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目的:国内外首次使用悬浮培养的转基因何首乌毛状根生物转化外源性底物麝香草酚并考察其在该转化体系中的生物转化情况。方法:麝香草酚和转化体系共培养5天后,通过薄层色谱和柱色谱分离其转化产物,通过物理化学和光谱学方法测定其化学结构。此外,利用高效液相色谱分析并测定麝香草酚在该转化系统的时效曲线,探明其生物转化情况。结果:分离纯化得到了麝香草酚糖基化产物(DMP),并测定其结构为5-甲基-2-(1-甲基乙基)酚羟基-β-D-吡喃葡萄糖苷;分析测定了其时效曲线,结果表明转化产物在培养物和培养基中的分布随着共培养时间的不同而变化,并在共培养时间为5天的情况下,产物主要分布在培养基中。结论:芳香性外源底物麝香草酚首次在转基因何首乌毛状根中成功地进行了生物转化,得到其糖基化产物;时效曲线阐明了其转化产物与共培养时间的对应关系。
OBJECTIVE: To firstly use the suspension culture of transgenic Polygonum multiflorum hairy roots to transform the exogenous substrate thymol and investigate its biotransformation in this transformation system. METHODS: Thymol and the transformation system were co-cultured for 5 days. The products were separated by TLC and column chromatography. Their chemical structures were determined by physicochemical and spectroscopic methods. In addition, high performance liquid chromatography (HPLC) was used to analyze and determine the aging curve of thymol in the transformation system to ascertain its biotransformation. Results: Thymol glycosylation product (DMP) was isolated and purified, and its structure was determined as 5-methyl-2- (1-methylethyl) phenol-β-D-glucopyranoside. The time-dependent curves were measured. The results showed that the distribution of the products in culture and medium varied with the time of co-culture. When the co-culture time was 5 days, the products were mainly distributed in the medium. CONCLUSION: Thymol, an aromatic exogenous substrate, was successfully transformed into the hairy root of Polygonum multiflorum for the first time, and its glycosylation product was obtained. The time-dependent curve elucidated the corresponding relationship between its conversion products and co-cultivation time.