Study On Biotransformation Of Ginsenoside Rg₁ And Its Key Enzymes

Ginsenoside F₁ （GF₁） is proved by modern pharmacology to have the functions of inducing the gap junction-mediated intercellular communication （GJIC） to reduce, inducing human body osteosarcoma cell U₂OS to death, reducing the cell death which UV-B causes and to prevent the human HaCaT keratinization cell which UV-B induces to perish weakly and anti-platelet aggregation. However, the content of naturally occurring GF₁ in wild ginseng is so little that the production of GF₁ is very difficult. Compared with GF₁, GRg₁ has one more glucose molecule at C₆ position, but it can be obtained easily. Therefore, a great interest exists in the preparation of GF₁ by limited hydrolysis of GRg₁ at C₆ position. So, how to obtain large quantity of purified GF₁ is an extremely vital economic problem with very high medicine value.This research was studied on the biotransformation of GRg₁ and its key enzyme. The biotransformation product from fermentation broth was confirmed as GF₁ by MS, ¹³C NMR and HPLC method. The active strain 2246 was identified as Penicillium sclerotiorum by morphological observation and ITS-5.8S sequence analysis.Theβ-D-glucosidase, which could transformate GRg₁ to GF₁ with strong substrate specificity, was determined as a constitutive enzyme. It was purified 94.65 fold and appeared on SDS-PAGE electrophoresis as a single band of MW 40 kDa. The enzyme was found that it consisted of multitude identical subunits （40kDa） with a native molecular mass of approximately 200kDa, and its maximal activity of hydrolysis of p-Nitrophenylβ-D-glucopyranoside （pNPG） occurred at 65℃and pH 4.5. Some metal ions Ba²⁺, Fe³⁺ and K⁺ can activate the enzyme activity strongly while some metal ions Mg²⁺ and Ag⁺ can inhibite its activity. The substrate specificity experiment indicated that the obtainedβ-D-glucosidase can only hydrolyze the C₆-glucose of GRg₁.The preliminary transformation processes were studied and transformation medium was defined as （g/100mL）: glucose 0.5g, carbon source A 2.0g, nitrogen source A 1.0g, yeast extract 1.0g, MgSO₄·7H₂O 0.3g, KH₂PO₄ 0.3g, pH 4.0, sterilizated at 121℃for 20min. The 250 mL flasks were filled with 80 mL media. The favorable transformation condition was determined: the age of inoculum 36h, inoculation amount 8%, transformation temperature 25℃-28℃, rotate speed 200 r/min, transformation time 48h after 0.3% substrate GRg₁ was feed to transformation broth.Eventually, the maximum biotransformation efficiency from GRg₁ to GF1 can reach 72%, which raised by 30% compared with the original culture and the production cost can be reduced by a large scale.