Hydrolysis On Protopanaxadiol Type Ginsenosides By Ginsenosidase Type ? From Sp.848 Strain

In this paper,the ginsenosidase type ? was fermented from sp.848 and purified.And reacted with a certain concentration of protopanaxadiol type ginsenoside solution in an equal volume.Calculated for each ginsenoside kinetic parameters,and studied how to hydrolysis and its hydrolysis pathways.Firstly,sp.848 was activated,then selected and fermented the strains which had preferred enzyme activity.Purification the crude enzyme with DEAE-cellulose DE-52 ion-exchange column chromatography,to get pure ginsenosidase type ?.And it weights about 74 kDa based on SDS-PAGE.Secondly,the ginsenosidase type ? with a certain concentration of Rb1,Rb2,Rb3,Rc,Rd,F2 and s-Rg3 solution was reacted with an equal volume and calculated each ginsenoside kinetic parameters shows:The Km and Vmax for hydrolysis on Rb1 20-O-?-(1?6)-glucopyranoside bond were 14.782 mM,and 58.69 mM/h;the Km and for hydrolysis on Rb2 3-O-?-(1?2)-glucopyranoside bond were 19.001 mM,and 38.048 mM/h;the Km and Vmax for hydrolysis on Rb3 3-O-?-(1?2)-glucopyranoside bond were 15.128 mM,and 10.822 mM/h,the Km and Vmax for 20-O-?-(1?6)-xylopyranoside bond were 15.127 mM,and 1.618 mM/h;the Km and Vmax for hydrolysis on Rc 3-O-?-(1?2)-glucopyranoside bond were 10.999 mM,and 6.606 mM/h;the Km and Vma.for hydrolysis on Rd 3-0-?-(1?2)-glucopyranoside bond were 0.364 mM,and 0.844 mM/h;the Km and Vmax for hydrolysis on F2 3-O-?-glucopyranoside bond were 2.335 mM,and 3.665 mM/h;the Km and Vmax for hydrolysis on s-Rg3 3-O-?-(1?2)-glucopyranoside bond were 0.355 mM,and 0.45 mM/h;the enzyme transformation velocity at 10 mM substrate was 23.683mM/h for Rb1,13.120mM/h for Rb2,4.307 mM/h for Rb3?C-Mx1,0.644 mM/h for Rb3?Rd,3.146mM/h for Rc,0.814mM/h for Rd,2.971 mM/h for F2,0.043mM/h for s-Rg3.The structures of Rb1,Rb2,Rb3,Rc,Rd and s-Rg3 were similar.They contain two sugar moieties,and both contain a glucosyl residue at 20-O-position;but at 20-O-position,Rb1 has terminal glucosyl,Rb2 has terminal arabinopyranosyl,Rb3 has terminal xylosyl,Rc has terminal arabinofuranosyl;Rd has one glucosyl,s-Rg3 has H,to be a hydroxy.Compared with Rd,F2 missed a glucosyl at 3-O-position.Compared the velocity of ginsenosidase type ? from sp.848 hydrolysis ginsenoside Rb1,Rb2,Rb3,Rc,Rd,F2 and s-Rg3 shows:the total rate of hydrolysis was Rb1>Rb2>Rb3>Rc>F2>Rd>s-Rg3;hydrolysis on 20-O-?-(1?6)-glucopyranoside bond was fastest,much further than hydrolysis on?-(1?6)-xylopyranoside bond.The rate of hydrolysis on 3-O-?-(1?2)-glucopyranoside bond was Rb2>Rb3(C-Mx1)>Rc>F2>Rd>s-Rg3.Analyzed the reasons may be that the number of sugar moieties and terminal sugar moiety are different:the more sugar moieties at 20-O-position,the faster hydrolysis velocity are,and the rate was arabinopyranosyl>xylopyranosyl>arabinofuranosyl;when the same number of sugar moieties at 20-O-position,the less sugar moieties at 3-O-position,the faster hydrolysis velocity are.By TLC and part of ginsenosides HPLC analysis,the hydrolysis mechanism of ginsenosides can be divided into:hydrolyzing ginsenoside Rb,,Rb3(Rb3*Rd),firstly hydrolyzed its sugar moiety at 20-O-position into Rd,then hydrolyzed two moieties at 3-O-position of Rd into F2,final to C-K.Hydrolyzing ginsenoside Rb2,Rb3(Rb3*C-Mx1),Rc,s-Rg3,firstly hydrolyzed two moieties at 3-O-position into intermediates saponins,then hydrolyzed the moiety at 20-O-position into final product C-K,as,Rb2?C-0?C-Y?C-K,Rb3?C-Mx1?C-Mx?C-K and Rc?C-Mc1?C-Mc?C-K.Hydrolyzing ginsenoside s-Rg3,it has not sugar moiety at 20-O-position,hydrolyzed two moieties at 3-O-position into Aglycone,as,s-Rg3?s-Rh2?PPD Aglycone.Finally,to obtain ginsenosidase type ? which sp.848 was mass fermented,and transformed 64g PPD at 45 C,desugared and decolorized by AB-8 and D-280,evaporated to dryness,obtained 46.47g hydrolysates,which mainly contained F2,C-K,with the yield was 72.61%,17.53g carbohydrate was taken off.Silica gel column chromatography used to separate monomer ginsenoside from 35g hydrolysates with chloroform:methanol:water(8.5:1.5:0.1)as the eluent.19.16g C-K was obtained with the yield of C-K was 54.74%;1.66g C-Mc was obtained with the yield of C-Mc was 4.74%;0.64g C-Y was obtained with the yield of C-Y wasl.83%;2.51g F2 was obtained with the yield of F2 was 7.17%.The purity of C-K,C-Mc,C-Y and F2 were 92.27%,58.23%,67.85%and 83.54%,with the same results by TLC.