Function Of Substrate Concentration Research Articles

Synergistic effects of heterotrophic and phototrophic metabolism for Haematococcus lacustris grown under mixotrophic conditions

AbstractMixotrophic cultivation of Haematococcus lacustris is one of the most promising strategies to produce natural astaxanthin. During mixotrophic growth, microalgae assimilate and metabolize organic carbon in addition to photosynthetic growth, resulting in increased biomass productivity. Several studies have evaluated the effect of different organic carbon sources on mixotrophic growth in various microalgae species. However, knowledge of detailed growth kinetics as a function of substrate concentration and light intensity is lacking. In this study, the growth kinetics of H. lacustris using four different carbon sources and the effect of light under mixotrophic and photoautotrophic conditions are described. Mixotrophic cultivation showed significant differences in respect to applied substrate and achieved maximum specific growth rates of 0.91 ± 0.13, 0.19 ± 0.05, 0.36 ± 0.05, and 0.23 ± 0.05 day−1, for acetate, methanol, glucose, and glycerol, respectively. Optimal growth at mixotrophic conditions using acetate was 1.8 times higher than the sum of hetero- and photoautotrophic growth. Furthermore, the optimum light intensity was 1.3 times higher for mixotrophic than for autotrophic growth. Thus, mixotrophy increases light intensity tolerance. These results indicate a strong interconnection between carbon metabolism and photosynthetic activity and lay the foundation for more detailed mathematical models describing the mixotrophic growth of H. lacustris. Graphical Abstract

Nanoarchitectonic approaches for measuring the catalytic behavior of a membrane anchored enzyme. From Langmuir-Blodgett to a novel Langmuir-Schaefer based nanofilm building device

Self-organized lipid monolayers at the air-water interface (Langmuir films, LF) are commonly used for measuring the catalytic properties of membrane-bound enzymes. This methodology allows to provide a consistent flat topography molecular density, packing defects and thickness. The aim of the present work was to show the methodological advantages of using the horizontal transfer method (Langmuir-Schaefer) with respect to the vertical transfer method (Langmuir-Blodgett) when mounting a device to measure catalytic activity of membrane enzymes. Based on the results obtained we can conclude that it is possible to prepare stable Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) films from Bovine Erythrocyte Membranes (BEM) preserving the catalytic activity of its native Acetylcholinesterase (BEA). In comparison, the LS films showed Vmax values more similar to the enzyme present in the vesicles of natural membranes. In addition, it was much easier to produce large amounts of transferred areas with the horizontal transfer methodology. It was possible to decrease the time required to mount an assay with numerous activity points, such as building activity curves as a function of substrate concentration. The present results show that LSBEM provides a proof of concept for the development of biosensors based on transferred purified membrane for the screening of new products acting on an enzyme embedded on its natural milieu. In the case of BEA, the application of these enzymatic sensors could have medical interest, providing drug screening tools for the treatment of Alzheimer's disease.

Physical limitations induced by a medium based on wheat bran for the production of biopesticides by Bacillus thuringiensis spp kurstaki

Biopesticides, vectors for the development of the bioeconomy, represent a realistic alternative to petroleum-based chemical pesticides and their known harmfulness to humans and the environment. Bacillus thuringiensis is a gram-positive, spore-forming bacterium used for the production of biocontrol agents in agriculture. This insecticidal activity is mainly due to the production of parasporal δ-endotoxins consisting of an assembly of crystalline proteins called Cry. The European project IPM-4-Citrus (MSCA RISE, No. 734921, 2017-2023) aims to optimise these bioproductions from 3 strains of Bacillus thuringiensis subsp kurstaki (Lip, BLB1 and HD1) in a complex medium (wheat bran). The biochemical composition and nutritional limitations induced by the medium were identified. The physical properties of these heterogeneous suspensions are necessary for the definition of operating conditions favourable to scale-up. In this study, the morpho-granulometry of substrates, the ability to separate solid-liquid suspension and to clarify fermentation broth, and their rheological behaviour were studied as a function of substrate concentration and granulometry.

