Separation And Preliminary Analysis Of The Structure Of Antibiotics From A Marine Actinomycete

In this study, different samples including sea water, sea sand, sea invertebrate, sea moss, sea mud and decayed wood were collected from Xiamen sea area. A variety of marine microorganisms were isolated，purified，preserved from different samples. As a result, a marine-derived actinomycete DY2242with broad-spectrum antimicrobial activity was screened from sea invertebrata collected from Xiamen intertidal zone. According to its morphological, cultural characteristics and physiology and biochemistry and16S rDNA analysis, this strain was identified as Nocardiopsis dassonvillei preliminarily.In order to increase the yield of antibacterial substances produced by DY2242, Response Surface Methodology（RSM）was employed to optimize culture medium of actinomycete DY2242.The influences of different media，carbon and nitrogen resource were investigated by single factor experiment. The plackett-Burman design was used to evaluate the effects of different components in the culture medium. NaCl, maltose and NaNO₃have significant influences on the antibacterial substance production. The path of steepest ascent was used to approach the optimal region of above three factors. The concentrations of three key factors were optimized subsequently using Box-Behnken design and response surface methodology. The optimum medium composition: NaCl2.08%, maltose3.08%，NaNO₃0.875%. As the result, the inhibition zone diameter of antibacterial substance increased from25mm to31mm with the optimal medium. The diameter is increased by24%. Optimization of fermentation conditions showed that initial pH was7.0, inoculation quantity5%, seed age72h, the optimum volume70mL （250mL flask）, the optimum temperature28℃and fermentation time5d. The validation experiment of optimization condition shown that, the inhibition zone diameter of antibacterial substances increased from25.5mm to33.5mm with the optimal medium. The diameter is increased by31.4%.The antibacterial substances in the ferment broth were stable to the ultraviolet light and pH. The antibacterial activity started to decline at100℃. Separated by Sephadex LH-20column chromatography, a strong antibacterial fraction was obtained. The sub fraction displaying a clear single spot in TLC anlysis was demonstrated by gas chromatography-mass spectrometry. The total ion chromatogram shown a single peak, which was preliminarily identified to be dibutyl phthalate （DBP） based on the mass spectrum comparing with Wiley Registry of Mass Spectral Data.