Restructuring F26g Furostanol Saponins To Lo Steroidal Saponins Biological Conversion Research

In this paper, The bioconversion from furostanol glycosides to spirostanol glycosides catalyzed by F26G was studied. The cDNA fragment encoding F26G was released through the digestion of pKG27 by BamH I and Xba I. The fragment was ligated to pET-22b pretreated by BamH I and Xba I. The expression vector pET-22b-F26G was successfully constructed which was confirmed by PCR and enzymatic digestion. The detection of furostanol glycosides and spirostanol glycosides by TLC and HPLC was achieved. On the basis, the reaction condition of bioconversion in vitro was established, and the enzyme activity of F26G was successfully detected in E.coli BL2I/ pET-22b-F26G. Some affecting factors were investigated such as pH,adding time and inducing time of IPTG in the process of F26G overexpress ion. The expressed F26G was applied to catalyze different kinds of furostanol glycosides.The results of the bioconversion were analyzed by TLC, HPLC and ESI-MS, and the selectivity and catalyzing activity of F26G were studied. The products of enzymatic reaction were prepared and purified, the structures of which were also confirmed. The conversion mechanism from furostanol glycosides to spirostanol glycosides was discussed.