Apparent Michaelis Constant Research Articles

Evaluation of Enzymatic Kinetics of GOx‐based Electrodes by Scanning Electrochemical Microscopy at Redox Competition Mode

AbstractGlucose oxidase (GOx) is an enzyme, which is used for the development of enzymatic biofuel cells. Therefore in this research redox competition mode of scanning electrochemical microscopy (RC‐SECM) was applied for the investigation of glucose oxidase (GOx) catalyzed reaction kinetics. The GOx was immobilized by glutaraldehyde on substrates of different electrical conductivity: (i) gold covered glass was used as conducting substrate and (ii) plastic poly(methyl methacrylate) was used as non‐conducting substrate. Current vs distance dependencies were registered by SECM at different concentrations of glucose in the absence of redox mediator. The potential of −750 mV vs Ag/AgCl(3 M KCl) was applied to the microelectrode (ME), which was used as a probe in SECM, in order to register oxygen reduction current. Consumption of oxygen by the GOx based layer was evaluated according to principles determined by Michaelis‐Menten kinetics. Apparent Michaelis constants KM(app.) were calculated from the dependencies of current vs glucose concentration. In both these cases the KM(app.) value increased when the distance between ME and enzyme modified surface was increasing from 10 to 30 μm, while the KM(app.) value decreased by increasing the distance from 30 to 60 μm.

Calcium-Induced Mitochondrial Permeability Transitions: Parameters of Ca2+ Ion Interactions with Mitochondria and Effects of Oxidative Agents.

We evaluated the parameters of Ca2+-induced mitochondrial permeability transition (MPT) pore formations, Ca2+ binding constants, stoichiometry, energy of activation, and the effect of oxidative agents, tert-butyl hydroperoxide (tBHP), and hypochlorous acid (HOCl), on Ca2+ -mediated process in rat liver mitochondria. From the Hill plot of the dependence of MPT rate on Ca2+ concentration, we determined the order of interaction of Ca2+ ions with the mitochondrial sites, n = 3, and the apparent Kd = 60 ± 12µM. We also found the apparent Michaelis-Menten constant, Km, for Ca2+ interactions with mitochondria to be equal to 75 ± 20µM, whereas that in the presence of 300µM tBHP was 120 ± 20µM. Using the Arrhenius plots of the temperature dependences of apparent mitochondrial swelling rate at various Ca2+ concentrations, we calculated the activation energy of the MPT process. ΔEa was 130 ± 20kJ/mol at temperatures below the break point of the Arrhenius plot (30-34 °C) and 50 ± 9kJ/mol at higher temperatures. Ca2+ ions induced rapid mitochondrial NADH depletion and membrane depolarization. Prevention of the pore formation by cyclosporin A inhibited Ca2+-dependent mitochondrial depolarization and Mg2+ ions attenuated the potential dissipation. tBHP (10-150µM) dose-dependently enhanced the rate of MPT opening, whereas the effect of HOCl on MPT depended on the ratio of HOCl/Ca2+. The apparent Km of tBHP interaction with mitochondria in the swelling reaction was found to be Km = 11 ± 3µM. The present study provides evidence that three calcium ions interact with mitochondrial site with high affinity during MPT. Ca2+-induced MPT pore formations due to mitochondrial membrane protein denaturation resulted in membrane potential dissipation. Oxidants with different mechanisms, tBHP and HOCl, reduced mitochondrial membrane potential and oxidized mitochondrial NADH in EDTA-free medium and had an effect on Ca2+-induced MPT onset.

Regulation of the catalytic behavior of pullulanases chelated onto nickel (II)-modified magnetic nanoparticles.

