Preliminary Research On The Construction And Application Of ?-Glucosidase Microreactors

The research aimed to immobilize the?-glucosidase on suitable supports?Fe₃O₄/Fe₃O₄@rGO?to construct enzymatic microreactors and their subsequent applicability in efficient inhibitor screening from Chinese Yam?Dioscorea opposita Thunb.?peel was stuied using high performance liquid chromatography and mass spectrometry?HPLC-MS?.The research in this thesis not only provides a theoretical basis for the further development and utilization of Henan genuine medicinal materials,but also provides a theoretical basis for the design and synthesis of hypoglycemic drugs in the future.The main contents are as follows:1.Simultaneously determination of eight compounds from Dioscorea opposita Thunb.peel extract by HPLC-MS.Eight compounds including trans-N-p-coumaroyltyramine,seven batatasins and their derivatives in DioscoreaoppositaThunb.peelextract?batatasin?,3,3?-dihydroxyaiaryl,batatasin?,2,4-dimethoxy-6,7-dihydroxyphenanthrene,batatasin V,batatasin I,3,5-dimethoxy-2?-hydroxyaiaryl?were simultaneously identified by HPLC-MS.Under the typically HPLC-MS conditions,the limit of detection was low?0.15¹.00?g/mL?,the linearity?0.9990⁰.9999?and recovery by addition standard?82.6%¹08.6%?were good.Also,the stability and repeatability were satisfying.The developed method not only can be used to simultaneously identify various known compounds but alsocan be used to analyse the unknown components from Dioscorea opposita Thunb.2.Constructionoftwo?-glucosidasemicroreactorsandtheirapplicationonDioscorea opposita Thunb.peel for inhibitors screening2.1.Fe₃O₄@?-glucosidase microreactorsAmine-functionalized Fe₃O₄ nanoparticles were synthesized according to a facile one-step solvothermal method and characterized by FTIR,XRD,TEM and SEM technologies,respectively.The results showed that the magnetic materials had uniform particle size?20 nm?,good saturation magnetization?89 emu/g?and dispersibility.Several parameters,including glutaraldehyde concentrations,immobilization times and enzyme amounts were investigated to achieve the optimum immobilization conditions.Under the optimum conditions?12%?6 h?80:1?,the immobilization amount of?-glucosidase on Fe₃O₄ was about 8.04?g?-glucosidase/mg carriers.Two?-glucosidase inhibitors were selectively extracted from Dioscorea opposita Thunb peel extracts and identified as batatasin I and2,4-dimethoxy-6,7-dihydroxyphenanthrene and the binding capacities were 15.6%,26.7%,respectively.The results of this work demonstrated that Fe₃O₄ MNPs prepared by one-pot solvothermal method are promising supports to immobilize?-glucosidase for the efficient inhibitors screening from natural products.2.2.Fe₃O₄@rGO@?-glucosidase microreactorsAmine-functionalized rGO@Fe₃O₄ nanocomposites were synthesized by a facile one-step solvothermal method and characterized by TEM,FTIR,XRD,and SEM technologies,respectively.The results showed that the nanomaterials had average particle size,good saturation magnetization and dispersibility.Several parameters,including proportion of FeCl₃·6H₂O to GO,crosslinking agent concentration,reaction time and enzyme amounts were investigated to achieve the optimum immobilization and the immobilization amount of?-glucosidase on rGO@Fe₃O₄ under the optimized conditions?2.5:1?10%?6 h?25:1,respectively?was about 40?g?-glucosidase/mg carriers which was about five times than thatofpureFe₃O₄ MNPs.Furthermore,thebindingcapacitiesofscreenedinhibitors,2,4-dimethoxy-6,7-dihydroxyphenanthrene and batatasin I,were 35.6%and 68.2%,respectively which was also higher than that of pure Fe₃O₄ MNPs.The data presented in this two parts provided strong evidence that the Fe₃O₄ MNPs and rGO@Fe₃O₄ nanocomposites exhibited significantly higher immobilization efficiency and binding capacities that could be employed as supports for the enzyme microreactors construction to screen the potential ligands from natural product resources.3.Investigation of the interaction of 2,4-dimethoxy-6,7-dihydroxyphenanthrene with?-glucosidaseApplying enzyme kinetics,spectroscopic,and molecular docking methods,the interaction properties of 2,4-dimethoxy-6,7-dihydroxyphenanthrene with?-glucosidase were systematically investigated.The?-glucosidase inhibitory activities(IC₅₀=0.40 mM)were significantly higher than that of acarbose?as control?and the spectrometric results revealed that the 2,4-dimethoxy-6,7-dihydroxyphenanthrene inhibited?-glucosidase in a reversible and noncompetitive manner,that is to say,the inhibitor bind to the inactive region of?-glucosidase and could be selectively screened from Chinese Yam peel extract and separated from the binding sites.Hydrogen bond was the key interaction force from the results of the molecular docking study,and binding energy was-27.754 kJ/mol.In the CD studies,the content of?-helix in?-glucosidaseincreasedfrom17.2%to17.8%withtheconcentrationvaringof2,4-dimethoxy-6,7-dihydroxyphenanthrene.The change of?-glucosidase secondary structure was further proved by the attenuated total reflectance-fourier transform infrared spectra?ATR-FTIR?measurements and the FTIR spectras of?-glucosidase resulted in obvious red shift with the addition of2,4-dimethoxy-6,7-dihydroxyphenanthrene.Allthemeasurementsprovedthat2,4-dimethoxy-6,7-dihydroxyphenanthrene interacted with?-glucosidase and conformationally changed the secondary structure of?-glucosidase.