Isolation And Identification Of Myxobacteria And Study On Its Antitumor Activity

Myxobacteria is a kind of gram negative bacteria,which is a class of complex multicellular population and belongs to the Delta Branch of the Proteobacteria.Myxobacteria can gliding movement,with predator characteristics to prey on bacteria.Myxobacteria can produce various bioactive metabolites,which have novel structure and various mechanisms of action.Myxobacteria has important research and application value in agriculture,medicine,industry and other aspects.Due to the important resources of natural products,myxobacteria has became the second largest category of medicine source development bacteria following the actinomycetes.Exploring metabolites from myxobacteria for anticancer drug lead compounds has become a hot topic of microbiologic and pharmaceutical technology research in the field.In this study,we separated and purified eight strains of myxobacteria from soil samples use the destroy living Escherichia coli induce fruiting body method and filter paper induce fruiting body method.We preliminary class and identify these strains belonging to two genera,five strains for Myxococcus and three strains for Sorangium,by observed they fruiting body and colony morphology.We identified and classified strain STXZ77 as Myxococcus stipitatus STXZ77 combined with a series of bacterial identification method of morphological observation,physiological and biochemical characteristics and 16S rRNA gene sequence homology analysis.This study enrich the resources of myxobacteria strains,which provided an important basis for excavating the secondary metabolite of myxobacteria.The antitumor activity of strain STXZ77 was studied by toxicity test.The supernatant of the fermentation broth had a highly effective toxicity effect on B16 and HCT-16 cells in dose-and time-manner.The fermentation supernatant lost antitumor activity in more than 60 ? water-bath heating for half an hour,indicated that the main active substance in the fermentation supernatant with thermal instability.The antitumor activity of precipitation from the fermentation supernatant protein by solid ammonium sulfate fractionation was tested.The results show interval of 40%?60%saturation has the strongest cytotoxicity,while interval of 80%?100%weakest.The protein crude extract was obtained by 80%saturation ammonium sulfate precipitation,and then the antitumor active compound was separated from the crude extract by anion-exchange chromatography.Fermentatde supernatant of strain STXZ77 was adsorbed by XAD-16 macroporous adsorption resin for 24 hours.XAD-16 resin crude extract was harvest by desorped the adsorbed XAD-16 resin with methanol.The antitumor activity of XAD-16 resin crude extracts was studied by CCK-8.Results reveal that the XAD-16 resin extract has toxicity to B16,4T1,SMMC-7721,HeLa and SW480 with IC50 values of 5.34 ?g/mL,13.50?g/mL,11.93 ?g/mL,28.70 ?g/mL and 48.09 ?g/mL,respectively.It has minor toxic to HUVEC cells,which IC50 value is 17.09 ?g/mL,less than B16,4T1 and SMMC-7721 cell lines.Four antitumor activity compounds,named AP-A,AP-B,AP-C and AP-D,was separated from the XAD-16 resin crude extract by HPLC.Mass spectrometry analysis the compound AP-C,its ESI-MS m/z 422.99[M+H]+.These compounds have an important value for development and utilization,which laid an important foundation for the search of new antitumor drugs.