Study On Biotransformation Of Ginsenosides By Glycoside Hydrolases

Ginsenosides are the principle components responsible for the pharmaceuticalactivities of ginseng. So far, about forty kinds of different ginsenosides have been isolatedfrom different parts of ginseng. Some minor ginsenosides exhibited various biological andpharmacological activities, including anti-tumor, immune-modulatory, anti-inflammatoryand anti-aging effects. These minor ginsenosides, however, exsited in very low content oreven did not naturally present in ginseng. The minor ginsenosides, which had sameginsengenin as the high-content ginsenosides, could therefore be prepared by hydrolyzingthe sugar moieties in high-content ginsenosides.Methods including heating, acid hydrolysis, microbial and enzymatic transformationwere used in the hydrolysis of high-content ginsenosides to prepare minor ginsenosides.Among these methods, microbial and enzymatic transformations are more potential due totheir high specificity, low side reaction and mild conditions. Biotransformation ofglycosides to get useful compounds was one hot spotpoint in glycoside application.Therefore, in this study, we focus on looking for suitable strains or enzymes which couldbe used in ginsenoside biotransformation. The main results are as follows:1. In this study, twelve kinds of commercial enzymes were tested for their ability totransform protopanaxadiol-type ginsenosides mixture and single ginsenoside Rb1. Amongthem, only one enzyme had no transformation activity against the substrates, the othereleven enzymes could transform protopanaxadiol-type ginsenosides mixture andginsenoside Rb1with different pathways and efficiencies. Among the eleven enzymes,eight kinds of enzymes transformed ginsenoside Rb1to Rd, the transformation pathwaywas Rb1→Rd; one enzyme transformed Rb1to ginsenoside F2, the pathway wasRb1→Rd→F2; two enzymes transformed Rb1to final product C-K, the transformationpathways were Rb1→Rd→F2→C-K and Rb1→Rd/GXVII→F2→C-K, respectively.2. A fungus, sp.68, which was found to be able to transform protopanaxadiol-typeginsenosides into bioactive C-K with high activity and specificity, was isolated from theginseng-cultivating soil. The strain was identified to be Penicillium oxalicum by18S rDNAand ITS sequencing. Then the crude enzyme was prepared by DEAE-cellulosechromatography and sulfate ammonium precipitation from the fermentation broth of P.oxalicum. The crude enzyme was used in ginsenoside transformation. Ginsenoside Rb1,Rb2and Rc were transformed to the final product C-K by the crude enzyme via differentpathways. The pathways are as following: Rb1→Rd→F2→C-K；Rb2→CO→CY→C-K； Rc→Mb→Mc→C-K. The conditions for C-K production by crude enzyme of P. oxalicumwere optimized, as well. Following optimization conditions were obtained: substrate,ginsenoside Rb1; pH,3.5; temperature,55oC; concentration of Rb1,0.5mg/ml.3. Three extracellular glycosidases GH1, GH3-1and GH3-2were purified from thefermentation broth of P. oxalicum. The substrate specificity results showed that GH1couldhydrolyze Rb1to sole product Rd, without hydrolyzing Rb2, Rc and Rd. GH3-1andGH3-2exhibited similar transformation pathways against Rb1, Rb2and Rc, the pathwaysare: Rb1→Rd→F2→C-K, Rb2→CO→CY→C-K, Rc→Mb→Mc→C-K. The enzymeswere characterized as well. These enzymes would be useful in ginsenosides transformation.4. Seven glycoside hydrolases were cloned, expressed and purified fromCellulomonas fimi. Among them, cfi-01, cfi-08, cfi-11and cfi-13belonged to glycosidehydrolase family3while cfi-02, cfi-04and cfi-10belonged to family31,42and1,respectively. The substrate specificities of these recombinant enzymes were symtemicallystudied. Among the glycoside hydrolase family3enzymes, cfi-01was bifunctional enzymewith both β-D-xylosidase and-L-arabinofuranosidase activities. The enzymes cfi-08andcfi-11were β-D-glucosidases. Cfi-13hydrolyzed p-nitrophenyl substrates, withouthydrolyzing disaccharides or oligosaccharides, therefore, cfi-13was aryl-β-D-glucosidase.The enzyme cfi-02, which belonged to glycoside hydrolase family31, was-xylosidase.Cfi-10exhibited β-D-galactosidase, β-D-glucosidase, β-D-fucosidase and β-D-xylosidaseactivities. Also, the transformation ability of these seven recombinant glycosidases againstginsenosides were studied. The results showed that cfi-08and cfi-10transformedginsenoside Rb1to give the sole product Rd. Other glycosidases exhibited no activityagainst tested ginsenosides. The substrate specificities of the glycoside hydrolases from P.oxalicum and C. fimi were symtemically studied and compared. These enzymes would beuseful for hydrolysis of glycoside compounds or oligosaccharides.