Kinetic Analysis Of Ginsenosidase Type ? Reaction

Compound-K(C-K)has important pharmacodynamic action,such as anti-cancer,liver-protection.C-K is absent in wild ginseng,it is metabolin in intestinal tract.Ginsenosidase type I produced from sp.48 and sp.848 strain can hydrolyze protopanaxadiol type ginsenoside(PPD)into C-K.In this paper,the kinetic of ginsenosidase type I from different source which hydrolyzed ginsenoside Rb1,Rc,Rd,F2 and Rg3 was investigated;and C-K was prepared largely.The substrates,pure Rbl,Rc and Rd were separated and purified by macroporous absorption resin and silica gel from total ginsenosides with the yields of 32.0%,15.2%,25.2%,and the purity were 96.5%,81.7%and 94.9%based on HPLC,respectively.Ginsenosidase type I from sp.48 strain was purified by DEAE.The purified enzyme formed a single band on the SDS-PAGE,and its molecular mass was 74 kDa.This results was checked by HPLC.The kinetic parameters of ginsenosidase type I from sp.48 strain induced by ginseng was as follows:Km for Rbl,Rc,Rd,F2 and Rg3 were 0.621,0.928,0.158,0.227,0.0170 mM,respectively;Vm for the above substrate were 14.6,20.7,7.38,8.14,0.557 mmol/L min,respectively.The kinetic parameters of ginsenosidase type I from sp.48 strain induced by bean pulp was as follows:Km for Rb1,Rc,Rd,F2 and Rg3 were 0.720,0.809,0.110,0.203,0.0170 mM,respectively;Vm for the above substrate were 14.6,27.0,5.02,7.15,0.854 mmol/L min,respectively.The enzyme from two sources exhibited little difference,and velocity for one substrate was close.The velocity order of reaction of ginsenosidase type I hydrolyzed PPD substrates was Rc>Rb1>F2>Rd>Rb2>Rb3>Rg3.After 24 h,the reaction was balanced.The transformation rate was not changed obviously with increasing time.Crude C-K product was transformed from 40.0 g PPD by ginsenosidase type I producing from sp.848 strain,the yield was 68.9%.This crude product firstly sepatated by AB-8 macroporous absorption resin.The resin washed by 40.0%,60.0%,70.0%,84.0%ethanol can separate the product roughly,after decoloured by D-296,the yield of C-K was 67.4%.Next,the product was further purified by silica gel.Monomor C-K and F2 was obtained,the purity was 93.1%and 80.3%,the yield was 26.5%and 17.3%,respectively.Finally,C-K was refined by crystallization,the purity was 98.9%,and the yield was 98.3%.