Epimedium Folium(EF) is a traditional Chinese herbal medicine, and its primary bioactive ingredients, such as icariin, are flavonoid glycosides. A rare EF flavonoid, baohuoside I, exhibits superior bioactivities and enhanced bioavailability compared to its metabolic precursor icariin. The biotransformation of icariin to baohuoside I can be effectively and specifically achieved by β-glucosidases. In this study, 33 candidate full-length β-glucosidase genes were screened from a previously built carbohydrate active enzyme (CAZyme) gene dataset derived from cow fecal microbiota. Thirteen of them exhibited β-glucosidase activity, with DCF-bgl-26 and DCF-bgl-27 showing relatively high expression levels and β-glucosidase activity. The maximum β-glucosidase activity of DCF-bgl-26 and DCF-bgl-27 was achieved at 45 °C and pH 6.0, with DCF-bgl-26 demonstrating better thermostability and pH tolerance compared to DCF-bgl-27. The activities of DCF-bgl-26 and DCF-bgl-27 were 123.2 U/mg protein and 157.9 U/mg protein, respectively, both of which are higher than those of many bacterial β-glucosidases. Structure analysis suggested that both β-glucosidases possess canonical (β/α)8-TIM barrel fold structure of GH1 family β-glucosidases. Thin-layer chromatography results showed that both enzymes could efficiently convert icariin to baohuoside I in 30 min, indicating they have potential application in the production of high value rare baohuoside I.
Lin Yu,Chen Wen-wen,Ding Bo,Guo Man,Liang Meng,Pang Hao,Wei Yu-tuo,Huang Ri-bo,Du Li-qin
Background Icaritin is an aglycone of flavonoid glycosides from Herba Epimedii. It has good performance in the treatment of hepatocellular carcinoma in clinical trials. However, the natural icaritin content of Herba Epimedii is very low. At present, the icaritin is mainly prepared from flavonoid glycosides by α-L-rhamnosidases and β-glucosidases in two-step catalysis process. However, one-pot icaritin production required reported enzymes to be immobilized or bifunctional enzymes to hydrolyze substrate with long reaction time, which caused complicated operations and high costs. To improve the production efficiency and reduce costs, we explored α-L-rhamnosidase SPRHA2 and β-glucosidase PBGL to directly hydrolyze icariin to icaritin in one-pot, and developed the whole-cell catalytic method for efficient icaritin production.Results The SPRHA2 and PBGL were expressed in Escherichia coli, respectively. One-pot production of icaritin was achieved by co-catalysis of SPRHA2 and PBGL. Moreover, whole-cell catalysis was developed for icariin hydrolysis. The mixture of SPRHA2 cells and PBGL cells transformed 200 g/L icariin into 103.69 g/L icaritin (yield 95.23%) in 4 h in whole-cell catalysis under the optimized reaction conditions. In order to further increase the production efficiency and simplify operations, we also constructed recombinant E. coli strains that co-expressed SPRHA2 and PBGL. Crude icariin extracts were also efficiently hydrolyzed by the whole-cell catalytic system.Conclusion sCompared to previous reports on icaritin production, in this study, whole-cell catalysis showed higher production efficiency of icaritin. This study provides promising approach for industrial production of icaritin in the future.
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