Isolation, Purification And Partial Characterization Of A Pyrimidine Nucleoside Antifungal-antibiotic

In recent years, the infection of deep fungus increases gradually, which threatens sufferers' health and lives. A strain of Bacillus which can produce a kind of pyrimidine nucleoside antifungal-antibiotic was screened. This material has high antifungal activity against Canidia albicans. In addition to the study of fermentation process, we focused on the purification of the antifungal antibiotics in this work. This would be useful for the further development and applications of this novel antibiotic.The fermentation conditions were optimized as follows under 250 mL shaking flask culture: initial pH 6.5, inoculation 8% (vol/vol), rotating speed 200 r/min, liquid volume 40 mL/250mL, 28℃, fermentation period 48h. Under the aforementioned condtions, antifungal activity of the broth reached its peak value as 940.35 U/mL. Furthermore, the process was scaled-up in 5 L continuous stirred tank reactor (CSTR). The potency of antifungal antibiotic arrived at its maximum value as 950.5 U/mL after 36 h cultivation period under the operating conditions as rotation speed 200 r/min, broth content 60%, aeration rate 2 wm . Compared with incubation in 250 mL shaking flask, the fermentation period was decreased 12 h and the potency of antifungal activity was increased about 1% in 5 L CSTR.The physical and chemical properties of the antibiotic fermentation broth were also investigated. The results indicated that the antibiotic was stable in hot and acid conditions, but not stable in an alkaline one. The broth had polarity and was insensitive of light.The process of isolation and purification of the antifungal antibiotic was studied primarily. Firstly, the culture broth was flocculated with chitosan, and its operating conditions were as follows: pH 2.0, the amount of chitosan 0.4 g/L, temperature 30℃. The flocculation rate (FR(%)) was 84.6%; Secondly, the metal ions and proteins were both removed by oxalate and hydrochloric acid under pH 4.0 and 2.0, repectively. Macroporous adsorption resin was used to absorb the pigment in the culture broth. The decolorization rate was 95.1% under the condition of pH 2.0 with flow rate of 2 BV/h. Finally, ion-exchange chromatography was chosen for further purification. The conditions were optimized as follows: initial pH 2.0, flow rate 2 BV/h for adsorption, 4 BV/h for desorption, 0.6 mol/L NH₃·H₂O as the mobile phase. The activity of the purified antifungal antibiotic was 5855.5U/mg and the recovery rate was 63.4 %.The crude antibiotic was analyzed by HPLC, which showed that the purity was 70.24% by normalization method.The retention time of the antibiotic was 6.069 min. After further purified by preparation HPLC, the purity reached 95.75% and the final activity was 8280.5 U/mg. The molecule weight of the antibiotic was 520.68 tested by LC-MS method. By UV full-wavelength scanning, its UV absorption peak was 298 nm. The purified antibiotic had good antifungal effects and the MIC against Candida albicans was 0.1807μg/mL.