Study On Biotransformation Of Ginsenosides By ?-glucosidase And The Interaction Mechanism

Ginseng,as a treasure of traditional Chinese medicine,has remarkable medical and health effects,and has been widely used in the development of drugs and new resources food.As the material basis for the production of pharmacodynamics,ginsenosides are the main active components of ginseng.The main ginsenosides are with high content in ginseng,such as ginsenoside Rb₁.When it is removed from some/all glycosyl groups,it is called rare ginsenoside,which has stronger activity and bioavailability.?-glucosidase can catalyze the hydrolysis of nonreductive?-D-glucoside bond at the end of the glycosyl chain,such as alkylglycoside and aryl glycoside,and can convert the main ginsenoside into rare ginsenoside.The resources of?-glucosidase were explored and the related properties were analyzed.The binding and interaction mode of?-glucosidase with ginsenoside substrate in the catalytic reaction were clarified.Exploring the structure-activity relationship of rare ginsenosides as nuclear receptor modulators is of great significance for understanding the mechanism of?-glucosidase acting on the atypical substrate ginsenoside Rb₁and expanding the physiological activity of ginsenosides.The main contents of this paper include the following aspects.(1)The gene of?-glucosidase was from Paenibacillus polymyxa in GH1 family(Gen Bank:M60211.1).The codon of the coding gene was optimized to make it easier for prokaryotic expression.The gene bgl B was synthesized by PCR.Then,bgl B gene was connected with pET-28a(+)vector to construct the recombinant plasmid pET-28a(+)-bgl B,and the recombinant plasmid was transformed into E.coli BL21(DE3)and cultured and induced by IPTG at 30?,220 rpm,then high expression products were obtained in the supernatant of broken cells.The crude enzyme solution was purified by Ni-NTA chromatography.The molecular weight of the recombinant?-glucosidase protein monomer was 52 k Da and the protein concentration was 0.479mg/m L as well as the purity was more than 95%.The physical and chemical properties of the?-glucosidase were analyzed by using a variety of bioinformatics software and online tools.The enzyme has no transmembrane domain,no signal peptide,and the overall performance is hydrophilic as well as the secondary structure is stable.The secondary structure of protein was analyzed.The tertiary structure of protein is reasonable,and the enzyme contains 4 phosphorylation sites.It is found that ORF2 sequence in 9 open reading frames contains the feature sequence of GH1 of glucosidase family,and the subcellular localization is located in cytoplasm.The results show that there are differences in the homology between the glycosidase and the active center of its catalytic function.In general,the?-glucosidase with clear sequence and structure information was obtained through gene synthesis and prokaryotic expression purification.Combined with bioinformatics analysis,it is helpful to understand the evolutionary relationship and related properties of the enzyme.(2)The interaction mode of?-glucosidase and ginsenoside Rb₁was studied by multi spectral method and molecular simulation,and the pure enzyme and the complexe of and?-glucosidase and ginsenoside Rb₁were characterized.The fluorescence spectrum analysis showed that the spontaneous fluorescence of?-glucosidase decreased with the increase of ginsenoside Rb₁concentration at different temperatures,and the microenvironment of the intrinsic fluorescent amino acid residues in?-glucosidase changed.The slight blue shift at 280 nm indicates that the enzyme was more stable at the ground state.The stern Volmer equation(y=0.00754x+1.0984,R²=0.9941)was obtained.The quenching constant K_SVwas 8.37×10³L/mol,the rate constant K_qwas 8.37×10¹¹L/mol×s.The K_SVincreased with the increase of temperature,indicating that it belongs to static quenching.The results showed that the fluorescence intensity of?-glucosidase was changed by amino acid residues when??=15 nm and??=60 nm.The three-dimensional fluorescence spectrum was consistent with the fluorescence spectrum.The UV visible absorption spectrophotometry analysis increased the concentration of Rb₁,which made Trp hydrophobic group in?-glucosidase more wrapped.A new conjugation system was formed between the enzyme Rb₁,and the non radiation energy transfer occurred between them,and a new?-?*transition was added.The secondary structure of?-glucosidase was changed by the addition and binding of Rb₁by circular dichroism.LSPR analysis showed that when?