Isolation And Purification Of A New Myxobacteria And Studies On Its Cytotoxicity

Myxobacteria is a kind of aerobic gram-negative bacteria and belongs to the delta branches of Proteobacteria.Myxobacteria are famous for their complicated multicellular behavior and excellent ability of producing secondary metabolites,which have important application in medicine,agriculture and ecological environment.Looking for antitumor drug molecules from myxobacteria has become a hotspot in microbial medicine technology.We isolated new strains of myxobacteria from the soil and tested their bioactivity tests against pathogenic bacteria and tumor cells.Furthermore we obtained the antitumor active substance from the fermentation supernatant of the new strain,the antitumor active substance was detected to be a new protein by MS.We isolated a myxobacteria S72 from soil sample via inactivated E.coli inducing method,S72 was demonstrated to have high biological activity and was identified and named as Myxococcus sp.STXZ72according to the homologous analysis of 16S r RNA and biochemical characteristic.16S r RNA gene sequences were submitted to the Gen Bank database under accession number:KP090855.This study enriched the resources of myxobacteria strains,which provided an important foundation for excavating the secondary metabolite of myxobacteria.The anti-bacteria activity of S72 was performed with agar plate diffusion method,S72 has anti-bacterial effect to Proteus vulgaris,salmonella typhimurium,staphylococcus aureus,Monilia albican.The anti-fungal activity was performed with Plate Confrontation Culture Method,It was found that S72 has anti-fungal effect to Pepper fusarium oxysporum,Pyricularia oryzae.We suggested that S72 was an inhibitor to various bacteria and fungel and the ability of anti-bacteria and anti-fungal showed obvious difference.The main emphasis of our research was the antitumor activity of S72.The fermentation supernatant has efficiently antitumor activity against various tumor cells.We found that the antitumor substance was a kind of protein or polypeptide by proteinase K treatment and many kinds of biochemistry test containing ammonium sulfate precipitation,temperature gradient,organic solvent treatment.The main antitumor active substances were extracted through HPLC from the fermentation supernatant,proteins were obtained by?NH₄?₂SO₄solution and the crude extraction were fractionated by Sephadex G-75.We collected three fractions and tested their bioactivity.Fraction 2 has antitumor activity and identified by MS to be a new protein.MTT method was utilized to detect the inhibition ability of tumor cells.It showed that the active component isolated from the fermentation of S72 can inhibite various tumor cells.The component inhibited the growth of He La,4T1,Hep-3,MDA-MB-231,B16,HT-29,HCT-116,H460 with IC₅₀ values of 84 ng/m L,81 ng/m L,73 ng/m L,71 ng/m L,63ng/m L,62 ng/m L,58 ng/m L,41 ng/m L.We found that after the active component treatment tumor cells were floating from the dish by inverted microscope observation.The mechanism of antitumor,performed with FCM technology,may be related to specifically associate with tumor cell membranes.This study played an significant role in exploitating antitumor active pharmaceutical.In order to increase the production of active material,we studied the influence of metal ions on its growth by adding Cu²⁺,Fe³⁺,Mn²⁺,Zn²⁺into the medium.The effect of Cu²⁺can make the biomass increasing significantly,but can not increase its antitumor activity.The study has great significance for further study on molecular structure of anti-tumor active material,confirmation of the antitumor molecula r mechanism and enhancement of the anti-tumor effect of S72.