【作者】 周婕；

【导师】 吴雪昌；

【作者基本信息】 浙江大学 ， 遗传学， 2004， 硕士

【摘要】 本实验室分离到一株抗生素产生菌AP19-1，该菌发酵所产的抗生素19-1，对包括耐甲氧西林金黄色葡萄球菌在内的革兰氏阳性菌有明显的抑制作用。为了进一步确定该抗生素的可开发性，初步研究了该抗生素的生物活性，以及如何从发酵液中分离得到高纯度的抗生素化合物。 该抗生素产生菌AP19-1的发酵上清液对金黄色葡萄球菌，藤黄微球菌，枯草芽孢杆菌，短小芽孢杆菌24h的最小抑菌浓度(MIC)分别为稀释1000倍，1600倍，3600倍，2400倍。对从临床上分离出来的五株不同的耐甲氧西林金黄色葡萄球菌B3892，B8208，B6172，B6189，B4524的24h最小抑菌浓度分别为稀释1280倍，600倍，1000倍，1000倍，400倍。以这五株耐甲氧西林金黄色葡萄球菌做供试菌，5μl的发酵上清液，5μg的氨苄青霉素，青霉素，环丙沙星，以及含量30μg的万古霉素商用纸片做为药品，进行抑菌圈试验，发现，氨苄青霉素，青霉素，环丙沙星均没有产生抑菌圈，含量30μg的万古霉素产生平均直径为18.1mm的抑菌圈，而发酵上清液产生平均直径为20.7mm的抑菌圈。 为了确定该抗生素的结构，从发酵液中分离出单一的抗生素化合物是最基本的基础。采用溶液萃取，柱层析分离，薄层层析纯化的方法，使用waters高效液相系统研究高效液相色谱的初步条件，然后在shimadu高效液相系统中，以Diamonsil C18柱(250X4.6mm，5μm)为柱材料，流动相使用：乙腈：20mM乙酸铵水溶液(pH 4.0)35：65(V／V)，流速为1ml／min，柱温25℃，紫外检测器276nm处检测，大量制备高纯度的抗生素化合物，达到了92.5％的纯度，符合了结构分析中的核磁共振，质谱的纯度要求，为继续研究该抗生素的分子式，元素组成以及结构奠定了基础。更多还原

【Abstract】 The 19-1 complex was isolated from the fermentation broth of Streptomyces sp. AP19-1, whose major component 19-1, showed a strong activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus(MRSA) in vitro. The study of the activity comparing to the other antibiotics and the methods of purification was presented.The supernate of the fermentation broth was highly active against Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Bacillus pumilus. The minimal inhibitory concentrations(MIC) of supernate against these bacteria were diluted 1000, 1600, 3600, 2400 fold, respectively. The MIC of 5 different MRSA, which were isolated from the clinical patients, B3892, B8208, B6172, B6189, B4524, were diluted 1280, 600, 1000, 1000, 400 fold, respectively. 5 supernate, 5g ampicillin, 5g penicillin, 5g ciprofloxacin, and 30p.g vancomycin commercial production were used to test whether there was an inhibitory effect against the MRSA. Ampicillin, penicillin, ciprofolxacin had no effect at all, while vancomycin produced 18.1mm inhibitory zone on average, the supernate produced 20.7mm inhibitory zone on average.The antibiotic substance was purified by silica gel column chromatography, thin layer chromatography(TLC), and high performance liquid chromatography(HPLC). The liquid chromatographic separation was performed using a Diamonsil C18, 5m(250 4.6mm,I.D.), reversed-phase column, and a mixture of 20mM ammonium acetate(pH 4.0)-acetonitrile(65:35, v/v) as mobile phase, delivered at 1ml/min, at 25. The compounds were detected by UV detector at 276nm. The separation yielded antibiotic principle with purity of 92.5%.更多还原