应用柱前衍生化高效液相色谱法开辟了一种简单、快速的DNJ产生菌的筛选途径

Efficient screening of 1-deoxynojirimycin producing strain by high performance liquid chromatography with pre-column derivatization

摘要:

【目的】应用柱前衍生化高效液相色谱法筛选产1-脱氧野尻霉素(DNJ)菌株, 并对其进行系统鉴定。【方法】利用芴甲氧酰氯(FMOC-Cl)柱前衍生化高效液相色谱法筛选DNJ产生菌; 通过形态特征、培养特征、生理生化特征以及16S rRNA 序列相似性分析等多项分类方法对DNJ产生菌进行鉴定; 在10 L发酵罐水平进行发酵研究, 利用FMOC-Cl柱前衍生化高效液相色谱法测定DNJ含量。【结果】从80株具有α-糖苷酶抑制剂活性的土壤放线菌中筛选得到一株DNJ产生菌, 菌株编号为PW409, 初步确定为戈壁三素链霉菌(Streptomyces gobitrici); 发酵研究表明, DNJ为链霉菌PW409的次级代谢产物, 发酵液中DNJ浓度为12.1 mg/L。【结论】首次将FMOC-Cl柱前衍生化高效液相色谱法应用于DNJ产生菌的筛选, 并首次报道从戈壁三素链霉菌发酵液中鉴定到DNJ。

Abstract:

[Objective] To screen the 1-deoxynojirimycin producing strains by employing high performance liquid chromatography with pre-column derivatization and then perform phylogenetic identification. [Methods] High performance liquid chromatography with 9-fluorenylmethyl chloroformate (FMOC-Cl) pre-column derivatization was a way of screening the DNJ producing strains. The polyphasic taxonomic characteristics, including morphological, physiological and biochemical characteristics, as well as comparative sequence analysis of 16S rDNA were used to identify strain. Then, a fermentation study was carried out on the scale of 10 L fermentor and DNJ content was determined by high performance liquid chromatography with 9-fluorenylmethyl chloroformat (FMOC-Cl) pre-column derivatization. [Results] A DNJ producing strain, namely PW409, which was subsequently identified as Streptomyces gobitricini, was obtained from 80 actinomycetes strains possessing α-glucosidase inhibitory activity. Fermentation study showed that DNJ belonged to a secondary metabolism of strain PW409, and the concentration of DNJ in fermentation broth was 12.1 mg/L. [Conclusion] This article first applies high performance liquid chromatography with 9-fluorenylmethyl chloroformat (FMOC-Cl) pre-column derivatization to screen DNJ producing strains as well as first identifies DNJ from the fermentation broth of Streptomyces gobitricini.