Kinetic Analysis Of Ginsenosidase Type ? Reaction

In this paper,the reaction kinetics of the cloned Ginsenosidase Type III on the PPD ginsenoside such as Rb1,Rb2,Rb3,RC,Rd,Rg3 and F2 were sudied.The main ginsenoside Rb1,Rb2,Rc,Rd,Re,Rg1 in ginseng have high thermal stability;after heating 3 to 5 hours at the temperature of 120 ?,the ginsenoside Rbi,Rb2,Rc,Rd,Re,Rgi solution were not decomposed.Protopanaxadiol type ginsenoside of Ginseng stems and leaves was separated using a silica column.Finally 4.74g Rd and 3.59g Rb1 were obtained from 40g PPD ginsenoside;the yield was 11.8%for Rd,and 9%for Rb1 respectively.The ginsenosidase Type III showed single-peak in the HPLC analysis to prove the enzyme was pure enzyme;the molecular weight of the ginsenosidase Type III was 72.4 kDa in the SDS-polyacrylamide gel electrophoresis.The optimum pH of enzyme reaction was 7.0,and the optimum temperature was 37 0C.The Ginsenosidase Type III preferentially and sequentially hydrolyzed the terminal and inner glucoside at the 3-0-(C-3)position of ginsenoside Rb,to gypenoside GypXVII,and GypXVII was further hydrolyzed to GypLXXV;the enzyme also hydrolyzed C-3 position glucoside of ginsenoside Rb2 to C-O,further hydrolyzed to C-Y.The enzyme also hydrolyzed C-3 position glucoside of ginsenoside Rd to ginsenoside F2,CK,and hydrolyzed terminal glucose at the C-3 position of ginsenoside Rc,Rg3,F2 to produce C-Mcl,Rh2,CK.Kinetic studies was performed with ginsenosidase Type III using ginsenoside Rbl,Rb2,Rb3,RC,Rd,Rg3 and F2 as substrate.The value of Km is 4.08mM/L,9.09mM/L,8.85mIM/L,3.85mM/L,9.7 mM/L,11.369.7 mM/L,12.85 mM/L respectively.The value ofVm is 16.64 mM/Lh,14.31 mM/Lh,14.6 mM/Lh,23.15 mM/Lh,1.395 mM/Lh,1.81 mM/Lh,0.41 mM/Lh respectively.The value of Km is smaller,and value of Vm is bigger,the enzyme reaction rate is rapider;therefore,the rate of enzymatic reaction is Rc>Rb1>Rb2>Rb3>Rg3>Rd>F2.The ginsenosidase Type III is much more effective in the conversion of substrate Rb1,Rb2,Rb3,Rc than that of the Rg3?Rd?F2 substrate.