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对格尔德霉素产生菌吸水链霉菌17997的发酵液乙酸乙酯提取物进行了硅胶板TLC初步分离和NaOH溶液喷涂显色,对显红色、具有抗革兰阳性菌活性的条带进行了HPLC分析,提示抗革兰阳性菌活性化合物可能为大环二内酯类抗生素洋橄榄叶素;以dTDP-葡萄糖-4,6-脱水酶(Tgd)基因保守区设计PCR引物,扩增了吸水链霉菌17997基因组DNA中的tgd并进行了序列分析,表明吸水链霉菌17997含有洋橄榄叶素生物合成基因簇中的tgd基因;对NaOH溶液喷涂显红色的化合物进行LC-(+)-ESI-MS分析,证实其为洋橄榄叶素。因此,吸水链霉菌17997产生洋橄榄叶素;同时,建立了一种快速鉴定洋橄榄叶素及其产生菌的方法,主要包括TLC硅胶板分离、NaOH溶液显色、HPLC和LC-MS分析,以及tgd的PCR检测与序列分析。
Ethyl acetate extract of the fermentation broth of Streptomyces hygroscopicus 17997 of geldanamycin was preliminarily separated by TLC on silica gel plate and sprayed with NaOH solution for color development. The bands that showed red color and had activity against Gram-positive bacteria HPLC analysis suggested that the active compound against Gram-positive bacteria may be macrolide-derived diploid olive oil. PCR primers were designed based on the conserved region of dTDP-glucose-4,6-dehydratase (Tgd) gene, Streptomyces 17997 genomic DNA tgd and sequenced, indicating that Streptomyces hygroscopicus 17997 contains the gene of tgd in the biosynthetic gene cluster of the diploid olivellin; spraying a bright red compound on NaOH solution for LC - (+) - ESI- MS analysis confirmed that it is foreign olive element. Therefore, Streptomyces hygroscopicus 17997 produced foreign olive leaves. At the same time, a rapid method for the identification of foreign olive leaves and its producing bacteria was established, including TLC silica gel plate, NaOH solution, HPLC and LC-MS. As well as PCR detection and sequence analysis of tgd.