Chelating of pullulanases onto nickel (II)-modified magnetic nanoparticles results in one-step purification and immobilization of pullulanase, and facilitates the commercial application of pullulanase in industrial scale. To improve the catalytic behavior, especially the operational stability, of the nanocatalyst in consecutive batch reactions, we prepared various iminodiacetic acid-modified magnetic nanoparticles differed in surface polarity and spacer length, on which the His6-tagged pullulanases were chelated via nickel ions, and then studied the correlation between the MNPs surface property and the corresponding catalyst behavior. When pullulanases were chelated onto the surface-modified MNPs, the thermostability of all pullulanase derivatives were lower than that of free counterpart, being not relevant to the protein orientation guided by the locality of the His6-tag, but related to the MNPs basal surface polarity and the grafted spacer length. After chelating of pullulanases onto MNPs, there were changes observed in the pH-activity profile and the apparent Michaelis constant toward pullulan. The changing tendencies were mainly dependent on the His6-tagged pullulanase orientation, and the changing extents were tuned by the spacer length. The reusability of pullulanase immobilized by N-terminal His6-tag was higher than that of pullulanase immobilized by C-terminal His6-tag. Moreover, the reusability of the immobilized pullulanase tested increased till grafting polyether amine-400 as spacer-arm, therefore the N-terminal His6-tagged pullulanase chelating MNPs grafted polyether amine-400 gave the best reusability, which retained 60% of initial activity after 18 consecutive cycles with a total reaction time of 9h. Additionally, the correlation analysis of the catalyst behaviors indicated that the reusability was independent from other catalytic properties such as thermostability and substrate affinity. All the results revealed that the catalyst behavior can be mainly controlled by the His6-tagged pullulanase orientation than by the MNPs surface property which can tune the catalyst function.

Metal-ion co-ordination assembly based multilayer of one dimensional gold nanostructures and catalase as electrochemical sensor for the analysis of hydrogen peroxide

In this study, we present for the first time an electrochemical biosensor for the quantification of hydrogen peroxide (H2O2) based on one dimensional gold nanostructures (1D-AuNs) and catalase (CAT) multilayer-by layer film fabricated on graphite electrode (GE) through metal-ion co-ordination assembly technique. Synthesis of 1D AuNs was performed by template directed synthesis employing cobalt nanoparticles as sacrificial templates. Characterization of the AuNs using UV–vis spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive analysis of X-rays (EDAX) clearly demonstrated the successful formation of nanostructures. Besides, the multilayer-by layer film was evaluated in terms of number of layers and electrochemical behavior using electrochemical quartz crystal microbalance (EQCM), UV–vis spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). In addition, the fabricated multilayer film was evaluated for electrocatalytic analysis of H2O2 using CV, differential pulse voltammetry (DPV) and chronoamperometry. CV assay indicates that a direct electron transfer is accomplished between the CAT and the electrode surface through metal-ion co-ordination assembly technique. Also, we determined the optimal operating potential and electrolyte pH to avoid possible interfering substances. The proposed biosensor showed superiority over previously reported electrochemical biosensors in the analytical characteristics such as rapid response (5s), wide linear range (0.05–19.35mM), high sensitivity (992μA−1mM−1cm−2) and low detection limit (0.98nM). The apparent Michaelis–Menten constant (Kmapp) which gives an evidence of enzyme affinity of the biosensor was estimated to be 0.21mM indicating high affinity for H2O2. The excellent anti-interference ability of the biosensor is also presented. Moreover, the proposed biosensor illustrated good repeatability, reproducibility, admirable electrochemical and long-term stability.

Real-time in vitro detection of cellular H2O2 under camptothecin stress using horseradish peroxidase, ionic liquid, and carbon nanotube-modified carbon fiber ultramicroelectrode

Single-walled carbon nanotubes (SWCNTs), horseradish peroxidase (HRP), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM·BF4) were employed to construct a cellular H2O2 sensor based on direct electron transfer. At a working potential of −0.35V, HRP-BMIM·BF4/SWCNTs/CFUME showed a dynamic range of up to 10.2μM with a low detection limit of 0.13μM (S/N=3) and a high sensitivity of 4.25A/Mcm2. The apparent Michaelis-Menten constant (Km,app) was estimated to be as low as 15.4μM, which suggested that HRP molecules entrapped in the BMIM·BF4 and SWCNTs immobilized at the carbon fiber ultramicroelectrode maintained a very high affinity. Because of the extremely small dimension and low working potential, HRP-BMIM·BF4/SWCNTs/CFUME enabled direct amperometric real-time monitoring of H2O2 in HeLa cells treated with the anticancer drug, camptothecin, without requiring complex data processing and extra surface coatings to prevent interference. HRP-BMIM·BF4/SWCNTs/CFUME testing clearly showed that the H2O2 level significantly increased in HeLa cells under camptothecin stress. When HeLa cells were cultured in medium without camptothecin, the H2O2 level remained stable during the whole measurement process. These results indicate that HRP-BMIM·BF4/SWCNTs/CFUME could be a powerful tool for real-time investigation of cellular H2O2 level, especially under anticancer drug stress. This method could provide in-depth insights regarding the occurrence, development, and apoptosis of tumors.

