Assembly And Application Of Enzymes Confined In Porous Nanostructured Materials

Cytochrome P450 enzymes?CYP450?are heme-containing transmembrane proteins,which metabolize a plenty of endogenous and xenobiotic molecules.As the most highly expressed xenobiotic enzymes in the liver,they metabolize approximately 73%of all clinical used drugs.Since the drug metabolized by the CYP450 enzyme may cause changes in the activity of the CYP450 enzyme,and interfer its metabolic behavior on other drugs,triggering a change in efficacy or genotoxicity,which threatens human health.Therefore,it is necessary and meaningful to fabricate simple and inexpensive CYP450 enzyme-mediated drug metabolism systems in vitro for studying the metabolic mechanism of CYP450 enzyme,the induction or inhibition effects of drugs on CYP450 enzyme,and the genotoxicity of the metabolites,which is of great significance and valuable for the design and synthesis of new drugs,and human health.For this purpose,in this paper,a variety of nano-materials with porous structure were synthesized.And the biocatalytic activity of the assembled enzyme,the synergistic catalysis efficiency and their applications in biosensing were investigated.Details were described as below.?1?A simple and accurate in vitro drug metabolism strategy was proposed by using poly?diallyldimethyl-ammonium chloride??PDDA?modified macroporous ordered siliceous foam?denoted as PMOSF?as a nanoreactor for confining the nanohybrid of CdTe quantum dots?QDs?and cytochrome P450 2D6?CYP2D6?.Tramadol was chosen as the model substrate.Under the irradiation of a white-light,the photocurrent of PMOSF/CYP2D6/QDs/ITO increased with the addition of tramadol in a wide linear range from 4.0?M to 100?M.The apparent Michaelis-Menten constant was measured to be 3.6?M.Such nanoreactor provided a suitable environment to confine a mass of enzyme,as well as to maintain their catalytic activities for highly effective drug metabolic reactions.This showed promising potential for immobilizing various enzymes and other biomolecules in biomimetic metabolism study.?2?The increasing demand for early screening of the genotoxicity of new chemicals and drugs is pushing the envelope of micro/nanoreactors for metabolic study.Herein,a novel light-driven enzymatic bio-nanoreactor was designed with the gold nanoparticles embedded carbon nanocage?Au@CNC?as a nanoreactor,and meso-tetrakis?4-carboxyphenyl?porphyrin?TCPP?as a photosensitizer for cytochrome P450-catalyzed drug metabolism.Profiting from the 3D hierarchical porous structure,large specific surface area,and fast electron transfer capacity of Au@CNC,both higher metabolic rate and faster reaction kinetics were realized when using7-ethoxytrifluoromethyl coumarin?7-EFC?as a target drug compared to conventional in vitro pharmaceutical metabolism system by NADPH,and the metabolic yield of7-EFC achieved 36%within 10 min visible-light irradiation.Moreover,a linear relationship between the concentration of substrate and the FL intensity value of the product over a range of 100³000 nM was obtained.Notably,the TCPP/Au@CNC nanoreactor exhibited a variety of advantages in terms of short assay time?10 min?,good specificity,remarkable repeatability,NADPH-free and low detection limit?33nM?.And the enzyme inhibition test was also achieved with the TCPP/Au@CNC photochemical bionanoreactor.All the results demonstrated that such a photochemical bionanoreactor had great potentials for the development of new drugs and screening of effective drug candidates.?3?Herein,a novel electrochemical platform for monitoring in vitro drug metabolism has been constructed by using nitrogen-doped carbon nanocages?NCNC?nanocomposite as matrix for immobilization of cytochrome P450 2C9?CYP2C9?enzymes through electrostatic interaction.The NCNC was prepared by an in-situ MgO template method.Profiting from the 3D hierarchical porous structure,large specific surface area,good hydrophilicity and fast electron transfer capacity of NCNC,in the presence of substrate tolbutamide,the electrochemical-driven CYP2C9mediated catalytic behavior toward the conversion of tolbutamide to o-demethyl-tolbutamide was achieved.Upon successive addition of tolbutamide into the electrolyte,the reduction current of the CYP2C9/NCNC/GCE increased with the increasing concentration of tolbutamide.The linear response range of tolbutamide concentration was from 3.0 to 70?M.High performance liquid chromatography method confirmed the product by electrochemical driven tolbutamide metabolism strategy.And the Michaelis-Menten constant was calculated to be 29.2?M.Furthermore,the inhibition effect of sulfaphenazole on CYP2C9 catalyze-cycle was also investigated.The immobilized CYP2C9 on NCNC displayed high enzymatic activity and strong affinity toward the substrate.This electrochemical platform may have potentials for the assessment of interactions between drugs and food.?4?Herein,a novel nanocarbon-based material?Au@NCNC?was synthesized by embedding gold nanoparticles?AuNPs?inside the pores of 3D hierarchical nitrogen-doped carbon nanocages?NCNC?through an in-situ chemical deposition method.The resultant Au@NCNC was employed as an electrochemical catalyst for oxygen reduction reaction?ORR?and an electrode material for supercapacitors.As a result,comparing to NCNC,the as-prepared Au@NCNC electrocatalyst exhibited high performance towards ORR,which manifested in high numbers of electron transfer,high kinetic current density,enhanced electrocatalytic stability and remarkable methanol durability.Moreover,the Au@NCNC displayed high specific capacitances,good rate capability and cycling stability with⁹7%of its initial capacitance retained at high current density of 10 A g^-1 after 5000 cycles.This could be attributed to the synergetic effect of ultrafine gold nanoparticles.By depositing AuNPs inside the pores of NCNC,the conductivity and hydrophilicity of Au@NCNC were much improved compared to those of pristine NCNC,which provided an easy accessibility to the active sites of reactants?such as hydrated O₂?and thus promoted the electrocatalytic performances.This work offers a new perspective for preparing excellent catalysts in fuel cells,ORR and supercapacitive by enhancing the conductivity and surface hydrophilicity of electrocatalysts.