Biotransformation Of Ginsenosides By Glycosidases And Their Catalytic Mechanism

Ginsenosides are the major active compounds of ginseng.To date,more than 150 natural ginsenosides have been isolated and identified.The contents of different ginsenosides in ginseng are different,the minor ginsenosides,such as Rg3,Rh2,CK,F1,F2,and Rh1 exist in smaller amounts but are more pharmacologically.They can be conveniently produced from the major ginsenosides?Rb1,Rb2,Rc,Rd,Re,and Rg1?.Several methods for the transformation including acid hydrolytic,heating,and biotransformation.Because of mild conditions,high efficiency,and specificity,glycosidase is an effective tool for the preparation of ginsenosides.It is of great theoretical and practical significance to find a variety of glycosidases with good specificity and high activity,which are convenient for the preparation of minor ginsenosides.After decades of research,some information about the hydrolysis mechanism of glycosidase have been accumulated.However,a comprehensive understanding of their substrate specificity is still lack,Therefore,the hydrolysis mechanism needs to be further revealed.Resolving the three-dimensional structures could provide important information for the modification of enzyme.The main results are as follows:1.Preparation and characterization of glycosidasesSix glycosidases CcBgl2,CcBgl3,CcBgl5,CcBgl6,CcBgl7 and BglPC28 were successfully expressed in E.coli BL21?DE3?.Characterization of recombinant glycosidase were performed with pNP?Glc substrate,the hydrolytic activity of them were stable within the pH range from 6.0 to 7.0 at 30 ^oC,Cu²⁺,Hg²⁺,and SDS significantly inhibited the enzymatic activity.The catalytic efficiencies(k_cat/K_m)value of Cc Bgl5 and CcBgl7 were57.16±3.37 mM^-1 s^-1 and 17.52±1.80 mM^-1 s^-1,higher than others.The specificity of substrate revealed that CcBgl2 and CcBgl3 have?-1,2/1,3/1,4 glucosidase activity,CcBgl5 have?-1,2/1,3 glucosidase activity,CcBgl6 have?-xylosidase activity,CcBgl7 have?-1,6/1,3/1,2glucosidase activity.CcBg5,CcBgl7 and BglPC28 were the most powerful hydrolytic agents,which could specifically hydrolyze C-3 outer glucose,C-20 outer glucose,and inner glucose of ginsenosides,respectively.2.Preparation of minor ginsenosides by combinatorial enzyme methodUsing a combination of Cc Bgl5,CcBgl7 and BglPC28 could convert major ginsenoside Rb1 into a series of minor ginsenosides.CcBgl5 could convert Rb1 into Gyp XVII.CcBgl5and CcBgl7 could convert Rb1 into F2.Cc Bgl7 and BglPC28 could convert Rb1 into Rg3,added Ccbgl5,the product was Rh2.CcBgl5 and BglPC28 could convert Rb1 into Gyp LXXV,added Ccbgl7,the product was CK.Furthermore,the optimum conditions for the preparation of rare ginsenosides were determined,substrate conversion was accomplished during 2-12 h using the appropriate buffer?pH6.0-7.0?and mild conditions?30 ^oC?.After purification,the purity of Gyp XVII,F2,Rg3,Rh2,Gyp LXXV,and CK could be achieved 90.5%,92.7%,95.8%,93.1%,91.3%,and 91.5%,respectively.The yields were 77.6%,73.4%,79.9%,78.3%,76.5%,and 75.3%,respectively.3.Structural analysis of glycosidasesThe structure of CcBgl7,CcBgl7-glycerol,CcBgl7-gentiobiose,CcBgl7-gentiobiose moiety of Rb1,CcBgl7-Rb1,and BglPC28-glycerol were refined to 2.02?,2.40?,2.40?,2.50?,2.40?,and 2.50?,respectively.CcBgl7 contained an??/??₈ barrel domain,an??/??₆sandwich domain,and a fibronectin type III-like domain.The co-crystal structure showed the residues at-1 subsite?Asp83,Arg146,Lys185,His186,and Asp264?played roles in trapping and constraining the non-reducing end glucose.The enzyme-substrate intermediate was fixed in the catalytic center of CcBgl7.It indicated the C1 atom of the oxocarbenium ion is covalent bonded with O?1 of the conserved nucleophilic residue Asp264.