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Ginsenoside Glycosidases from Panax Ginseng

Author: ZhangChunZhi
Tutor: AnLiJia;JinFengZuo
School: Dalian University of Technology
Course: Biochemical Engineering
Keywords: Ginsenoside -β- glucosidase Ginsenoside -α- Arab refined glycosidase Ginsenoside glycosyl hydrolase Enzyme purification Enzymatic hydrolysis Ginsenoside Rh2 Ginsenoside Rd
CLC: TQ461
Type: PhD thesis
Year: 2002
Downloads: 621
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Abstract


Specific hydrolysis ginsenoside-specific glycosylation, separation and purification of ginsenosides glycosyl hydrolase, nature study, and ginsenoside Rg3 and Rc directed enzymatic reaction proved Enzymatic directed transformation of ginsenoside structure possibilities. Buffer extraction, ammonium sulfate precipitation, ion exchange chromatography two ginsenoside glycosyl hydrolase - Ginsenoside-β-glucosidase and ginsenoside-alpha-arabinosidases enzyme isolated and purified from ginseng roots. Ginsenoside-β-glucosidase was purified 16-fold, a yield of 14.4%, ginsenoside-α-arabinoside enzyme was purified 18.5-fold, a yield of 4.6%. PAGE analysis of the purified enzyme a single band. Of ginsenoside-α-glucosidase hydrolysis the ginsenoside Rg3 generation ginsenoside Rh 2 , the optimum temperature was 55-60 ℃, the optimum pH was 5.0 Ca activation of the enzyme, Cu inhibition. Enzyme is stable below 60 ℃, pH4.0-7.0, SDS-PAGE and measured molecular weight of 59kDa. N-terminal amino acid sequence of this enzyme is SLDANYVPKYVTLPL, no homology with known sequences of the β-glucosidase, ginsenoside-β-glucosidase in the hydrolysis characteristics different from the traditional β-glucosidase (EC3. 2.1.21). The article also ginsenoside-β-glucosidase catalytic the Rg3 reaction of the Michaelis-Menten equation. Ginsenoside-α-arabinosidases hydrolytic and ginsenoside Rc generated ginsenoside Rd optimum temperature of 50 ° C, the optimum pH was 5.0, Cu enzyme inhibition. Enzyme is stable below 60 ℃, pH4.0-6.0, SDS-PAGE and measured molecular weight of 86kDa. The ginsenoside Rg3 ginsenoside glycosyl hydrolase hydrolysis substrate concentration of 10mg/ml, pH5.0, 55 ℃ conditions, reaction The 24h of ginsenoside Rg3 conversion rate can reach 60%. Saturated n-butanol extraction, silica gel column, hydrolysates Rh 2 purity of up to 90%, and the yield was 32%. Ginsenoside-β-glucosidase hydrolysis of 20 (S)-Rg3 and 20 (R)-Rg3 respectively generate 20 (S)-Rh 2 and 20 (R)-Rh 2 , roughly the same rate of hydrolysis. Magnetic resonance imaging ( 1 HNMR, 13 CNMR) and mass spectrometry (MS) detection results show that the resulting product of the enzymatic hydrolysis 20 (S)-Rg3 20 (S)-Rh < sub> 2 , system name is 3-O-(β-D-glucopyranosyl-yl) - dammar -24 - ene-3β, 12β, 20 (S) - triol; enzymolysis 20 (R )-Rg3 resultant product 20 (R)-RH 2 , system name is 3-O-(β-D-pyran-glucopyranosyl) - dammar -24 - ene-3β, 12β, 20 (R) - triol, molecular formula is C 36 H 62 O 8 , a molecular weight of 622. Ginsenoside glycosyl hydrolase substrate concentration 30mg/ml, pH5.0, 50 ℃ conditions hydrolysis ginsenoside Rc, the reaction 24h, RC the conversion rate reached 60%. Saturated n-butanol extraction, silica gel column, the hydrolysates Rd purity of 90%, a yield of 34%. Nuclear Magnetic Resonance ( 1 HNMR, 13 CNMR) and mass spectrometry (MS) detection results show that hydrolysates of 20 (S) - ginsenoside Rd, and the name of the system for 3 - O-(β-D-glucopyranosyl - (1 → 2)-β-the D-glucopyranose yl)-20-O-(β-D-pyran-glucopyranosyl) - dammar -24 - Summary alkenyl and p, 126,20 o Bu triol, molecular weight formula C48H82Ol to 946. In the prove enzymatic directional conversion of ginseng soap with the preparation of rare soap bitter feasibility, laid the experimental foundation for the further study of sugar hydrolase soap bitter glycosyl hydrolases.

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