解淀粉芽孢杆菌素Amylocyclicin W5的纯化及其抑菌机理

为研究解淀粉芽孢杆菌素Amylocyclicin W5的抑菌机理,本实验通过硫酸铵沉淀、离子交换以及?KTA蛋白纯化系统对Amylocyclicin W5进行分离纯化,并对Amylocyclicin W5进行三羟甲基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳;此外,以蜡样芽孢杆菌(Bacillus cereus LMG...

Purification and Antimicrobial Mechanism of Amylocyclicin W5 Produced by Bacillus amyloliquefaciens DH8030

In this study, amylocyclicin W5, a bacteriocin produced by Bacillus amyloliquefaciens DH8030, was purified by sequential use of ammonium sulfate precipitation, ion exchange chromatography and ?KTA purification system, and it was analyzed by Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Bacillus cereus LMGT2805 was employed as an indicator strain to evaluate the antibacterial effect of amylocyclicin W5. The changes in cell morphology and the intracellular structure induced by the bacteriocin were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results showed that the optimal degree of saturation of ammonium sulfate precipitation was 70%. In addition, 0.5 mol/L NaCl could significantly elute adsorbed bacteriocin from HiPrep SP XL 16/10 column. The eluate was fractionated by using ?KTA purification system into two fractions (F1 and F2). From bacteriostatic experiments, it was found that F2 but not F1 had a bacteriostatic effect against Bacillus cereus LMGT2805. The molecular mass of F2 was determined to be about 12.3 kDa. Amylocyclicin W5 at high concentration (four-fold higher than the half-minimal inhibitory concentration (MIC50) could completely inhibit B. cereus LMGT2805, and its antimicrobial mechanism was by destroying the cell wall to form holes and thus causing the leakage of intracellular contents, abnormal metabolism and finally cell death. These results provide data supporting the development of amylocyclicin W5 and its application in the field of food safety.