Optimization Of Fermentation, Purification And Antibacterial Mechanism Of Bacteriocin CAMT2Proudced By Marine Bacillus Amyloliquefacien

Bacteriocin CAMT2, produced by Bacillus amyloliquefacien ZJHD-06isolated fromintestine of Epinephelus areolatus, shows broad-spectrum antibacterial, thermotolerant andhigh pH stability. Bacteriocin CAMT2fermentation culture medium was optimized toobtain high antibacterial fermented liquid.Then, the fermented supernatant was purified inorder to obtain bacteriocin CAMT2of high purity. At last, the bacteriocin CAMT2of highpurity was researched on antibacterial mechanism.(1) Optimization of Bacillus amyloliquefacien of fermentation medium producingbacteriocin CAMT2: the titer was used as evaluation index against Bacillus flexus. Fristly,the TGE medium was selected as basic fermentation medium from seven fermentationmediums. Then, the notable factors (glucose, FeSO4, Vc) were screened byPlackett-Burman experiment design on the basis of16affecting factors. And, the centerlevels of Box-Behnken experiment design were respectively determined as7.0g/L,0.09mg/L,0.65g/L by the steepest uphill experiment. The results of the experiment wereanalysised by JMP software. Finally, it is concluded that the optimal proportion wasglucose7.15g/L, FeSO40.11mg/L, Vc0.69g/L, tryptone10g/L, yeast extract10g/L.The antibiotic titer of fermentation supernatant produced by the optimized fermentationculture medium was2.1times than fermentation supernatant of original TGE mediumthrough verification experiment.(2) Purification of bacteriocin CAMT2produced by Bacillus amyloliquefacien: thepurification of bacteriocin CAMT2included four steps: ammonium sulfate precipitation,SephadexG-50column chromatography, SDS-PAGE electrophoresis and identification of aprotein strip. The best precipitation aturation of fermented supernatant was60%accrodingto20%~80%saturation of ammonium sulfate precipitation. The protein precipitation wasdissolved by phosphate buffe. Then, it was dialysised by SephadexG-50columnchromatography.The eluent was0.01mol/L pH7.0phosphate buffer, and the elution curvewas drawed acroding to the absorption values under wavelength of275nm of each tubecollecting liquid. The two elution peaks were showed in the elution curve. The antibacterial activity of the first peak collecting liquid (F1) and the second peak collecting liquid (F2)were tested. The results showed that F1was no-antibacterial activity, F2was obviouslyantibacterial activity. F2collection was freeze dried in vacuum afer dialysised withultrapure water. The dry powder dissolved in phosphate buffer was analyzed bySDS-PAGE electrophoresis. The electrophoresis result showed only a protein band ofabout20kDa molecular weight. After treatment, the protein band was delivered to BGI fortotal mass spectrometry analysis and protein identification. The results showed that therewere5kinds of proteins. Through the comparison and analysis, the no.2protein,molecular weight of19608.57Da, N-terminal amino acid sequence asMKIARTAIGSCLALSLTIPF and the no.3protein, molecular weight of20265.572Da,N-terminal amino acid sequence as MKKWLLFLTTITLILSLGTA were likely to bepurpose proteins.(3) Research of antibacterial mechanism of bacteriocin CAMT2produced by Bacillusamyloliquefacien：growth inhibition of Bacillus flexus, cell morphological changes andeffects on cell membrane permeability were discussed in the experiment. The result ofgrowth inhibition curve of Bacillus flexus showed that Bacillus flexus was inhibitedobviously and had partial bacteriolysis phenomenon.The significant change of cellmorphology effected before and after was observed by scanning electron microscopy(SEM). The effected cell became tapered on both ends of the cell, the cell edge becamejagged, intracellular material leaked because of cells rupture. In terms of the cellmembrane permeability, the experiment was undertaked by testing extracellularconductivity, intracellular and extracellular K+concentration, the concentration of lactatedehydrogenase and ultraviolet absorption substance. It is found that after the action ofbacteriocin CAMT2, Bacillus flexus extracellular conductivity and extracellular K+concentration was quickly increasing within0.5h, concentration of intracellular K+wasreducing rapidly within0.5h, too. This indicatied that bacteriocins CAMT2can causedamage to cell membranes more quickly and efficiently. Extracellular lactatedehydrogenase and ultraviolet absorption material also had the obvious rising trend after3h, suggesting that damage degree of bacteriocins CAMT2to the cell membrane can alreadymake intracellular macromolecular material leaking, causing cell death.