Isolation And Identification Of The Strain SXW-05 From Three Gorges Reservoir Area Soil And Analysis Of Its Activity Production

Reserch background:For the past few years, with the wide use of antibiotics and the antibacterial agent's bacteria resistance to many antibiotics is increasing seriously, it is necessary to make effort to seek new and effective antibiotics immediately; And, owing to its specific heredity background and biosynthetic pathways, the fungi is extremely rich in secondary metabolites and is supposed to be the important resource of natural active products. Since Fleming discovered the first broad-spectrum antibiotic-penicillin, looking for novel active secondary metabolites has been regarded as a significant research by scientists working for biology and medicine over the decades.Reserch destination:Isolate and cultivate fungus from soil of Wushan of Three Gorges reservoir area,and screening for strains with antibiotics and anti-tumor activity.Select the fungi possessing the most widely Antibacterial spectrum or strongest antitumor activity as the objective strain.We proceed initial investigation of the the objective strain's fermentation production and separation,purification and structure characterization of activity agents in order to Looking for and enriching leading compounds from Three Gorges reservoir area.Reserch methods:We isolated fungal strains from treated soil sample of Three Gorges reservoir area Wu Shan using the method of plate diliquation.And then we screened antibiotics and anti-tumor activity strain with the method of Oxford cup double-deck plate and MTT (Mono-nuclear cell direc cytotoxicity assay) employing Staphylococcus aureus, Escherichia Coli, Candida albicans, Sarcina lutea, Bacillus subtilis as the tested strain and colon carcinoma cell SW480 as the tested cell. The integrated studies on morphological characteristics, cultural characteristics on three identification mediums (PDA medium, Czapek medium. Martin medium), and 18S rDNA sequence analysis combining together with the handbook of fungal identification (Jingchao Wei) to definite the objective fungi's genera; the strain SXW-05 was cultivated in PDA yeast medium under the condition of 210r/min at 25℃in a rotary shaker for 8 days, after that its activity production's separation and purification from the acetic ether extracts of fermentation was explored initially.by using the method of TLC(thin-layer chromatography), column chromatography, HPLC (high performance liquid chromatogram) and recrystallization. Through the spectrum resolution of MS (mass-spectrum), UV(ultraviolet spectra), NMR (nuclear magnetic resonance) we determined the acvtive compound FB44 and it was evaluated for its cytotoxicity against the tested lung cancer cell A549 and colon carcinoma cell by the MTT method.Reserch results:We obtained 20 fungal strains from treated soil sample of Three Gorges reservoir area.Through antibiotics and anti-tumor.activity screening, the strain nominated SXW-05 was selected as the further studied strain whose fermentation broth not only possessing apparent anti-tumor activities but also showing strong broad-spectrum antibacterial activities. The integrated studies on morphological characteristics, cultural characteristics, and 18S rDNA sequence analysis combining together with the handbook of fungal identification (Jingchao Wei) showed that the strain SXW-05 was accordance with Aspergillus terreus, with 98%homology and has intimate genetic relationship with Aspergillus terreus FJ878634.1.When the observation of colony of strain SXW-05, we discovered its growth condition is better than other mediums. Accordingly, the strain SXW-05 was cultivated in PDA yeast medium under the condition of 210r/min at 25℃in a rotary shaker for 8 days. After that we obtained 5kg acetic ether extracts of fermentation both and obtained a pure compound denominated FB44 with anti-tumor activity after separation and purification of the extracts and then the compound FB44's structure was determined as 4,5-dihydroxy-3-propenyl-2-cyclopenten-1-keone. It was evaluated for its cytotoxicity and the result indicated that FB44 displayed significant activity against colon carcinoma cell SW480, cervical cancer cell, lung cancer cell A549 and colon carcinoma cell SW480 with its IC50 was 0.03mM,0.23mM,9μM respectively.Conclusions:,We obtained the Aspergillus terreus fungi SXW-05 Which posseses anti-tumor activity and isolated one of its main metabolites, 4,5-dihydroxy-3-propenyl-2-cyclopenten-1-keone, which is the same with terrein, accordingly we established foundation of research for the compound's other activity and mechanism and enrich the natural leading compound of San Xia reservoir area.