Luong Tran My Linh BT060042 Pham Dinh Hai Yen BT060074

Contents
Introduction Materials and Methods Results Discussion Conclusion

Introduction

Ginseng is a traditional herbal medicine that has been used for thousands of years in Asian countries. It has various medicinal functions including tonic, adaptogenic, immunomodulatory, and anti-aging effects.

Many of its medicinal effects are attributed to triterpene glycosides known as ginsenosides

The ginsenoside Rg3 have strong antitumor activity but difficult and quite expensive to obtain from natural products. It is much easier to acquire ginsenoside Rb1, about 13%, and has a structure similar to that of ginsenoside Rg3. By enzymatically hydrolyzing the two glucose molecules at C-20, ginsenoside Rg3 can easily be transformed from Rb1.

In this study, they used a novel ginsenoside-hydrolyzing -glucosidase that specifically transforms ginsenoside Rb1 to Rg3 was purified from Paecilomyces Bainier sp. 229. The purification method was greatly simplified by the application of MCC column chromatography. Biological properties of purified enzyme were also studied.

Materials and Methods

Materials

p-nitrophenyl--D-glucoside (pNPG) p-nitrophenyl--D-glucoside (pNPG) p-nitrophenyl--D-xyloside (pNPX) Q-sepharose FF, phenyl-sepharose, CL-4B, and sephacryl S-300 HR CHT ceramic hydroxyapatite Microcrystalline cellulose (MCC) Ginsenoside Rb1, Rb2, Rb3, Rd, Rg3, F2, and Rh2, CK

Isolation of ginsenoside-transforming fungal strain (Paecilomyces Bainier sp.)

Preparation of crude enzyme:

Paecilomyces Bainier sp was cultured in an aerobic fermentor in medium containing 3% soybean powder, 3% sucrose, 0.2% calcium chloride, 0.1% magnesium sulfate, 0.1% ammonium sulfate, and 0.1% ginseng general ginsenosides The cells were harvested by centrifugation and washed twice with cold acetate buffer (pH 5.0). The pellets were then freeze-dried. The disrupted cells were suspended in acetate buffer (pH 5.0)-> stirr for 1h -> remove cell pellets -> get the supernatant as the crude enzyme solution.

Purification methods of Glucosidase

General method 5 steps

Improved method 3 steps

Crude enzyme Ammonium sulfate precipitation Q-sepharose FF Phenyl-sepharose CL-4B CHT ceramic hydroxyapatite Sephacryl S-300 HR

Crude enzyme Ammonium sulfate precipitation Q-sepharose FF Microcrystalline cellulose (MCC)

Electrophoresis and molecular weight estimation

The molecular weight of the glucosidase was estimated by both SDSPAGE and gel filtration on a sephacryl S-300 HR column.

Assay of enzyme activity

-glucosidase activity was determined by a colorimetric method using pNPG as a substrate. To check the ginsenoside biotransforming activity, a reaction mixture containing ginsenoside, enzyme solution, formate buffer (pH 3.5) was incubated for 0.572 h at 45oC. The reaction was stopped by extraction with nbutanol. The n-butanol fraction was then analyzed by high-performance TLC (HP-TLC) using chloroform-methanol-water (65:35:10 v/v, lower phase)

Effects of pH and temperature on enzyme activity and stability.

To determine the optimal pH, pNPG-hydrolyzing activity was studied at 45oC in the following buffers: 50mM formate buffer (pH 2.54.5) 50mM acetate buffer (pH 4.06.0) 50mM phosphate buffer (pH 6.08.0) The pH stability of the enzyme was measured according to the residual activity after the enzyme was preincubated in the above buffers (pH 2.58.0) at 45oC for 4 h. To determine the optimal temperature, -glucosidase activity was studied at 2565oC in 50mM formate buffer (pH 3.5). Thermal stability was examined according to residual activity after pre-incubating the enzyme in 50mM formate buffer (pH 3.5) at 2565oC for 4 h.

Results & Discussion

Effects of pH and temperature on enzyme activity and stability

Conclusion

They successfully simplified the purification method for a novel ginsenoside-hydrolyzing glucosidase from Paecilomyces Bainier sp. 229 by the application of MCC as a novel chromatographic matrix. The properties of the purified enzyme were different from those of previously reported -glucosidases. This is the first report on the application of MCC column chromatography in -glucosidase purification and of a ginsenoside-hydrolyzing glucosidase that converts ginsenoside Rb1 to Rg3 specifically and efficiently.