薄层层析法(TLC)测定螺菌黄质系类胡萝卜素的相对极性和稳定性

The relative polarity and stability of each carotenoid of spirilloxanthin biosynthesis pathway by thin-layer chromatography (TLC)

摘要:

【目的】阐明不产氧光合细菌螺菌黄质系类胡萝卜素(Car)之间的相对极性和稳定性关系及规律。【方法】以沼泽红假单胞菌CQV97为材料，采用薄层层析(TLC)，结合图像灰度分析、吸收光谱和HPLC等方法，探求了螺菌黄质系Car 合成途径中各Car组分之间的相对极性、光谱特性和光稳定性规律。【结果】TLC能将CQV97菌株积累的7种Car分离，分别为Lycopene (C1)、Rhodopin (C2)、3,4-Didehydrorhodopin (C3)、Anhydrorhodovibrin (C4)、Rhodovibrin (C5)、OH-spirilloxanthin (C6)和Spirilloxanthin (C7)；其相对极性由低到高顺序是C1、C4、C7、C5、C6、C2、C3；依据特征性吸收光谱，这7种Car分为3个群，分别为C1和C2，C3、C4和C5，C6和C7，Car合成过程中脱氢反应使Car分子共轭体系增大，引起光谱较大幅度的红移(约10 nm)，而水化和羟基甲基化反应则引起较小幅度红移(0?4 nm)；光照条件下，7种Car在TLC板上均不很稳定，其半衰期在54?137 min，稳定性顺序依次为C2>C3≈C7>C5≈C4≈C1≈C6。黑暗条件下，7种Car均在90 min内稳定，稳定性顺序与光照条件基本一致。【结论】TLC能够良好地展示出螺菌黄质系途径中积累的7种Car。TLC板上的Car组分对光敏感，黑暗时短期稳定。本研究将有助于APB Car的TLC快速鉴别，为快速筛选高稳定性Car提供思路和参考。

Abstract:

[Objective] To elucidate the relative polarity and stability of seven carotenoids accumulated in normal spirilloxanthin biosynthesis pathway of anoxygenic phototrophic bacteria (APB) by thin-layer chromatography (TLC). [Methods] We investigated the changes in polarity, characteristic absorption spectra and stability of seven carotenoids accumulated in spirilloxanthin biosynthetic pathway of Rhodopesudomonnas palustris CQV97 by TLC, image gray intensity analysis, absorption spectroscopy and HPLC analysis. [Results] Strain CQV97 accumulated seven types of carotenoids including lycopene (C1), rhodopin (C2), 3,4-didehydrorhodopin (C3), anhydrorhodovibrin (C4), rhodovibrin (C5), OH-spirilloxanthin (C6) and spirilloxanthin (C7), the seven carotenoids were clearly separated by TLC. The relative polarity of seven carotenoids on TLC plate changed in the order of C1C3 ≈C7>C5≈C4≈C1≈C6. In darkness, the seven carotenoids were stable within 90 min. [Conclusion] TLC was capable of resolving clearly all seven carotenoids accumulate in spirilloxanthin biosynthetic pathway of APB. The seven carotenoids on TLC plate were sensitive to illumination and short-term stable in darkness. The results will be helpful for rapid identification of carotenoids by TLC.