吡咯伯克霍尔德氏菌JK-SH007抗菌蛋白的分离纯化

Isolation and Purification of Antifungal Protein from Burkholderia pyrrocinia JK-SH007

摘要:

对拮抗细菌吡咯伯克霍尔德氏菌(Burkholderia pyrrocinia) JK-SH007菌株抗菌蛋白进行了初步研究。研究发现, JK-SH007菌株的抗菌粗蛋白对热和蛋白酶K不敏感, 碱性环境不利于抑菌蛋白活性的发挥。JK-SH007菌株的无菌发酵滤液经硫酸铵沉淀、透析(3.5 kD)、冷丙酮沉淀(-20°C)、Sephadex G-50葡聚糖凝胶分子筛层析及DEAE-Sephadex A-25离子交换层析分离纯化, 得到了蛋白组分A-Ⅱ-2, 经SDS聚丙烯酰胺凝胶电泳检测不是单一组分, 该复合组分具有明显抑制3种杨树溃疡病病原真菌金黄壳囊孢(Cytospora chrysosperma)、拟茎点霉(Phomopsis macrospore)、七叶树壳梭孢(Fusicoccum aesculi)生长的作用。

关键词:杨树溃疡病, 吡咯伯克霍尔德氏菌, 抗菌蛋白, 分离纯化, 拮抗细菌

Abstract:

A preliminary study on the antagonistic protein produced by Burkholderia pyrrocinia JK-SH007 was carried out. The results showed it was stable to heat, not sensitived to proteinase K and partially sensitive to alkali. Cell culture was obtained after centrifuging and filtering (0.22 μm) of ferment liquid of B. pyrrocinia JK-SH007 by shake-flask fermentation. The crude proteins were obtained by fractionation with (NH4)2SO4 (20%-60%). Active proteins(A-Ⅱ-2) were obtained after purification of the crude proteins by 3.5 kD dialysis bag dialysis, cold acetone precipitation (-20°C), Sephadex G-50, DEAE-Sephadex A-25 aion-exchange column chromatography. A-Ⅱ-2 were shown three bands by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins showed good inhibition activity to three pathogens Cytospora chrysosperma, Phomopsis macrospore and Fusicoccum aesculi which caused poplar canker.

Key words:Poplar canker, Burkholderia pyrrocinia, Antifungal protein, Isolation and purification, Antagonistic bacteria