5. The enzyme activity was stimulated by Na^＋ , K^＋ , Pb^2＋ and EDTA, partly inhibited by Ca^2 ＋ , Ba^2 ＋ , Al^3 ＋ , Zn^2 ＋ , Mn^2 ＋ , Hg^2 ＋ and SDS, and heavily inactivated by PMSF. It is, therefore, reasonable to conclude that the protease was a serine protease. An in vitro assay showed that the purified protease expressed inhibition activity on conidia germination and hyphal growth of F. oxysporum f. sp. vasinfectum, which was the pathogen caused cotton fusarium wilt.

关 键 词：枯草芽孢杆菌 蛋白酶 纯化 抗真菌作用 

分 类 号：S432.4[农业科学—植物病理学] S476[农业科学—农业昆虫与害虫防治]