Construction Of A Double-Enzyme Co-Expression Strains And Study On The Hydrolysis Of Icariin

The traditional Chinese medicine Epimedium has a variety of pharmacological effects.Its main active ingredient icariin is metabolized into anhydroicaritin through a series of biochemical reactions before it is intakedand absorbed.The oral utilization of icariin is relatively low,while the anhydroicaritin is easier to be absorbed with smaller molecular weight and has the higher biological activity.At present,the production method of anhydroicaritin is relatively immature,and the production of anhydroicaritin by enzymatic hydrolysis is a relatively green way.In this study,we first used the laboratory’s existingα-L-rhamnosidase SPRHA2 whole cell to catalyze icariin and analyze the hydrolysate.In addition,we selected two enzymes from the laboratory’s existing β-glucosidase to synergistically catalyze Β-glucosidase with good effect of icariin,and then construct a double-enzyme co-expression recombinant strain in E.coli and yeast respectively,and use the recombinant strain to study the hydrolysis of icariin and icariin crude extract.First of all,recombinant strains E.coli/p QE30-sprha2 was used to whole-cell catalysis icariin glycosidein optimal conditions(p H 7.0,55 ℃,220 rpm).After wet weight of 5 g/L cells and 4 g/L of icariin glycoside(purity ≥ 95%)was reacted for 1 h,the hydrolysis rate which is analysed by HPLC was over 95%.After purification,the hydrolysis product was analysed as icariside Ⅰby NMR.Then using the existing β-glucosidase in the laboratory combined with SPRHA2 to catalyze the flavones of icariin under various catalytic conditions,it is concluded that PBGL has a better synergistic catalytic effect of icariin.The higher yield of anhydroicaritin was studied when SPRHA2 and PBGL were added at an enzyme amount of 2:1.The recombinant E.coli/p QE30-sprha2-pbgl was constructed in the form of co-expression of polycistrins in E.coli,and was named as polycistrins SP.The optimal conditions for polycis SP whole-cell catalysis icariin were p H 8.0(boric acid borax buffer),55℃,and 220 rpm.Under the optimal conditions,the hydrolytic rate of 5 g/L wet weight bacteria reacted with 2 g/L icariin for 1 h was above 90%,and the product was purified and analyzed as anhydroicaritin by NMR.PGAPZa A was used as the carrier to construct the co-expressed recombinant yeast strains in Pichia pastoris GS115.The enzymatic activity of the recombinant yeast strain was higher when the rotation speed was 200 rpm,and the recombinant yeast strain could completely hydrolyze the icariin in the 2%(W/V)icariin crude extract with the purity of 20%.In this study,the two enzymes were co-expressed in E.coli and Pichia pastoris GS115.Both co-expressed recombinant strains achieved the purpose of hydrolyzing icariin to prepare anhydroicaritin for enzymatic preparation of high purity anhydroicaritin provides a new idea.