Kinetic Study and Modeling of Wild-Type and Recombinant Broccoli Myrosinase Produced in E. coli and S. cerevisiae as a Function of Substrate Concentration, Temperature, and pH

The myrosinase enzyme hydrolyzes glucosinolates, among which is glucoraphanin, the precursor of the anticancer isothiocyanate sulforaphane (SFN). The main source of glucoraphanin is Brassicaceae; however, its natural concentration is relatively low, limiting the availability of SFN. An option to obtain SFN is its exogenous production, through enzymatic processes and under controlled conditions, allowing complete conversion of glucoraphanin to SFN. We characterized the kinetics of wild-type (BMYR) and recombinant broccoli myrosinases produced in E. coli (EMYR) and S. cerevisiae (SMYR) in terms of the reaction conditions. Kinetics was adjusted using empirical and mechanistic models that describe reaction rate as a function of substrate concentration, temperature, and pH, resulting in R2 values higher than 90%. EMYR kinetics differed significantly from those of BMYR and SMYR probably due to the absence of glycosylations in the enzyme produced in E. coli. BMYR and SMYR were subjected to substrate inhibition but followed different kinetic mechanisms attributed to different glycosylation patterns. EMYR (inactivation Ea = 76.1 kJ/mol) was more thermolabile than BMYR and SMYR. BMYR showed the highest thermostability (inactivation Ea = 52.8 kJ/mol). BMYR and EMYR showed similar behavior regarding pH, with similar pK1 (3.4 and 3.1, respectively) and pK2 (5.4 and 5.0, respectively), but differed considerably from SMYR.

A guide to enzyme kinetics in early drug discovery.

Drugs interact with their target of interest to bring about the desired phenotypic outcome that results in disease alleviation. Traditionally, most lead optimization exercises were driven by affinity measures (like IC50 ) to inform structure-activity relationship (SAR)-guided medicinal chemistry. However, an IC50 value is a thermodynamic estimate measured under equilibrium conditions that can vary as a function of substrate concentration and/or time (the latter especially for nonequilibrium modalities). Further, like other thermodynamic estimates, it is a state-function that is indifferent to the path traversed from the initial state to the final state. This can be a cause for concern in drug discovery given the predominance of nonequilibrium interactions and the open thermodynamic nature of the human system. Under such situations, employing rates along with equilibrium constants (or IC50 values) would be far more relevant to capture the time evolution of the small molecule's interaction with the target of interest. These rates are generally typified by the rate of association, rate of dissociation and the residence time of the small molecule on the target (target occupancy). These parameters, when combined with the concept of target vulnerability, therapeutic window, pharmacokinetic profile of the small molecule, estimates of endogenous ligand and target turnover, will shed critical insights into the kinetics and dynamics of a small molecule's interaction with the protein, and allow realistic modelling of the system to enable optimizations and dosing decisions. With that aim, this guide will attempt to introduce the traditional role of mechanistic enzymology within drug discovery and emphasize the importance of kinetics in guiding SAR-based optimizations. It will also present initial ideas on how kinetic investigation should be positioned relative to the temporal span of a drug-discovery pipeline to leverage maximal utility from the investment in time and effort.

Assessment of substrate load and process pH for bioethanol production – Development of a kinetic model

In the present work, four levels of initial substrate concentration (2%, 4%, 7%, and 10% w/v glucose), and six pH values (4.0, 4.5, 5.0, 5.5, 6.0, and 6.5) at 2% substrate load were used to assess their impact on bioethanol production from Saccharomyces cerevisiae, in order to develop a kinetic model. A synthetic medium, containing glucose as the only carbon and energy source was used as the fermentation substrate. An innovative kinetic model was developed to fit the obtained data since several bioethanol production inhibition models reported in literature failed to sufficiently describe the experimental results. To this end, the logistic model was used to express the inhibition of biomass growth and the Monod model to describe the limitation of growth due to substrate depletion. In addition, Smax was imported as the parameter that affects the yield of ethanol production rate to biomass growth rate (Ye/x), as a function of substrate concentration. Two more parameters were used to describe substrate (glucose) consumption for maintenance purposes (Kcm) and simultaneous bioethanol production (Ycm) during the biomass stationary phase, where bioethanol is still being produced. The proposed model showed a high coefficient of correlation, R2 = 0.9694. Both the yeast growth and bioethanol production were not substantially affected by the pH, within the range of pH values tested in this study.

