Purification And Characterization Of Ginsenoside Rb₁-hydrolyzing β-D-glucosidase From Fulvia Fulva Ciferr

Ginseng, the root of Panax ginseng C. A. Meyer (Araliaceae), is an ancient and famous herb that has been widely used in our country for over 4,000 years. Many studies have demonstrated that ginseng has remarkable pharmacological activities towards central nervous system and immunity system, and so on.Ginsenosides, the major active components of ginseng, exist in nearly all parts of ginseng. So far, there are almost 40 kinds of ginsenosides that had been isolated and identified from ginseng. Recent studies have demonstrated that some minor ginsenoside, such as ginsenoside Rd, has remarkable pharmacological activities. However, the production of ginsenoside Rd is difficult since the content of naturally occurring ginsenoside Rd in ginseng is only about 0.1%., However, ginsenoside Rb₁, the content of which is the highest in ginseng, has just one more glucose residue at C-20 position compared with ginsenoside Rd. Therefore, preparation of ginsenoside Rd by limited hydrolysis of ginsenoside Rb₁ gets more and more attention in recent years. So far, many methods, such as heating, acid treatment and enzymatic conversion, can be used to convert ginsenoside Rb₁ to ginsenoside Rd. Compared to other methods, enzymatic conversion is much more advantageous due to its gentle conditions, simple processes, easily separation of products, and no by products existed. Therefore, now many studies have focused on enzymatic conversion to preparation minor ginsenosides.Through screening many phytopathogenic fungi, we found a goodβ-glucosidase producer Cladosporium fulvum, which could secretedβ-glucosidase to the fermentation broth. This enzyme could hydrolyze ginsenoside Rb₁ to Rd. In this paper, we studied the isolation and purification ofβ-glucosidase produced by Cladosporium fulvum, and giving some properties of the purified enzyme.The results are as follows:1. Exploration on the isolation and purification of ginsenoside Rb₁-hydrolyzing enzymeFirst, we tried many methods to isolation and purification of this glucosidase, using pNPG as substrate to measure the enzyme activity. The fermentation broth of Cladosporium fulvum were treated using superfiltration, (NH₄)₂SO₄ precipitation, DEAE-Sepharose CL-6B chromatography, Sepharose CL-6B chromatography, Q-Sepharose fast flow chromatography, Mono Q HR 5/5 anion exchange chromatography coupled with HPLC, finally we got a fundamental, single protein band in SDS-PAGE. From the studies we got the conclusion that the enzyme activity of the fermentation broth was very low after cultured for 7days, and the enzyme activity was partially lost in superfiltration step, so in subsequent research, we will look for good methods to solve these problems.2.Isolation and purification of ginsenoside Rb₁-hydrolyzing enzymeAfter the small experiment, we made some improvement in the large-scale preparation of theβ-glucosidase.The result showed that the maximumβ-glucosidase activity of 4.77 U/ml was reached at 84 h. According to the result of theβ-glucosidase activity, the fermentation was, therefore, stopped at 84 h. The culture was treated using DEAE-cellulose chromatography, DEAE Sepharose CL-6B chromatography, Sepharose CL-6B chromatography, Phenyl sepharose CL-4B chromatography, Mono Q HR 5/5 anion exchange chromatography coupled with HPLC, Bio-Scale CHT20-1 hydroxyapatite chromatography. Finally we got a single symmetrical peak in gel filtration, and a single protein band in SDS-PAGE. We also use these two methods to detecting the purity and the molecular weight of theβ-glucosidase, the results showed that the purity of the enzyme could reach 98%. The molecular weight of theβ-glucosidase was 105 KDa, measured by SDS-PAGE, and 210 KDa, measured by gel filtration, which indicated that the enzyme was a homodimer protein. The purification factor of the purified enzyme was 540-fold compared with the crude enzyme solution, while the enzyme recovery was 7%, the specific activity of the purified enzyme was 23768 U/mg.3.Enzymatic properties study of the purified ginsenoside Rb₁-hydrolyzing enzymeThe enzymatic properties studies of the purifiedβ-glucosidase indicated that the optimal temperature was 45℃, while the enzyme was steady below 40℃. The optimal pH value of the purified glucosidase was 5.5 while it was stable at pH5.0-pH6.5. Ca²⁺, Mg²⁺ and Ba²⁺ had certain inhibitition of the enzyme activity in 0.25M ,the enzyme activity descends to 80%,79% and 78% respectively。Na⁺, K⁺ and Mn²⁺ ions had no obvious influence on enzyme activity between 0-0.25M while EDTA, Cu²⁺ and Zn²⁺ had strong inhibitition of the enzyme activity. SDS could inhibit the enzyme activity in 0.25M as well. When adding mercapto protective agent, the inhibition of SDS to the enzyme activity decreased obviously, which indicated that there was disulfide bond existed in the enzyme, and the disulfide bond must play an important role in the enzyme active center. The study of the substrate specificities demonstrated that the enzyme was specific to glucoside bond. The order of hydrolysis abilities was cellobiose>lactose> sucrose>pNPG>ginsenoside Rb₁. The results of kinetic parameters were as follows: the Km value was 0.18 mM and the Vmax value was 5.52 mM/min. The study of the amino acid sequence analysis indicated that the enzyme we obtained was a new protein, and it was homologous with the fungiβ-glucosidase belonging to Family 3 in glycoside hydrolase families.In conclusion, in this paper we isolated and purified an effective ginsenoside Rb₁-hydrolyzingβ-glucosidase, and measured the enzymatic properties as well. By using this enzyme to preparation ginsenoside Rd from ginsenoside Rb₁, it has good specificity and high yield, and there is no by-products generate in the reaction process. Therefore, it would lay a foundation in the industrial application.