Purification And N-Terminal Sequence Identification Of Bacillus Thuringiensis BRC-XQ6 Bacteriocin

Listeria monocytogenes(Lm)is a potentially lethal foodborne pathogen commonly found in the environment.Both human and animal can be infected.Application of antibiotics on controlling Lm has caused a series of safe and environmental problems.Bacteriocin is different from antibiotics although they are both excreted by bacteria.Bacteriocin is non-toxic,no adverse effects and can be degradated by some proteases.In the present study,bacteriocin BRC-XQ6 produced by Bacillus thuringiensis(Bt)BRC-XQ6 was purified and characterized,and its primary structure was studied.The microscopic examination shows it's vegetative phase,sporogonium,phase and lysis phase were at 14h,30h and 48h after incubation,respectively.The vegetative cell is rhabdoid in a single or twin short chains.Sporogonium contains transparent elliptic spore and purple rhomboid parasporal crystal.The cry genes were characterized by PCR analysis.The result showed that Bt BRC-XQ6 harbored cry1 and cry1I genes.And the molecular weight of the main ICP were determined as about 130 kDaand 700-100kDa.Kinetics of bacterioncin production during the growth of Bt BRC-XQ6 showed that the fermentation solution had no activity on the indicator strain Lm 100526 during the logarithmic growth phase(0-8 h).The activity existed during the stationary phase(10-34 h)and disappeard after 36h.The activity got highest value on 26 h.The addition of cell-free supernatants or living cells of BRC-XQ6 and Lm 100526 cannot induce the production of BRC-XQ6 bacteriocin,respectively.According to the antibacterial activities of bacteriocin BRC-XQ6 precipitated by different concentration ammonium sulfate,the 70%concentration ammonium sulfate got the best precipitation effect.The molecular weight of bacterioncin were determined as about 10 kDa by in situ gel assay.The crude bacterioncin was further purified by sephedex G-50,cellulose DEAE-52 anion-exchange and C18 HPLC chromatography.The activitive fraction was 43 ?g/mL and its activity was 224 U with 12.7%recovery rate.8 amino acids of the bacterioncin's N-terminal were measured by Edman degradation method.The sequence was DQ(N,T)EW(S,A)VP.The sequence was compared in the Non-Redundant Database(NRDB)through BLASTp.Since no similarity was found,bacteriocin BRC-XQ6 should be a new kind of bacterioncin.Futher study to this bacteriocin's structure should be conducted and it will be helpful for the development of new natural antibacterial material.