Enhancement of amperometric response of glucose biosensor by electrodeposition of silver nanoparticles onto chitosan-modified electrode

A highly sensitive biosensor based on silver nanoparticles (AgNPs) was fabricated for glucose detection in aqueous phase. Firstly, a platinum (Pt) electrode was modified with the mixture of glucose oxidase and chitosan. AgNPs were electrodeposited into the modified electrode by single pulse potentiostatic method at –0.4 V. The electrochemical performance of the modified electrode was evaluated by cyclic voltammetry and amperometry. The fabricated biosensor had a high sensitivity of 58.6 μA mM−1 cm−2 and detection limit of 4.4 μM glucose at a signal to noise ratio of 3. In addition, the biosensor showed a short response time less than 5 s and a wide linear range of 0.05-11.5 mM. The apparent Michaelis–Menten constant (KM) was found to be 9.14 mM. In addition, thermal stability and anti-interference ability of the biosensor were investigated. The results demonstrated that AgNPs enhanced the analytical performance of the biosensor.

Fabrication of enzyme reactor utilizing magnetic porous polymer membrane for screening D-Amino acid oxidase inhibitors

In this work, a unique D-amino acid oxidase reactor for enhanced enzymolysis efficiency is presented. A kind of magnetic polymer matrices, composed of iron oxide nanoparticles and porous polymer membrane (poly styrene-co-maleic anhydride), was prepared. With covalent bonding D-Amino acid oxidase on the surface of the matrices and characterization of scanning electron microscope and vibrating sample magnetometer, it demonstrated that the membrane enzyme reactor was successfully constructed. The enzymolysis efficiency of the enzyme reactor was evaluated and the apparent Michaelis-Menten constants of D-Amino acid oxidase were determined (Km was 1.10mM, Vmax was 23.8mMmin−1) by a chiral ligand exchange capillary electrophoresis protocol with methionine as the substrate. The results indicated that the enzyme reactor could exhibit good stability and excellent reusability. Importantly, because the enzyme and the substrate could be confined into the pores of the matrices, the enzyme reactor displayed the improved enzymolysis efficiency due to the confinement effect. Further, the prepared enzyme reactor was applied for D-Amino acid oxidase inhibitors screening. It has displayed that the proposed protocol could pave a new way for fabrication of novel porous polymer membrane based enzyme reactors to screen enzyme inhibitors.

An amperometric glucose oxidase biosensor based on liposome microreactor-chitosan nanocomposite-modified electrode for determination of trace mercury

A biosensor for trace mercury ions based on glucose oxidase (GOD) immobilized on liposome microreactor and chitosan (CS) nanocomposite through layer-by-layer method is described herein. The GOD liposome microreactors (GLMs) were characterized using Fourier transform infrared (FTIR), scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and amperometric methods. The results indicated that GLMs were prepared by encapsulating the enzyme GOD in l-α-phosphatidylcholine liposome resulting in spherical bioreactor with a mean diameter of 5.83 ± 0.75 μm. The encapsulation efficiency and drug loading content of the GLMs were about 53.58 ± 0.91 and 41.15 ± 0.95%, respectively. Cyclic voltammogram (CV) was further utilized to explore relevant electrochemical activity on (CS/GLM)8 nanocomposite-modified glassy carbon electrode (GCE). The biosensor based on the (CS/GLM)8-GCE composite films was applied to detect Hg2+ with a broad linear range from 0.5 to 5.00 μmol/L, and the detection limit was brought down to 0.076 μmol/L. The apparent Michaelis-Menten constant, K m, for the enzymatic reaction was 0.37 mmol/L (S/N = 3). Furthermore, the biosensor showed good stability and reproducibility. Such new biosensor based on encapsulation of GOD within liposome microreactors shows great promise for rapid, simple, and cost-effective analysis of Hg2+ in environmental samples.

