生防芽孢杆菌T2胞外蛋白酶的纯化及其抗真菌作用

 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⁺Pb²⁺、EDTA对酶有激活作用,Ca²⁺、Ba²⁺、Al³⁺、Zn²⁺、Mn²⁺、Hg²⁺、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。

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

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⁺, Pb²⁺ and EDTA, partly inhibited by Ca²⁺, Ba²⁺, Al³⁺, Zn²⁺, Mn²⁺, Hg²⁺ 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.