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

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(3.5kD)、冷丙酮沉淀(-20℃)、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。

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℃）,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.