Antibacterial And Anti-tumor Activity Studies Of Bacillus Amyloliquefaciens X030

Bacillus has a series of characters, such as wide distribution, easy isolation, short growth cycle and fast breeding. Bacillus can produce abundant metabolites during the growth and reproduction process,which have biological activities including antimicrobial, insecticidal and anti-tumor. 339 strains of Bacillus were isolated from soil samples all over the country. Among them, a new Bacillus strain named Bacillus amyloliquefaciens X030(referred BaX030, CCTCC NO:M 2014159)was identified according to morphological observation, physiological and biochemical characteristics, and the homologous analysis of 16 S rRNA sequences. A kind of polypeptides with antibacterial activity and a type of small molecules with anti-tumor activity were obtained by multistep separation.After cultivation for 48 h in LB, sterile supernatant of BaX030 strain was obtained by centrifugation and filtered handle. Antibacterial component B2 and antitumor active BAH1 were obtained from the sterile supernatant by dextran gel chromatography and C18 reverse phase chromatography. LC-MS/MS identification showed that the molecular sizes of B2 and BAH1 were 182.09 Da and 122.14 Da respectively.The fermentation supernatant of BaX030 was treated with different temperatures and pH, then bacteriostasis experiment was performed through filter paper method against Staphylococcus aureus,Candida albicans and other seven pathogens; confrontation culture method was executed against Chili pointed cell anthrax, gloeosporium eriobotryae speg and other six plant pathogenic fungi. Results showed that the fermentation supernatant has antibacterial activity againstStaphylococcusaureus, Candidaalbicans, Chili pointed cell anthrax and gloeosporium eriobotryae speg; it has a best activity at room temperature, neutral pH and treatment with acid, its activity decreased in the high temperature and treatment with alkali; Chili pointed cell anthrax and gloeosporium eriobotryae speg basically stopped growing and formed a encirclement around the plant pathogen, when Ba X030 underwent a confront culture study with the plant pathogen for ten days.The morphology of B16, Hela, Hep-3B and HUVEC cell were observed by inverted microscope after treating with BAH1; the subcellular structures of tumer cells were analyzed by Confocal observation after immunofluorescence staining with Hochest33342,tubulin Alexa Fluor 488 and Mito Tracker Red CMVRos respectively.Results showed that the morphology and structure of these three tumor cell have undergone significant changes after treatment with BAH1 including cytoskeletal damage, cell shrinkage rounded, irregular nucleus and mitochondrial membrane lysis; the morphology of HUVEC cells has little changes after treating with BAH1 while compared to the control. MTT results showed that BAH1 could inhibit the proliferation of the three tumor cell, which reduced tumor cell activity; and BAH1 also has some effect on HUVEC cell proliferation.These results revealed that BAH1 showed tumor cytotoxicity by disrupting the cytoskeleton and mitochondria, and its toxicity was relatively weak to normal cells.