Isolation And Characterization Of Ginsenoside Rb₁-Hydrolyzing Glycosidase From Cylindrocarpon Destructans

Ginseng (Panax ginseng C.A.Meyer) is a famous medicinal plant. It contains many active components. The major active components of ginseng are ginsenosides. Up to now, more than 40 ginsenosides have been isolated and characterized from various ginseng species. Minor ginsenosides( e.g. Rg₃, Rh₂, C-K) usually have more potential biological and pharmacological activities.Minor ginsenosides were difficult to be prepared by isolating from ginsengs because of its low content. At present, we usually transformed major ginsenosides to minor ginsenosides by changing glucosyl residues, because chemical structure of some minor ginsenosides have just less several glucosyl residues compared to major ginsenosides (e.g. high content ginsenoside Rb₁). There are several methods for transformation of glucosyl residues of major ginsenosides, e.g. chemical method and method of biological convertion. Enzymatic conversion is thought to be the most potential method to prepare minor ginsenosides due to many advantages, such as high selectivity, fewer by-products, and less pollution. This work mainly studied the following contents:1. Exploration on fermentation and culture conditionWe evaluated culture and fermentation condition of Cylindrocarpon destructans including selection of culture media, selection of scraping spores amount, determination of optimal fermentation time, ginsenoside induction trials, spores reusing trials. The results are as follows: V8 juice medium; scraping 10 flats of spores into every 150 ml liquid medium; fermentation time: 84 h. Ginsenoside induction and spores reusing trials did not enhance enzymatic activity.2. Isolation of ginsenoside Rb₁- hydrolyzing enzymeFermentation broth was filtrated and centrifuged after culture for 84 h. The supernatant was isolated in sequence by DEAE-cellulose anion exchange chromatography, 30%-80% (NH₄)₂SO₄ precipitate, Sepharose CL-6B chromatography, DEAE-Sepharose fast flow, Mono Q HR 5/5 anion exchange chromatography (HPLC). At last, partially purified enzyme was obtained. The enzyme was purified to 313 folds compared to the crude extract. the yield of enzyme activity was 12%.3. Enzymatic properties of ginsenoside Rb₁-hydrolyzing enzymeWe studied enzymatic properties of the partially purified enzyme. The results showed that optimal pH of the partially purified enzyme was 5.0. It was stable at pH 3.0-11.0. Optimal temperature of the partially purified enzyme was 55°C. It was stable at between 20°C to 55°C. Metal ions such as Mg²⁺ could lightly activated the enzyme activity; Na+, K+, Ca²⁺, EDTA, Cu²⁺, Zn²⁺ had different inhibition on enzyme activity; Among them Cu²⁺ intensely inhibited enzyme activity; Research of substrate specificity testified that the enzyme was specific toβ-D-glucosidic bond. It was a kind ofβ-glucosidase. The enzyme could transform high content ginsenoside Rb₁ to more active minor C-K as final product. Conversion rate is about 70% according to TLC results suggested. The pathway of transformation is Rb₁→Rd→F₂→C-K.In conclusion, we studied condition of culture and fermentation, isolation and properties of extrocellularβ-glucosidase from Cylindrocarpon destructans. The partially purified enzyme could transform high content ginsenoside Rb₁ to more active minor ginsenoside C-K as final product.