The Primary Study On Separation And Characterization Of An Novel Antibiotic From Fungus DT06

Fungal DT06 belongs to the variant of Simplicillium lanosoniveum. Previous study found that DT06 can produce unknown antibiotic-like metabolites(ALM) strongly inhibiting gram-positive bacteria. Study on activity natural products from S.lanosoniveum is rare. This is the first report on the antibacterial substances as an antibiotic-like metabolite of symbiotic fungi associated with cyanobacteria, the main results are as follows :In the optimization experiment of adsorption conditions of antibiotic about CT-12 macroporous resin, the growth status of DT06 conidia was observed. Then, we selected three factors which influence the adsorption effect of macroporous resin, and estimated L9(33) orthogonal experiment. The best adsorption condition was as follows: pH=3; D/H=1/5; flow rate=1.2mL/min. Under these coditions, antibiotic recovery rate reached to 95.11%. After determineing the optimal adsorption conditions, we found that the best elutions was 95% ethanol and 50% ethanol solution can remove a lot of pigment by concentration gradient elution.In order to further purification of antibiotics, three kinds of weakly basic anion exchange resin D392, D380 and D301 T were selected. The results reveal that D301 T resin, which was acidificated by acetic acid solution, adsorpted pigment in the sample liquid, but antibiotics were not adsorpted. We considered ion exchange resin D301 T as decoloring agent. In the purification process of antibiotics, the pigment caused great disturbance. In this paper, we conducted a preliminary study of pigment. The results revealed that the pigment belongs to polypeptide substances.This study laid a foundation for the further study about the symbiotic relationship between DT06 and cyanobacterium.In the experiment of the physical and chemical properties of antibiotic, carbon tetrachloride was determined as the best precipitant agent. HPLC analysis indicated that ALM was distinct from the expected cephalosporin C, with the different retention times of 4.1917 min and 3.1083 min, respectively. Furthermore, the negative Biuret tests confirmed that antibiotic unlike polypeptide or lacking peptide bond; The negative Ninhydrin tests confirmed that unlike cephalosporin C, ALM lack-NH3 and –COOH; Sulfuric acid ethanol solution color results indicated that antibiotics are likely to belong to the terpenoids or steroid material. Absorption spectrum revealed that ALM has a major and a minor absorption peaks around 228 nm and 277 nm, suggesting the possible existence of aromatic side chains. Thus, these results demonstrated that antibiotic is a new antibiotic, which may be of application potential in bio-industry and help explain the complex interactions between photoautotrophic and heterotrophic microorganisms in aquatic ecosystem. This study laid a foundation for the industrial purification and crystallization process of this unique antibiotic.