快速鉴定格尔德霉素产生菌——吸水链霉菌17997中的洋橄榄叶素

Rapid identification of elaiophylin from Streptomyces hygroscopicus 17997,a geldanamycin producer

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。

To identify the anti-bacterial compound（s） from Streptomyces hygroscopicus 17997,a geldanamycin producer,silica gel thin layer chromatography（TLC） TLC was used to separate the secondary metabolites of S.hygroscopicus 17997.Compound（s） from the silica gel TLC with anti-Gram positive bacteria activity and becoming red upon color reaction by 2.0 mol/L NaOH was analyzed by HPLC.The UV absorption profile and the retention time of a peak of HPLC were identical to those of authentic elaiophylin.A conserved region of dTDP-glucose-4,6-dehydratase（Tgd） gene was amplified by PCR from the genomic DNA of Streptomyces hygroscopicus 17997.DNA sequence analysis of the amplified DNA fragment indicated that it should be the tgd gene of elaiophylin biosynthetic gene cluster.These results implied that the compound in the peak of HPLC was elaiophylin,a macrodiolide antibiotic.The compound was then confirmed to be elaiophylin by LC-（＋）-ESI-MS,which revealed that Streptomyces hygroscopicus 17997 was an elaiophylin producer.At the same time,a fast procedure,which consisted of silica gel TLC,color reaction,HPLC,PCR detection and DNA sequence analysis of tgd gene,and LC-（＋）-ESI-MS,was established for rapid identification of elaiophylin and its producer.