Optimization Of Fermentation Conditions And Purification Of Antimicrobial Compounds From Streptomyces Xinghaiensis

Streptomyces xinghaiensis is a novel Streptomyces species isolated from marine sediment in Xinghai Bay in Dalian, China. The fermentation broth of Streptomyces xinghaiensis displayed certain antimicrobial activities against test strains including Staphylococcus aureus, Psedomonas aeruginosa, Escherichia coli, Bacillus subtilis, Candida albicans.In this thesis, the antimicrobial activities of the culture broth of S. xinghaiensis against drug-resistant microorganisms were investigated, and the fermentation conditions of the active compounds were optimized. The active compound(s) against methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates were purified. The main experimental results were as follows:The fermentation broth of Streptomyces xinghaiensis presented antimicrobial activities against drug-resistant Acinetobacter baumannii and Staphylococcus aureus. Cultured at rich nutriment and with no addition of NaCl, the fermentation broth had the highest antimicrobial activity on the 7th day.The antimicrobial compounds in the fermetation broth were stable at 20℃to 100℃within 30 min and under pH 1 to 14 within 1h. The N-hexane, acetone, acetoacetate extracts of the fermentation broth had no antimicrobial activity, the active compounds were hydrophilic. SPE experiment showed that the active components against drug-resistant Staphylococcus aureus are alkaloid compounds.The fermentation broth exhibited activity against drug-resistant Staphylococcus aureus after macroporous adsorptive resin HP-20 absorption. Active fractions were collected from Sephadex LH 20 chromatography. HPLC analysis revealed that the active components had no specific UV absorption, which correlates with the characteristics of aminoglycoside antibiotics.Thin Layer Chromatography (TLC) analysis and ninhydrin coloration experiment were further performed with the active fractions from Sephadex LH 20 experiment, prunosus ninhydrin coloration further supported the hypothesis that the active compounds likely belong to a minoglycoside antibiotics. The compounds with positive reaction in ninhydrin coloration were isolated well with the chromatography solvent system of methanol-chloroform-aqueous ammonia (5:2:4). The biggest Rf value of the compound with the deepest ninhydrin colour was found using the chromatography solvent system of methanol-chloroform-aqueous ammonia (3:1:2). The antimicrobial compound(s) was (were) able to migrate on silica gel plate based on the antimicrobial test. The genomic sequence of S. xinghaiensis was searched for possible aminoglycoside antibiotic gene cluster, and a ribostamycin-like gene cluster was identified. However, TLC analysis indicated that components of the active compounds are different from ribostamycin sulfate.