圆孢芽孢杆菌A95抗菌蛋白的分离纯化及性质研究

对圆孢芽孢杆菌(Bacillus globisporus)A95产生的抗菌蛋白进行了分离纯化,并研究了其部分性质。菌株摇瓶发酵液离心后的上清液经硫酸铵40%~60%分级盐析获得粗提物。粗提物溶液经Sephadex G-100柱层析、DEAE-Sepharose Fast F low离子交换层析、Sephadex G-50脱盐纯化获得的抗菌蛋白,通过性质检测、电泳及MALDL-TOF-MS质谱检测,表明为分子量1.464~1.529 kD的一组环肽类抗菌蛋白。该抗菌蛋白对多种家蚕病原真菌具有较好的抗菌活性,参照美国国家临床试验标准化委员会(NCCLS)提出的标准,用微量液基稀释法分别测定了对绿僵菌、白僵菌和黄曲霉的最小抑菌浓度(MIC)和最小杀菌浓度(MFC)。该抗菌蛋白对热稳定,对胰蛋白酶、蛋白酶K、氯仿有一定的耐受性,对酸部分敏感,对碱稳定。

Purification and Characterization of Antagonistic Protein From Bacillus globisporus A95

The antagonistic protein produced by Bacillus globisporus A95 was isolated and purified,and it's partial properties were studied.Cell-free culture was obtained after centrifuging of ferment liquid of Bacillus globisporus A95 by shake-flask fermentation.The crude proteins were obtained by fractionation with(NH_(4))_(2)SO_(4)(40%~60%).Active protein was obtained after purification of the crude proteins by Sephadex G-100,DEAE-Sepharose Fast Flow and Sephadex G-50 column chromatography.The result of MALDL-TOF-MS test showed its molecular weight was 1.464~(1.529 kD).The protein showed good antagonistic activity to many silkworm pathogenic fungi.With the broth microdilution testing procedure proposed by NCCLS,antifungal activity of active ingredients was determined.The minimum inhibitory concentration(MIC) and minimum fungicidal concentration(MFC) of the antagonistic protein to Nomuraea rileyi,Beauveria bassiana and Aspergillus flavus Link were measured.It is stable to heat,and not sensitive to trypsin,proteinase K and chloroform.It acts partially sensitive to acids but stable to alkalies.