The Idenfication Of Bacillus Subtilis Pda0426 And Research Of Antimicrobial Metabolites

It is important to exploit new antimicrobial drugs, as the strains with drug resistance appear. The important sources of selecting new antimicrobial drugs are the active antibiotics produced by microbial.In this thesis, one bacteria named pda0426 was separated based on activity of inhibiting staphylococcus aureus. The strain was identified to be Bacillus subtilis by morphology and 16S rDNA sequence and phylogenetic analysis. It was named Bacillus subtilis pda0426.The conditions of fermentation of Bacillus subtilis pda0426 were optimized according to activity of broth. The best constitutes of medium were peptone, glucose and NaCl. The best condition of fermentation was described as below: initiative pH is 8.0, temperature is 30°C, rotate speed is 180 r/min and it has been cultured for three days. At the same time, the method of crude separation has been builded based on the recovery rate of fraction, and confirmed to be LH-20 sephadex gel chromatography and reverse phase gel chromatography.Nine main compounds have been obtained by crude separation and purification of active fraction. The compounds, 4-11-1, 4-11-2, R6-5-13.14-1 and R7-1.2.3-13.18-5 were confirmed by 1H-NMR, 13C-NMR, 2D-NMR and MS. The results indicated that the compound named 4-11-1 was cyclic peptide and compound named R6-5-13.14-1 was macrolatin as same as 4-11-2.The activity of compounds has been studied by high throughput and MTT assay. The minimum inhibition concentration (MIC) and 50 % inhibition concentration (IC50) of compounds inhibiting test strains and tumor were confirmed. The MIC of compounds named 4-11-2 and R6-5-13.14-1 inhibiting staphylococcus aureus was 2.0μg/mL. The IC50 of inhibiting PC3 was 41.0μg/mL. The compounds could not inhibit fungi. So the compound named 4-11-2 was the main constitute of inhibiting staphylococcus aureus in broth. The active compounds inhibiting fungi needs to be separated, because the broth can inhibit fungi.