新型抗真菌抗生素分离与纯化

Isolation and purification of novel antifungal antibiotic from streptomyces ahygroscopicus

对1株不吸水链霉菌(Streptomyces ahygroscopicus ZJUIB001)产生的新型抗真菌抗生素进行分离纯化研究.通过发酵液经离心去沉淀后,采用氯仿和石油醚萃取.萃取后的水相调节至pH 2并采用Dowex 50×16阳离子交换树脂和XAD7HP大孔吸附树脂分离其中的抗真菌代谢物,对分离样品进行反相高效液相色谱分析.Dowex50×16层析条件优化如下:pH=2的萃取浓缩液以2倍床层体积/h的流速上柱,在去离子水冲洗后,用0.6mol/L氨水以1倍床层体积/h流速洗脱;Amberlite XAD7HP大孔吸附树脂层析条件为:体积分数为10%和70%的乙醇水溶液阶跃洗脱.将纯化得到的样品用高效液相色谱(HPLC)系统检测,色谱柱为YMC-C18(5μm,Φ4.6mm×250mm)氰基柱,流动相为0.01mol/L的乙酸铵缓冲液(pH=4.3),流速为1mL/min,检测波长为220nm.HPLC检测结果显示,分离的抗生素纯度和效价分别达到93.0%和21 632U/mg.

A technically feasible purification process for a novel pyrimidine nucleoside antifungal antibiotic produced by a Streptomyces strain（Streptomyces ahygroscopicus ZJUIB001） was conducted.The centrifugated culture broth was extracted with chloroform and then with petroleum ether.The water phase solution was adjusted to pH=2 and further isolated by cation-exchange chromatography,which was followed by macroporous resin adsorption.Dowex 50 × 16 was screened as the most suitable cation-exchange resin for removing the impurity from the crude extract under the optimized operating conditions as follows： sample loading flow rate of 2 bed volume（BV） per hour and eluting flow rate of 1 BV per hour using 0.6 mol/L NH3·H2O as the mobile phase.Amberlite XAD7HP was selected as the most suitable macroporous adsorption resin for the further purification of the antibiotic.The optimized desorbing condition of macroporous resin adsorption chromatography was multi-step gradient elution with ethanol solutions of 10% and 70% volume fraction,respectively.Then the purified antifungal compound was subjected to high-performance liquid chromatographu（HPLC） system with a YMC-C18（5 μm,Φ4.6 mm×250 mm） column.The mobile phase was 0.01 mol/L ammonium acetate buffer（pH 4.3）.The flow rate was 1 mL/min and the elution was detected at 220 nm.HPLC results show that the purified antibiotic compound has a purity of 93.0% with antifungal activity of 21 632 U/mg.