부산대학교 도서관

The roots of Panax ginseng C. A. Meyer (Araliaceae), known as Korean ginseng have been used a valuable and important folk medicine in the East Asian countries, such as China, Korea and Japan for thousands of years and has recently been popularized in many western countries during recent decades. Dammarane-type triterpenoid saponins, namely ginsenosides, are the principal compounds responsible for the pharmacological effects and health benefits of ginseng. The ginseng saponins are classified into two major groups, protopanaxadiol (PPD)-type and protopanaxatriol (PPT)-type ginsenoside. Among these, PPD-type ginsenosides such as G-Rb1, G-Rb2, G-Rc and G-Rb2 account for 80-90% of total ginsenosides in ginseng roots. However, the naturally occurring major ginsenosides (G-Rb1, G-Rb2, G-Rc and G-Rd) are known to be hydrolyzed into minor ginsenosides such as C-K, G-Rh2, C-O and C-Y by bacterial β-glycosidases in human intestinal tract. However, The minor ginsenosides, C-K, G-Rh2 and C-Y are more biologically active than major ginsenosides such as G-Rb1, G-Rb2 and G-Rc and are more easily absorbed into the blood stream. Accordingly, various methods such as mild acid hydrolysis, alkaline cleavage, microbial fermentation and enzymatic hydrolysis have previously been reported for producing minor ginsenosides from major ginsenosides. Among these methods, an enzymatic hydrolysis method is considered more favorable than any other methods due to their performance under milder reaction conditions, more simple reaction steps, and high regioselectivity. However, in previous studies, some attempts to produce minor ginsenosides from major ginsenosides by enzymatic method were limited by complexity of enzyme isolation, low yields, or microbiological food safety problems. In this study, minor ginsenodsides, C-K and C-Y were found to be produced with a high yield from G-Rb1, G-Rb2, G-Rc, PPD-type ginsenoside fraction and total ginsenoside fraction by crude enzyme preparations isolated from the liquid culture of basidiomycetes, Formitella fraxinea under submerged culture condition. β-Glycosidases, which has a hydrolytic activities of PPD-type major ginsenosides was purified by suitable purification process. The results were summarized as follows. 1. In the preliminary experiment, the crude enzyme preparations isolated from broth and mycelium of liquid cultured F. fraxinea showed very similar hydrolytic activity toward G-Rb1 and G-Rb2 used substrates. These results indicate that β-glycosidases present in the broth and mycelium of cultured F. Fraxinea have similar enzymatic properties to each other.2. Two ginsenoside-hydrolyzing β-glycosidases (BG-Ⅰ and BG-Ⅱ) from F. fraxinea broth was homogeneously purified by ammonium sulfate (30-80%) precipitation, DEAE-cellulose anion exchange and Sephadex G-150 chromatography. The molecular weights of BG-Ⅰ and BG-Ⅱ on SDS-PAGE and native PAGE were 118.3 kDa and 131.9 kDa with specific activity of 32.3 U and 11.2 U/mg protein, respectively. Purification of two isozymes were 78.8 and 27.3 fold with yield of 2.44% and 1.09%, respectively.3. Both β-glucosidases showed optimal activity and stability at pH 4.5-4.8 toward p-nitrophenyl-β-D-glucopyranoside used as a substrate and was stable between pH 4 and 5. The enzymes became vulnerable to inactivation below and above this pH range. The optimum temperatures of both enzymes were 60 oC, respectively, and the enzymes were stable under 50 oC, but the activities decreased sharply above 70 oC. Both enzymes showed very similar hydroysis pattern toward G-Rb1, G-Rc and G-Rb2 used as substrates.4. F. fraxinea β-glycosidases showed a high hydrolytic activities toward G-Rb1 with hydrolysis pathway of G-Rb1 → G-Rd → G-F2 → C-K, and with two hydrolysis pathways, G-Rc → G-Rd → G-F2 → C-K and G-Rc → C-Mc1 → C-Mc → C-K toward G-Rc. The optimal conditions to transform G-Rb1 and G-Rc into C-K were reaction time 8-24 hr and pH 4-5, respectively. 5. The β-glycosidasea were also exhibited potent hydrolytic activities toward G-Rb2. The optimum conditions for C-Y formation from G-Rb2 by these enzymes were a reaction time 8-24 hr and pH 4.0−5.0. The pathway for C-Y formation from G-Rb2 was G-Rb2 → C-O → C-Y but the hydrolysis pathway of G-Rb2 → G-Rd → G-F2 → C-K did not observed. 6. F. fraxinea β-glycosidases produced G-Rg1 by hydrolysis of the PPT-type ginsenoside, G-Re but showed no hydrolysis activity from G-Rg1 to G-Rh1. These results indicated that F. fraxinea β- glycosidases are multi-enzyme that also have α-L-rhamnosidase activity as well as the activities of β-glucosidase, α-L- arabinopyranosidase and α-L-arabinofuranosidase.7. Also, the hydrolysis of PPD-type ginsenoside or total ginsenoside fraction by crude enzyme preparation from F. fraxinea were remarkably increased the amounts of minor ginsenosides such as C-K and C-Y in the reaction mixtures. These results suggest that crude enzyme preparation isolated from cultured F. fraxinea under submerged culture condition can be used to produce biologically active minor ginsenosides, C-K from G-Rb1 and G-Rc, C-Y from G-Rb2, and to prepare mixture of minor ginsenosides with high yield from PPD-type ginsenoside fraction or total ginsenoside fraction.