-glucosidase and ginsenoside Rb₁formed a stable1:1 complex,the equilibrium dissociation constant(KD)was 5.24×10^-4(±2.35×10^-5)mol/L,Ka was 29.7(±6.62×10²/mol×L^-1×s),Kd was 1.56×10^-2(±2.17×10^-5)/s.The structure changes of?-glucosidase and ginsenoside complex were analyzed by FTIR.The hydrodynamic radius of?-glucoside enzyme and the Zeta potential of the solution were analyzed by dynamic light scattering.The binding conformation of ginsenoside Rb₁with?-glucosidase(8.9 kcal/mol),binding site,hydrogen bond and hot amino acid residues were analyzed by molecular docking.The results showed that the total binding free energy of the system was-50.86 kcal/mol,indicating that the two compounds had better binding ability.(3)In the study of the enzymatic properties of recombinant?-glucosidase and the transformation of ginsenoside Rb₁,the specific activity of the pure enzyme was increased by 15 times,the yield was 51.0%and the specific activity of hydrolyzed p NPG reached 12.4 U/mg.The K_mand V_maxvalues of recombinant?-glucosidase were 0.743 mmol/L and 3.14×10⁴?mol/min/mg,respectively.K_cat/K_mwas3.813×10³/s/mmol/L,which indicated that it has higher specificity and catalytic efficiency for typical substrate p NPG,and explores the substrate specificity of the enzyme and the influence of catalytic environment on recombinant?-glucosidase.The results show that the enzyme had activity in pH range of 3.0 to 9.5,the highest activity was at pH 6.5,and the temperature range was from 20?to 70?,and the highest activity was at 40?.The effects of metal ions and chemical reagents on enzyme activity were studied.The results showed that the activity of recombinant?-glucosidase was slightly affected by the monovalent metals as Na⁺?K⁺?Li⁺and divalent metal ions as Ba²⁺?Ca²⁺?Mg²⁺?Mn²⁺?Zn²⁺.The addition of trivalent metal ions as Al³⁺and heavy metal ions as Pb²⁺and Ag⁺plasma inhibited the enzyme activity to a great extent.Alcohol and DMSO had little influence on the enzyme activity,while SDS and EDTA could inhibit 80%to reduce the activity of the enzyme.The understanding of enzymology properties laid a foundation for the development and application of enzymes.The HPLC method for simultaneous determination of ginsenoside such as Rb₁,Rd,F₂and CK was established,and the composition and structure of ginsenoside were determined by triple quadrupole linear ion trap tandem mass spectrometry.The recombinant?-glucosidase with 10 U activity could transform ginsenoside Rb₁,and the C-3 glucose group and C-20?-d-pyranoglucosyl were hydrolyzed under the optimal conditions(pH 6.5;temperature 40?;ginsenoside Rb₁concentration 1 mg/m L).The yield of CK was 41.85%,the yield of F₂was 20.57%and the total conversion rate of ginsenoside Rb₁was 95.70%at 72 hours.(4)As the main metabolites of ginsenoside and the structural basis,20(S,R)-protopanaxadiol[PPD(S,R)]and 20(S,R)-protopanaxatriol[PPT(S,R)]were used as the research objects to investigate the structure-activity relationship of ginsenoside as an estrogen receptor agonist.The recombinant plasmid p GEX-4t-1-ER?-LBD was transformed into E.coli BL21 cells.The target protein ER?-LBD was purified by the GST affinity chromatography column after IPTG induction.The soluble protein was62.8 k Da by SDS-PAGE analysis,which was the corresponding GST fusion protein containing the binding domain of ER?.In the field of fluorescence polarization in vitro binding and competition,ginsenoside of the subjects showed dose-dependent binding with hER?-LBD.Kd value was between 1260.64?mol/L and 1716.19?mol/L.The order of binding strength was PPD(S)>PPD(R)>PPT(S)>PPT(R).In the dual luciferase reporter gene assay,pc DNA3.1(t)-hER?plasmid was constructed.The ER?reaction original ER was loaded on the ERE-luc,and p RL-TK was used as the internal parameter,and the fluorescence signal ratio(Fluc/Rluc)was used as the standardized ER?transcription activity.The results of the double luciferase reporter gene showed that the level of hER?was activated by PPD(S,R)and PPT(S,R)in dose-dependent manner,and it could up regulate the transcription of ER?mediated gene,which increased by 4 times.The ginsenosides can activate ER?transcription at the micro molar concentration,showing a weaker effect than E₂(Nanomole concentration).The interaction and binding modes of receptor ligand in ginsenoside-hER?-LBD complex were analyzed by molecular docking technology.The results of molecular docking have good correlation with the IC₅₀value(R²=0.94),which indicates that molecular dynamics simulation can be used to predict the binding effect of new bioactive compounds to target receptors.