Biotransformation Of Flavonol Glycosides In Epimedium Folium Based On Enzymatic Hydrolysis And Enzyme Immobilization

Epimedium Folium is rich in effective components of flavonol glycosides,and icariin,epimedin A,epimedin B,and epimedin C are the main flavonol glycosides in Epimedium Folium.The absorption and metabolism of different flavonol glycosides in Epimedium Folium were studied by bio-pharmaceutical evaluation system,and it was found that there were significant differences in absorption and metabolism among different flavonol glycosides.The absorption of secondary flavonol glycosides was significantly higher than that of original flavonol glycosid,and they also had higher intestinal permeability.However,the content of secondary flavonol glycosides in Epimedium Folium is low,and it is difficult to prepare them in large quantities using conventional extraction and separation methods.Therefore,the transformation of the abundant original flavonol glycosides into more active and rare secondary flavonoids is an important pathway,which is now a research hotspot.This paper focuses on studying the conversion of a large amount of original flavonol glycosids in the Epimedium Folium extract into more active secondary flavonol glycosids through enzymatic catalysis,recombinant expression,and immobilized enzyme methods to improve the stability and reusability of enzymes.The aim is to lay the foundation for the efficient preparation of secondary flavonol glycosids in industrial practice.The research work mainly includes the following three parts:(1)Hydrolysis of Epimedii Folium extracts by β-glucanaseFirstly,the conditions for the hydrolysis of Epimedii Folium extract by β-glucanase were optimized by one factor at a time strategy,and scale-up trial of enzymatic hydrolysis was performed.The β-glucanase was immobilized by adsorption-crosslinking method on mesoporous silica SBA-15.The results showed that the conversion rates of icariin,epimedin A,epimedin B,and epimedin C in Epimedii Folium extract were all over 93% when hydrolyzed by β-glucanase in HAc-Na Ac buffer(p H4.5)at 60℃ with a mass ratio of β-glucanase/substrate of 1.2:1 for 80 mins.In addition,when the hydrolysis system was scaled up 50 and 100-fold,the conversion rates of the original flavonol glycosids were still above 92%.In the immobilization process,β-glucanase was adsorbed onto SBA-15 and crosslinked with chitosan using glutaraldehyde.Under the immobilization conditions of buffer p H4.5,adsorption time16 hrs,enzyme addition amount 16.0 mg,and crosslinking time 40 mins,the enzyme activity recovery of the immobilized enzyme obtained was 14%.The optimal temperature of the immobilized enzyme was 70°C and the optimal p H was 4.5.(2)Studies on hydrolysis of Epimedii Folium extracts and immobilization of the recombinant enzyme Bgp ARecombinant β-glucosidase from Terrabacter ginsenosidimutans(Bgp A)was expressed and used for hydrolyzing primary glycosides in Epimedii Folium extracts to obtain secondary flavonol glycosids.Bgp A was immobilized using functionalized mesoporous silica as the carrier.The results showed that after Bgp A hydrolyzed for 10 mins,the conversion rate of primary glycosides reached 90%,and after 20 mins,the original flavonol glycosids were almost completely converted.In the immobilization process,Santa Barbara Amorphous 15(SBA-15)was modified with five silane coupling agents and used as the carriers to immobilize Bgp A.Among them,the enzyme activity recovery rate of R-SBA-15@Bgp A(Bgp A immobilized on N-aminoethyl-γ-aminopropyl trimethoxy silane modified SBA-15)was23.53%,significantly higher than that of other carriers.It is worth noting that Bgp A was fully captured by R-SBA-15,achieving simultaneous purification and immobilization.Through investigation of the influencing factors in the preparation process of R-SBA-15@Bgp A,the highest activity recovery and loading efficiency were achieved when the enzyme solution addition amount was 2.4 m L,adsorption time was 15 mins,temperature was 37℃,and buffer p H was 7.0.The optimal p H of R-SBA-15@Bgp A was 7.0,and the optimal temperature was20℃ higher than that of the free enzyme,increasing from 42°C to 62°C.After being incubated at 42°C for 8.0 hrs,it still retained over 70% of the relative activity.In addition,it had more advantages in storage stability than free enzymes.After being stored at 4°C for 33 days,the relative enzyme activity remained above 50%,and no significant enzyme leakage was observed during storage.(3)Studies on hydrolysis of Epimedii Folium extracts by R-SBA-15@Bgp ABy constructing an organic reagent-phosphate buffer-immobilized enzyme hydrolysis system,it was used for enzymatic hydrolysis of Epimedii Folium extracts.Firstly,the organic reagent resistance of SBA-15@Bgp A was investigated.Then,using epimedin A as the substrate,the immobilized enzyme hydrolysis system was applied to the conversion of individual original flavonol glycosids and the reuse performance was investigated.Afterwards,the system was applied to the enzymatic hydrolysis of Epimedii Folium extracts,which is a complex mixture rich in original flavonol glycosids.The results showed that SBA-15@Bgp A could remain stable in 15% DMSO phosphate buffer for a long time.When R-SBA-15@Bgp A/extract mass ratio was 1:2 and hydrolysis time was 20 mins,epimedin A could be completely converted in the enzymatic hydrolysis.After being reused 14 times,the relative conversion rate of epimedin A could still be maintained at nearly 80%.In the enzymatic hydrolysis of Epimedii Folium extracts,with R-SBA-15@Bgp A/extract ratio of 1:2 and at 42℃in 15% DMSO phosphate buffer(p H7.0),all of the original flavonol glycosids(including icariin,epimedin A,epimedin B,and epimedin C)could be completely converted after 30 mins of hydrolysis.