| During the research work of activated products from microorganism extracted from a definite condition, only a small number of microbial strains could produce activated metabolism.Only a small number of microbial strains to produce a corresponding activity, and they also can be directly applied to the study of secondary metabolites, the vast majority of strains in the corresponding screening models showed no corresponding activity. How to transform these non-active strains of secondary metabolism, thus making it effectively converted to the active strains, and produce corresponding active secondary metabolites, is the focus of this study need to study. Ribosome engineering and associated antibiotic resistance selection techniques have been successfully applied in the regulation of the secondary metabolic transformation of prokaryotic microorganisms in the field of microbiology, and aminoglycoside antibiotic resistance through the active filter will not be converted into an active wild type strains of fungi mutants, have tried to apply only in specific habitats between intertidal fungal G59 in our previous work on this basis of this study, the antibiotic-resistant fungal cell technology imported to another no activity of the wild strains of deep fungal origin, it activated the conversion occurs, a stable resistant mutants having activity through resistance have proved to be stable and resistant to the introduction, and then obtain a new separation produced by fermentation activity compounds and verified, and finally clarifies the new active product produced mutants.In this research, from the collection at 174.2130 °E, 24.3444 °S in the South Pacific in Fiji surrounding ocean, at the depth of 800 meters inactive mud fungal ZBY-3 was extracted, taxonomic studies identified by the strain of Aspergillus versicolor. Neomycin resistance screening methods and with different concentrations of DMSO under ultrasound-mediated acquisition of mutant strains and a mutant anti-bacterial, anti-tumor activity screening. On this basis, the activity of the mutant in which a new production activity was found.MTT assay was used to test human leukemia cell K562 cells by primary and secondary screening, at 100 μg/m L sample concentration no anti- tumor effect. This strain was inactive strains secondary development of research exploring inactive strains provides an effective original strain.Ultrasound and DMSO mediated different methods, a spore suspension containing different concentrations of neomycin treatment, confirmed DMSO mediated neomycin resistance screening method can obtain mutants. Through the bacterial suspension processing time, DMSO volume concentration ratio, neomycin concentration and other factors to inspect, to further improve and supplement the neomycin resistance screening method to import non-active fungal mediated DMSO, using aminoglycoside antibiotics against fungi be resistance screening, which will translate into active non-active fungal strain resources.Neomycin resistance by DMSO mediated screening method to create the deep source of non-active fungi Aspergillus versicolor ZBY-3 treatment, there were isolated the difference in morphology resistant mutants 33, wherein fermentation products acetate extract 12 when 100 μg/m L of human chronic myeloid leukemia K562 cells derived greater than 30% inhibition of the growth of proliferative activity( the total number of trees for the purpose of resistance mutations of 36.4%), TLC and by TLC analysis, the activity of secondary metabolites can be seen that the mutant strain, the generation of the parent strain could not ZBY-3 metabolism of substances, these results show that the methods section expressed by DMSO mediated under the guidance of neomycin import inactive deep fungi, can really transform the original strain, and get new active secondary metabolites, thereby expanding research activity strains available source products.On the other hand, high concentrations of neomycin resistance by ultrasound screening method established for non-mediated activity of the fungus Aspergillus versicolor deep source ZBY-3 treatment process, there were isolated the morphological difference of 30 resistant mutants, through a series of anti-tumor cell activity test, the antibacterial activity test, 17 secondary metabolites in the resistant mutant 100 μg/m L concentration of K562 cells inhibited by more than 30%, the conversion rate of active up 56.7%, of which 10 secondary metabolites of K562 cells greater than 50% inhibition rate stability, in addition to the seven secondary metabolites inhibition rate of K562 cells stably between 30% to 50%; six of mutant secondary metabolites measured in paper antibacterial activity experiment, under 750 μg/slip concentrations have six resistant mutants of Escherichia coli ATCC® 25922 has antibacterial activity, the active transformation rate of 20 %. Combined with TLC and HPLC-PDAD-UV comparison chart, as well as combining the strength of resistant mutants of activity, based on the richness of the product, we chose 200-200 3h 1-2 Secondary Metabolites of the object as a next step.Activity against mutant 200-200 3h 1-2, we carried out verification experiments resistance, under the same conditions of the original strain and the target strain ZBY-3 200-200 3h 1-2 simultaneously resistance test to verify the 200-200 3h 1-2 neomycin resistance were successfully imported. Further extensive fermentation antagonistic mutant and track the activity of the original strain using any separation mode control, compared to the original strains six new compounds were produced in the mutant strain differences in metabolite Purification: 3β, 5α, 9α-Trihydroxy-(22E, 24R)-ergosta-7,22-dien-6-one(6), Cyclo(D-Pro-D-Phe)(1), Cyclo-(D-tyrosyl-D-proline)( 2), Phenethyl 5-oxoprolinate(3), Cyclo(L-Ile-L-Pro)(4), Cyclo(L-Leu-L-Pro)(5), wherein the compound 3 is a new natural product. These products were 100μg/m L for K562, HL60, BGC-823, Hela cells were or weak or strong anti-tumor activity. Wherein the IC50 value for the compound 1,2,6 K562 cells was 399.5 μM(1), 303.7 μM(2), 221.0 μM(6), IC50 values for compounds of 1,2,3,6 HL-60 cells were to 225.6 μM(1), 254.7 μM(2), 215.1 μM(3), 89.1 μM(6), the compound 1,2,6 for BGC-823 cells with IC50 values of 242.2 μM(1), 328.7 μM(2), 177.7 μM(6), the compound 3,6 of Hela cells with IC50 values of 194.5 μM(3), 224.3 μM(6), and verified by HPLC-MS six compounds do not produce the original strain, but the new production of resistant mutants.In conclusion, in this study, with no deep source of the original activity of the wild strain of strain introduced on the basis of fungi and neomycin resistance DMSO on the ultrasound mediated by screening in vitro screening model, achieved without active strains the activity of the transformation, and received active mutants, and sheds light on the activity of the mutant strain of mutant 200-200 3h 1-2 parts of a new production of active metabolites. Through this series of studies have shown that non-active resistance to the import of the wild mutants; get active resistant mutants, and its great number of fermentation, the discovery of new active compounds as well as new active natural products. This study established a complete way, no activity can be transformed into a wild strain of active strains, making it no confined to the eyes of wild strains activity has effectively opened up new resources microbial strains drug source activity, is to study the feasibility and practical application value. |
PDF Full Text Request