An Amperometric Cytochrome P450-2D6 Biosensor System for the Detection of the Selective Serotonin Reuptake Inhibitors (SSRIs) Paroxetine and Fluvoxamine

Paroxetine is the second most prescribed selective serotonin reuptake inhibitor (SSRI) antidepressant drug, characterized by extensive inter-individual variation in steady state plasma concentrations resulting in drug toxicity amongst patinets. A nanopolymeric biosensor for studying the biotransformation of paroxetine is presented. The bioelectrode system consists of cytochrome P450-2D6 enzyme encapsulated in nanotubular poly (8-anilino-1-napthalene sulphonic acid) electrochemically deposited on gold. The biosensing procedure involved the determination of the extent of modulation of fluvoxamine responses to the P450-2D6 enzyme electrode after incubation in paroxetine standard solutions. Paroxetine inhibited the activity of cytochrome P450-2D6 (CYP2D6) resulting in a decrease in the fluvoxamine signal of the biosensor. The biosensor gave a linear analytical response for the paroxetine in the interval 0.005 and 0.05 μM, with a detection limit of 0.002 μM and a response time of 30 s. Electrochemical Michaelis–Menten kinetics of the reversible competitive inhibition of the fluvoxamine responses of the biosensor by 0, 0.05 and 0.1 μM paroxetine gave apparent Michaelis–Menten constant (KMapp) values of 1.00 μM, 1.11 μM and 1.25 μM, respectively. The corresponding value for the maximum response, IMAX was 0.02 A. The dissociation constant, KI, value evaluated from Dixon analysis of the paroxetine modulation data was estimated to be-0.02 μM while Cornish-Bowden analysis confirmed the competitive inhibitory characteristics of the enzyme.

Influence of the cytochrome P450 2D6 *10/*10 genotype on the pharmacokinetics of paroxetine in Japanese patients with major depressive disorder: a population pharmacokinetic analysis.

Although the reduced function of the cytochrome P450 2D6*10 (CYP2D6*10) allele is common among Asian populations, existing evidence does not support paroxetine therapy adjustments for patients who have the CYP2D6*10 allele. In this study, we attempted to evaluate the degree of the impact of different CYP2D6 genotypes on the pharmacokinetic (PK) variability of paroxetine in a Japanese population using a population PK approach. This retrospective study included 179 Japanese patients with major depressive disorder who were being treated with paroxetine. CYP2D6*1, *2, *5, *10, and *41 polymorphisms were observed. A total of 306 steady-state concentrations for paroxetine were collected from the patients. A nonlinear mixed-effects model identified the apparent Michaelis-Menten constant (Km) and the maximum velocity (Vmax) of paroxetine; the covariates included CYP2D6 genotypes, patient age, body weight, sex, and daily paroxetine dose. The allele frequencies of CYP2D6*1, *2, *5, *10, and *41 were 39.4, 14.5, 4.5, 41.1, and 0.6%, respectively. There was no poor metabolizer who had two nonfunctional CYP2D6*5 alleles. A one-compartment model showed that the apparent Km value was decreased by 20.6% in patients with the CYP2D6*10/*10 genotype in comparison with the other CYP2D6 genotypes. Female sex also influenced the apparent Km values. No PK parameters were affected by the presence of one CYP2D6*5 allele. Unexpectedly, elimination was accelerated in individuals with the CYP2D6*10/*10 genotype. Our results show that the presence of one CYP2D6*5 allele or that of any CYP2D6*10 allele may have no major effect on paroxetine PKs in the steady state.

