Bacillus Licheniformis Zju12 Bacteriocin Produced Isolation, Purification And Physicochemical Properties And Gene Cloning Research

Bacillus licheniformis ZJU12, which was isolated from soil, could produce bacteriocin-like peptides that exhibited a broad spectrum of antagonistic activity against various species of Gram-positive bacteria and fungal pathogens, but not against Gram-negative bacteria tested except Xanthomonas oryzae pv. oryzae, a rice pathogen. The bacteriocins were sensitive to proteinase K and trypsin. The activity was stable during temperature exposure up to 100 ℃ for 30 min, but lost completely at 121 ℃ for 15 min. The cell-free supernatant of B. licheniformis ZJU12 was shown to retain the activity within the pH range of 2-9, and the optimum pH for the activity was about 6.5. The purification of bacteriocin was obtained by following ammonium sulfate precipitation (65%), ion-exchange chromatography (DEAE DE52) and gel-filtration (superdex G-75). Then Tricine-SDS-PAGE and HPLC were applied to judge the purification. The molecular mass 3.5KDa was obtained from Tricine-SDS-PAGE. Three amino acid sequences MHENVEEYLPMN, QPATEYLYNTDY, LLPLLVDTY were calculated from LC-MS/MS. No homology with other sequences was found when searching in Gene Bank (NCBI). No amino acid residues were detected upon direct N-terminal analysis. No adverse effect of the antagonistic compound to mice was observed in acute toxicity tests with the dose of 0.8 mg/20 g.Antibacterial activity of nisn and cell-free supernatant of Bacillus licheniformis ZJU12 (CFSb), separately and in combination, against three food-borne bacteria: Micrococcus flavus, Bacillius cereus and Staphylococcus aureus was determined. M. flavus was more sensitive to nisin and CFSb than other two indicator strains in the MIC tests. Synergetic activity of nisin and CFSb was found against all the three indicator strains in the checkerboard microtire test and the time-kill test. The combination had the most notable antibacterial activity against B. cereus as determined by the Fractional inhibitory Concentration index (FICI). The antibacterial activity of nisin and CFSb separately began within the first 30 min, while theirsynergetic activity was observed after 4 h.The polyclonal antibody of bacteriocin produced by Bacillus licheniformis ZJU12 was prepared by immunizing New Zealand rabbit with purifed bacteriocin from peak 2 of gel -flitration (Superdex 75) step. The sensitivity of antigen-antibody reaction analysis was detected at antigen (500AU/ml) with antibody (1:2000) by dot-ELISA and at antigen (lOOAU/ml) with antibody (1:8000) by antagonistic activity assay. Therefore, antagonistic activity assay was more sensitive than dot-ELISA in the antigen-antibody reaction analysis. No plasmid was extracted from B. licheniformis ZJU12. Two bacteriocin suspected gene was obtained by screening genome DNA liabrary based on the antagonistic acitvity and polyclonal antibody probe. Moreover, it was found that L. lactis subsp. lactis and B. licheniformis ZJU12 could co-culture with no antagonistic activity against each other. One PCR product with ca. 1500bp was detected in PCR using DNA of B. lichenformis ZJU12 as template and partial Nisi sequence as primers.