Purification And Immobilization Of Microbioenzyme For Biotransformation Ginsenoside Rb₁

Ginsenosides are the main active components of ginseng,which can be available anti-tumor,immune regulation,prevention and treatment of cardiovascular diseases,inhibition of cancer cell proliferation,and other activities.The pharmacological activity of minor ginsenosides is generally stronger than those of major ginsenosides.Generally,the minor ginsenosides are obtained by converting major ginsenosides.Traditional chemical conversion methods have many by-products and it is difficult to purify the target products.Microbial conversion is more and more used in the field of ginsenosides conversion because of its strong reaction selectivity,mild reaction conditions,environmental friendliness,few by-product,and simple post-treatment.In this paper,American ginseng red ginseng powder was added to PDA medium to cultivate bacteria,and after primary screening,two high-quality strains A-1 and R-1that could be transformed with Rb₁were selected.Two-phase aqueous extraction technology can replace conventional organic solvent extraction.Moreover,the reaction conditions of the duplex phase are m ild,which can avoid the inactivation or denaturation of the bioactive substance s in the extraction process.In this paper,three kinds of polyethylene glycol 4000-potassium phosphate,polyethylene glycol 4000-potassium hydrogen phosphate were prepared from A-1 and R-1 from three kinds.The extracted microbioenz-ymes were used to transform ginsenoside Rb₁and evaluate the enzyme activit-y with nitrophenyl-D-pyranoside（pNPG）as the reaction substrate.The experim-ental results showed that the 4000-potassium phosphate and PEG 2000-potassiu-m phosphate duplex were more active,the activity of R-1 was 44 U and 41U in them,respectively,and that of A-1 was 37 U and 46 U in both systems,respectively.Conversion of ginsenoside Rb₁to Rg3 and Rd using species A-1extra-cted from polyethylene glycol 4000-potassium phosphate duplex water an d species R-1,The main product was ginsenoside Rg3,The yield were 28.23%and 32.3%,respectively,8.14%and 10.97%,respectively;Conversion of ginseno side Rb₁from the polyethylene glycol 2000-potassium phosphate duplex strain A-1 and R-1 to Rg3 and Rd,The major product,ginsenoside Rg3,The yield w ere 33.40%and 31.10%,respectively,13.31%and 9.77%improvement over micr obial fermentation,respectively.The immobilized enzyme technique can increase the repeated use of enzy me.In this thesis,amino-functionalized silica gel serves as the carrier to immob-ilizeβ-glucosidase by biotin-streptavidin method for the transformation of gins-enoside Rb₁.The results showed that the immobilizedβ-glucosidase converted gi nsenoside Rd,F2,Rg3,Rh2 in the yields of 42.40%,22.17%,34.13%,and 3.11%,respectively,and the conversion of Rb₁was 92.10%.The kinetic study of immob ilized enzyme showed that the maximum reaction rate and Michaelis constant was 0.74 mmol·L^-1·min^-1,0.53 mmol·L^-1,respectively.The result of K_m>>[C]indi cate that the conversion reaction of immobilized enzyme in the small column i s a first-order reaction.The immobilized enzyme showed good stability,which th e enzyme activity maintain 94.28%of its original after running 10 times.