Role of Carbon Nanostructured Platform in Electron Transfer of Flavin-Dependent Oxidases

Carbon nanostructures, such as carbon nanotubes (CNT) and graphene have gained considerable attention during the last years, especially due to their remarkable electronic and mechanical properties, which have made them extremely attractive for a wide range of sensing applications from structural materials to nanoelectronic components. The ability of carbon nanostructure-modified electrodes to promote electron transfer reactions has been documented in connection with important biomolecules [1]. Our goal was to study the influence of carbon nanostructured platform as an electrode material in facilitating the electron transfer between the enzyme molecule and the electrode surface in bioelectrocatalytic systems for oxidation reaction of glucose and lactate that is of potential utility for bioelectronic devices such as biosensors and biofuel cells. Our research focuses on the direct electrochemical performance of flavin-dependent oxidases such as glucose oxidase (GOx) and lactate oxidase (LOx) immobilized on 4-(pyrrole-1-yl) benzoic acid (PyBA) modified CNT (CNT/PyBA) and/or (for comparison) on hybrid material composed of electrochemically reduced graphene oxide (ERGO) and CNT/PyBA (ERGO/CNT/PyBA). GOx and LOx are enzymes containing tightly bound flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) redox centers, respectively, embedded deeply in the protein shell [2]. Modification of carbon nanotubes allows to obtain thin and organized films, causing a rapid and effective transfer of electrons between the active center of the biocatalyst (GOx or LOx) and the electrode surface. The presence of PyBA in our hybrid systems significantly improves their stability and introduces new functional groups that have great importance in the enzyme immobilization process onto the modified electrode [3]. Moreover, graphene oxide (GO) has a unique ability to form stable aqueous dispersions, highly bio-compatible and possesses good electrocatalytic properties. In addition, its reductive product graphene is a unique material with potential applications in diverse fields and one of the hottest materials of interest. It was demonstrated that it is possible to prepare a water dispersible GO/CNT hybrid material via non-covalent π-π stacking interactions [4]. Dispersing CNT via non-covalent approaches cannot depress its intrinsic electrical, mechanical, and optical properties. The incorporation of GO with CNT render this hybrid material as a versatile platform for the electrocatalytic applications. Conversely, the insulating property of GO caused by the aliphatic sp3 hybridized domain, limits its conductivity. Therefore for improving the conductance, it can be reduced to graphene either by chemical, thermal or electrochemical methods. Recent studies reveal that the electrochemical reduction of GO to graphene (ERGO) was appreciably improved after the incorporation of CNT. In this work stable immobilization and direct electron transfer of oxidase enzymes were achieved on the hybrid system modified glassy carbon electrode. The resulting electrode gave a well-defined redox peaks with a formal potential of about -458 mV for GOx and -446 mV for LOx (vs. Ag/AgCl) in 0.1 phosphate buffer solution pH=7.0. Furthermore, the method for detecting of glucose and/or lactate was proposed based on the decrease of oxygen caused by the enzyme-catalyzed reaction between enzyme and substrate. The low calculated apparent Michaelis-Menten constants (KM app) were 13.3 mM for GOx and 8.25 mM for LOx, implying high enzymatic activity and affinity of immobilized enzyme for glucose and lactate, respectively. The calculated heterogeneous electron transfer rate constants were twice higher due to the combined contribution of GO with CNT/PyBA. It can reasonably be expected that this observation might hold true for other noble carbon nanostructure-electroactive protein systems, providing a promising platform for the development of biosensors and biofuel cells.

Preparation of a ferrocene mediated bioanode for biofuel cells by MWCNTs, polyethylenimine and glutaraldehyde: glucose oxidase immobilization and characterization

AbstractFerrocene groups introduced polyethylenimine was adsorbed onto multi‐wall carbon nanotubes attached carbon cloth before crosslinking via glutaraldehyde to develop an enzyme electrode (anode) for biofuel cell applications. Glucose oxidase was covalently immobilized onto this electrode by leftover aldehyde groups on the surface. The apparent Michaelis constant of immobilized enzyme was measured as approximately fivefold higher than that of the free enzyme. Thermal stability of immobilized enzyme was improved as estimated. Activity half‐life of the immobilized glucose oxidase was determined as approximately 8 times longer than that of the free one; 2.4 mA/cm2 electrical current was obtained by using this electrode by forming a basic biofuel half‐cell. Copyright © 2016 Curtin University of Technology and John Wiley & Sons, Ltd.

