Screening Of Soil Microorganisms And Study On Their Secondary Metabolites

Because of their rich sources, structures, and other unique advantages, microbial-derived lead compounds have an immeasurable role in drug development. However, bacterial resistance is becoming increasingly prominent with antibiotics being widespreadly used. In the past 50 years, only two new classes of antibiotics, linezolid and daptomycin, have been introduced to the market, which highlights an urgent need for new chemical classes of antibacterial agents.Based on this background, With soil as the material, microorganisms with antibiotic activity are screened out in this study. Then target products are obtained through fermentation, extraction and purification. Finally, the products and the microorganisms are both identified.In this paper, five soil samples from different areas of mainland china are collected. 197 strains in total are isolated from the samples, including 96 bacterial strains, 43 fungal strains and 58 actinomyces strains. After the culturing and fermentation for the obtained strains, cylinder-plate method is used to evaluate the antibiotic activity of the extract from strains. In this way, 21 strains with antibiotic activity are selected. Accompanied by the HPLC spectrum, SIPI-2014406 is determined as the mainly studied strain.The fermentation product is purified by macroporous absorptive resin and reverse C18 chromatography to get the compound 018(98.3% purity) and 009(99.6% purity).It is shown in the antibiotic tests that compound 009 is the one with the similar antimicrobial spectrum as the fermentation extract, which is with broad spectrum, while compound 018 is more sensitive for Gram-negative bacteria. Constant trace amount of broth is used to determine the MIC value of compound 018 and 009 for sensitive strains. In this experiment, compound 009 shows weak antibiotic activity for all kinds of tested strains while compound 018 is effective on Gram-negative bacterials but works weak on Gram-positive and fungus.Amino acid determination proves that both compound 009 and 018 contain amino acids D-Ser, L-Thr, L-Asp, D-Asp, L-Glu, L-Pro and D-Tyr. As can be concluded among amino acid composition, Mass Spectrum, secondary mass spectrum and NMR analysis, together with related reference, compound 009 is normal-C14 bacillomycin D( C48H74N10O15) and the structural formula is determined. While compound 018 with the formula C50H78N10O15 is not determined yet.Based on the phylogenetic analysis on 16 S rDNA sequence, it is shown that the strain SIPI-2014406 is almost homologous with Bacillus sp. DOA6. This two share the morphological and chemical characteristics so the strain SIPI-2014406 is classified as Bacillus, and named as Bacillus sp. SIPI-2014406.The present study demonstrated that bacillomycin compounds could be produced by Bacillus sp. SIPI-2014406. At the same time the in vitro antibiotic activity test demonstrated that C16 bacillomycin D had a new effective effect on Gram-negative bacteria.