| Fungal infections have a wide spread in clinical, the common antifungal drugs include polyene macrolides, azoles, echinocandins, allylamines and pyrimidine. However, they are toxic, and can cause serious side effects. Study found that the side effects of polyene macrolides are the lowest when compared with other types of drugs, and it is also the last drug to treat fungal infections clinically. Use a large amount for a long time can also lead to renal failure and thrombophlebitis, so it is very important to look for a antibiotic with lower toxicity.Streptomycese netropsis SD-07 is a new strain found by our laboratory in 2007, which can produce polyene macrolide antibiotics. When compared with amphotericin B, it’s has a higher antifungal activity. This strain has two high-yielding mutant strains, named 163 and 180. In order to verify three strains after five years still have high yield, where there is a change of structure or activity of antibiotics. The present study produced by the comparison with three strain’s antibiotic production and activity, meanwhile different purification methods of activity, stability and toxicity were compared.The laboratory found that Ca2+ can greatly increase the conjugation frequency of the genetic transfer systems of S.netropsis SD-07. When observed Streptomycese netropsis SD-07 by electron microscopy, we can find there are more extracellular polysaccharide (EPS) around the strain when add Ca2+. In order to better clarify the relationship between extracellular polysaccharide, Ca2+ and conjugation, the present study had the related research of the fermentation and the analysis of extracellular polysaccharide structure.The results of the study are as follows:1. The experimental results of antibiotics180, the mutant strain of Streptomycese netropsis SD-07 is a high-yielding strain of antibiotics, the mutant strain 163 is lower, compared with the original strain SD-07, 180 antibiotics yield is 3.33 times and 163 is 1.28 times.The experiment results of HPLC, LC-MS and antibacterial proved that there is no major components difference between the three kinds of antibiotics.Different methods of preparation showed that the antibiotics have the most stable activity by extraction method, and the most unstable method is made by molecular sieve.By the antibacterial experiment which conducted after three months proved that the most stable antibiotics is produced by 180 strain, antibacterial activity increased with the extended time, that may be related to the solubility.Cytotoxicity test showed FPLC is a good way to reduce toxicity and improve antibacterial activity when purified antibiotic.Mutagenicity test by SOS/umu-lacZ system showed that in vitro antibiotics produced by Streptomycese are had no mutagenicity. Regardless of the mutant strains types and preparation methods.2. The experimental results of EPSWhen the fermentation time was 48h, the yield of extracellular polysaccharide was highest. We choose medium R2YE as fermentation medium, Ca2+ has obvious promoting role in the formation of polysaccharides, while Cu2+ inhibitory effect is most obvious, K+ and Na+ have almost no impact on polysaccharides, low concentration of Mg2+、Fe3+ and Mn2+ can promote the production of extracellular polysaccharide.Through alcohol precipitation and sevege method, the protein in EPS was removed, after separating EPS by DEAE, and the molecular sieve named SephadexG-75.Multi-angle laser light scattering analysis showed that the average molecular weight of the main component with polysaccharides is about 32.29kDa.Monosaccharide composition analysis results show the EPS of SD-07 is composed by four monosaccharide:Fuc, Gal, Glc and Man, and Fuc:Gal:Glc:Man= 1.33:1.00:8.67:27.33.FTIR results show that the polysaccharide contains mannose and glucose, and their configuration is β-D-glucose and (3-D-mannose.Research results:this research screened the stable high yielding and highly active strain 180, its composition antibacterial activity and other properties of antibiotics as the same as original strain SD-07, suggesting that 180 is the suitable strains in the future when production of antibiotics. The production of EPS is related to Ca2+,and EPS structure provide the reliable data for the relationship between EPS and the genetic transfer systems. |
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