Confining nanohybrid of CdTe quantum dots and cytochrome P450 2D6 in macroporous ordered siliceous foam for drug metabolism

In this work, a simple and accurate in vitro drug metabolism strategy was proposed by using macroporous ordered siliceous foam (MOSF) as a nanoreactor for confining the nanohybrid of CdTe quantum dots (QDs) and cytochrome P450 2D6 (CYP2D6). After CYP2D6 was coupled to QDs through the carbodiimide coupling chemistry, the resulting nanohybrid of CYP2D6/QDs exhibited fluorescence emission at 645nm, which could be used as a photocatalyst for drug metabolism. The energy level of CYP2D6 was located between the conduction band (CB) and valence band (VB) of QDs, which provided the possibility of the electron transferring from the CB of QDs to CYP2D6. Tramadol was chosen as the model substrate. Under the irradiation of a white-light, the photocurrent increased with the addition of tramadol in a wide linear range from 4.0μM to 100μM. The apparent Michaelis–Menten constant was measured to be 3.6μM. The high performance liquid chromatography coupled with mass spectrometry (HPLC–MS) confirmed the production of metabolite of o-desmethyltramadol. 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.

Effective immobilization of tyrosinase via enzyme catalytic polymerization of l-DOPA for highly sensitive phenol and atrazine sensing

The facile preparation of poly(l-DOPA)-tyrosinase (PDM-Tyr) composite and its application both in substrate (phenol) and inhibitor (atrazine) sensing is reported here for the first time. Effective immobilization of enzyme is realized via in-situ entrapping Tyr in poly(l-DOPA) (PDM), which is formed by Tyr catalytic polymerization of l-DOPA. The Tyr modified electrode is simply prepared by dipping the PDM-Tyr composite on an Au electrode and then covered by Nafion. The thus-prepared Tyr-immobilized electrode exhibits excellent performance superior to most Tyr-based electrochemical biosensors, the sensitivity to phenol is as high as 5122 μA mM−1 in the linear range of 10nM~1.25 μM, the apparent Michaelis-Menten constant (KMapp) determined as low as 3.13μM indicates strong substrate binding and high catalytic activity of the immobilized Tyr. The biosensor also works well in atrazine biosensing, with a linear detection range of 50ppb~30ppm and a low detection limit of 10ppb obtained. In addition, the biosensor shows excellent stability, precision, high sensitivity and fabrication simplicity.

Optimal design of amperometric biosensors applying multi-objective optimization and decision visualization

This paper presents a method combining mathematical modeling, multi-objective optimization and multi-dimensional visualization intended for the design and optimization of amperometric biosensors. An approach for optimizing the biosensor parameters is based on the availability of mathematical model of a catalytic biosensor (bioelectrode). A multi-objective visualization of trade-off solutions and Pareto optimal decisions is applied for the selection of the most favorable decision by a human expert when designing the biosensor. The proposed method is applied to the industrially relevant optimization of a glucose dehydrogenase-based amperometric biosensor utilizing the synergistic substrates conversion. The following three objectives were optimized: the apparent Michaelis constant, the output current and the enzyme amount.

Mechanisms of Enhanced Catalysis in Enzyme-DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis.

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme-DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9- and 2.4-fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4-aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems.

Amine-functionalized magnetic nanoparticles as robust support for immobilization of Lipase

Preparation of magnetic nanoparticles with controlled size and shape along with modulation of their surface properties via introduction of functional groups holds great prospect in the field of nanotechnology. Superparamagnetic, aqueous dispersible iron oxide nanoparticles (Fe3O4) with amine-functionalized surface were prepared through solvothermal method, using poly(ethylene imine) (PEI), ethanolamine (EA), and 2,2 ′-(ethylenedioxy)bis(ethylamine) (EDBE) as amine precursors. These aminated nanoparticles were used as support for the immobilization of lipase, an important industrial enzyme. Lipase was immobilized via glutaraldehyde coupling agent. These functionalized nanoparticles were characterized by XRD, FTIR, TEM, FESEM and VSM analysis. The maximum activity was obtained for the lipase immobilized on EDBE modified Fe3O4 nanoparticles. The lipase immobilized on EDBE-Fe3O4 depicted 83.9% relative activity with respect to the same amount of free lipase. Moreover, lipase immobilized on EDBE-Fe3O4 nanoparticles demonstrated good thermal and storage stability, and easy reusability. The kinetic parameters of lipase immobilized on EDBE-Fe3O4 were compared with those of free lipase and the apparent Michaelis-Menten constant of immobilized lipase was found to be nearly same as that of free lipase.

