Hydrolysis Pathways Of Protopanaxadiol Type Ginsenosides By The Enzyme From Aspergillus Sp.48

Ginseng is a famous Chinese herbal medicine,and ginsenosides are the active ingredients.The content of the rare ginsenosides which are absorbed by human digestive system is very low in the natural medicine,while with the special effect is one in a million.Biotransformation is one of the most important ways to prepare for rare ginsenosides.This paper focuses on the enzyme reaction kinetics of hydrolyzing ginsenosides to rare ones by microbial enzyme.Aspergillus sp.48 was chosen as the enzyme producing strain duing to its higher producing enzyme activity.The enzyme was acquired by incubating the strain in the liquid medium.The optimal enzyme reaction conditions were confirmed as follows:the substrate concentration was 5%,the temperature was 45?,and the reaction time was 48 h.The enzyme was isolated and purified by DEAE-CelluloseDE-52,and the telative molecular mass of pure enzyme was 74.0 kDa based on SDS-PAGE.The enzyme reaction kinetics of pure enzyme hydrolyzing 3-0-,20-O-glucosyl of the monomers was elaborated,and the ginsenoside monomers Rb1,Rb2,Rb3,Rc,Rd,F2 and 20(S)-Rg3 were respectively listed as substrates.Rbl was hydrolyzed to GypXVII,while the enzyme hydrolyzes 3-O-Glc of Rb1 and the and Vmax were 14.64 mM and 19.10 mM/h,respectively.Another way was that Rbl was hydrolyzed to Rd with 20-O-Glc hydrolyzed,and the Km and Vmax were 15.42 mM and 65.44 mM/h,respectively.Rb2 was hydrolyzed to C-O with 3-O-Glc hydrolyzed,and the Km and Vmax were 6.53 mM and 24.46 mM/h,respectively.Rb3 was hydrolyzed by two ways:hydrolyzing 3-O-Glc to C-Mx1,and the Km and Vmax were 24.89 mM and 33.64 mM/h,respectively;hydrolyzing 20-O-Xyl to Rd,and the Km and Vmax were 8.78 mM and 57.44 mM/h,respectively.Rc was hydrolyzed to C-Mcl with 3-O-Glc hydrolyzed,and the Km and Vmax were 4.70 mM and 23.31 mM/h,respectively.Rd was hydrolyzed to F2 with 3-O-Glc hydrolyzed,and the Km and Vmax were 0.79 mM and 11.84 mM/h,respectively.F2 was hydrolyzed to C-K with 3-O-Glc hydrolyzed,and the Km and Vmax were 1.13 mM and 3.75 mM/h,respectively.20(S)-Rg3 was hydrolyzed to 20(S)-Rh2 with 3-O-Glc hydrolyzed,and the Km and Vmax were 0.16 mM and 0.22 mM/h,respectively.When the substrate concentration was 10 mM,the hydrolysis rates were confirmed as follows:VRb1?Gyp17=7.75 mM/h;VRb1?Rd=25.74 mM/h;VRb2?C-O=14.80 mM/h;VRb3?C-Mx1=9.64 mM/h;VRb3?Rd=30.59 mM/h;VRc?C-mc1=15.86 mM/h;VRd?F2=10.97 mM/h;VF2?C-K=3.37;V20(S)-Rg3-20(S)-Rh2=0.22 mM/h.The sort of hydrolysis rate of 3-O-Glc was Rc>Rb2>Rd>Rb3>Rbl>F2>20(S)-Rg3.The hydrolysis rate of 20-O-glucosyl of Rb1 and Rb3 was higher than 3-O-Glc,and the hydrolysis rate of 20-O-Xyl of Rb3 was higher than 20-O-Glc of Rb1.In a word,the hydrolysis rate of the enzyme from Aspergillus sp.48 was Rb3>Rbl>Rc>Rb2>Rd>F2>20(S)-Rg3.64.0 g PPD was hydrolyzed by the enzyme from Aspergillus sp.48,and the hydrolysates were desugared by AB-8 macroporous adsorption resin and decolored by D280 anion exchange resin.The weight of the crude product was 44.4 g,and the yield was 69.3%.36 g crude product was isolated and purified by silica gel chromatography,the weight of the component C-K with purity of 98.36%was 4.33 g,and the yield was 12.03%.