Characterization of β-glucosidase from Aspergillus terreus and its application in the hydrolysis of soybean isoflavones.

An extracellular β-glucosidase produced by Aspergillus terreus was identified, purified, characterized and was tested for the hydrolysis of soybean isoflavone. Matrix-assisted laser desorption/ionization with tandem time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) revealed the protein to be a member of the glycosyl hydrolase family 3 with an apparent molecular mass of about 120 kDa. The purified β-glucosidase showed optimal activity at pH 5.0 and 65 °C and was very stable at 50 °C. Moreover, the enzyme exhibited good stability over pH 3.0-8.0 and possessed high tolerance towards pepsin and trypsin. The kinetic parameters Km (apparent Michaelis-Menten constant) and Vmax (maximal reaction velocity) for p-nitrophenyl-β-D-glucopyranoside (pNPG) were 1.73 mmol/L and 42.37 U/mg, respectively. The Km and Vmax for cellobiose were 4.11 mmol/L and 5.7 U/mg, respectively. The enzyme efficiently converted isoflavone glycosides to aglycones, with a hydrolysis rate of 95.8% for daidzin, 86.7% for genistin, and 72.1% for glycitin. Meanwhile, the productivities were 1.14 mmol/(L·h) for daidzein, 0.72 mmol/(L·h) for genistein, and 0.19 mmol/(L·h) for glycitein. This is the first report on the application of A. terreus β-glucosidase for converting isoflavone glycosides to their aglycones in soybean products.

Mesoporous carbon-containing voltammetric biosensor for determination of tyramine in food products.

A voltammetric biosensor based on tyrosinase (TYR) was developed for determination of tyramine. Carbon material (multi-walled carbon nanotubes or mesoporous carbon CMK-3-type), polycationic polymer—i.e., poly(diallyldimethylammonium chloride) (PDDA), and Nafion were incorporated into titania dioxide sol (TiO2) to create an immobilization matrix. The features of the formed matrix were studied by scanning electron microscopy (SEM) and cyclic voltammetry (CV). The analytical performance of the developed biosensor was evaluated with respect to linear range, sensitivity, limit of detection, long-term stability, repeatability, and reproducibility. The biosensor exhibited electrocatalytic activity toward tyramine oxidation within a linear range from 6 to 130 μM, high sensitivity of 486 μA mM−1 cm−2, and limit of detection of 1.5 μM. The apparent Michaelis–Menten constant was calculated to be 66.0 μM indicating a high biological affinity of the developed biosensor for tyramine. Furthermore, its usefulness in determination of tyramine in food product samples was also verified.Graphical abstractDifferent food samples were analyzed to determine tyramine using biosensor based on tyrosinaseElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9612-y) contains supplementary material, which is available to authorized users.

Electrochemical, continuous-flow determination of p-benzoquinone on a gold nanoparticles poly(propylene-co-imidazole) modified gold electrode

ABSTRACTA novel continuous flow biosensor based on gold nanoparticles and poly(propylene-co-imidazole) was developed for the online determination of p-benzoquinone. The amperometric response was measured as a function of p-benzoquinone concentration at an applied potential of −50 mV. The hydrogen peroxide concentration was optimized and fixed at 1 mM in samples. The mass transfer resistance of the copolymer film was minimized, and the flow cell was regenerated quickly at 1 mL/min. The resulting device provided good analytical performance based on a linear dynamic range from 5–100 µM, a short response time of 3 s, a detection limit of 3.3 µM, excellent repeatability with a relative standard deviation of 0.82%, long-term stability of 95% after four weeks, and an accuracy of 105%. The gold nanoparticles enhanced the electron transfer rate on the electrode. The apparent Michaelis-Menten constant was 4 mM, showing that the enzyme retained catalytic specificity and provided high activity for p-benzoquinone.