An antibody-based amperometric biosensor for 20S proteasome activity and inhibitor screening.

The 20S proteasome enzyme complex is involved in the proteolytic degradation of misfolded and oxidatively damaged proteins and is a focus of medical research for the development of compounds with pharmaceutical properties, which are active in cancer cells and/or neurodegenerative diseases. The present study aims to develop a biosensor for investigating the 20S proteasome activity and inhibition by means of electrochemical methods. The 20S proteasome is best immobilized at the electrode surface through bio-affinity interactions with antibodies that target different subunits on the 20S proteasome, enabling the investigation of the effect of an enzyme's orientation on biosensor response. The enzymatic activity is analyzed by fixed potential amperometry with the highest sensitivity of 24 μA cm-2 mM-1 and a LOD of 0.4 μM. The detection principle involves the oxidation of an electroactive probe that is released from the enzyme's substrates upon proteolysis. The most sensitive biosensor is then used to study the multicatalytic activity of the 20S proteasome, i.e. the caspase-, trypsin- and chymotrypsin-like activity, by analyzing the biosensor's sensitivity towards different substrates. The behavior of the immobilized 20S proteasome is investigated as a function of substrate concentration. The kinetic parameters are derived and compared with those obtained when the enzyme was free in solution, with K0.5 values being one to two orders of magnitude lower in the present case. Two 20S inhibitors, epoxomicin and bortezomib, are investigated by analyzing their influence on the 20S biosensor response. The proposed analytical method for proteasome activity and inhibitor screening has the main advantage of being cost-effective compared to the ones typically employed.

Shear viscosity of two-state enzyme solutions.

We discuss the shear viscosity of a Newtonian solution of catalytic enzymes and substrate molecules. The enzyme is modeled as a two-state dimer consisting of two spherical domains connected with an elastic spring. The enzymatic conformational dynamics is induced by the substrate binding and such a process is represented by an additional elastic spring. Employing the Boltzmann distribution weighted by the waiting times of enzymatic species in each catalytic cycle, we obtain the shear viscosity of dilute enzyme solutions as a function of substrate concentration and its physical properties. The substrate affinity distinguishes between fast and slow enzymes, and the corresponding viscosity expressions are obtained. Furthermore, we connect the obtained viscosity with the diffusion coefficient of a tracer particle in enzyme solutions.

Microbial fuel cells: A fast converging dynamic model for assessing system performance based on bioanode kinetics

In this work, a dynamic computational model is developed for a single chamber microbial fuel cell (MFC), consisting of a bio-catalyzed anode and an air-cathode. Electron transfer from the biomass to the anode is assumed to take place via intracellular mediators as they undergo transformation between reduced and oxidized forms. A two-population model is used to describe the biofilm at the anode and the MFC current is calculated based on charge transfer and Ohm's law, while assuming a non-limiting cathode reaction rate. The open circuit voltage and the internal resistance of the cell are expressed as a function of substrate concentration. The effect of operating parameters such as the initial substrate (COD) concentration and external resistance, on the Coulombic efficiency, COD removal rate and power density of the MFC system is studied. Even with the simple formulation, model predictions were found to be in agreement with observed trends in experimental studies. This model can be used as a convenient tool for performing detailed parametric analysis of a range of parameters and assist in process optimization.

Modeling and Parameter Identification for a Biofilm in a Microbial Fuel Cell

AbstractA dynamic physical model of a microbial fuel cell (MFC) anode is presented and parameterized. It describes the evolution of current density and biofilm mass over time as a function of substrate concentration. The model is particularly useful for process monitoring or control purposes because it reproduces the dynamics of the MFC anode and contains comparatively few parameters. Parameters are identified using data from the response of the MFC to a substrate concentration pulse. Theoretical and practical identifiability of the parameters is evaluated, and parameter confidence intervals are determined.

Functional effects of active site mutations in NAD+-dependent formate dehydrogenases on transformation of hydrogen carbonate to formate.

Conversion of hydrogen carbonate to formate by mutants of Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) formate dehydrogenases (FDHs) was studied. Hydrogen carbonate is not the primary substrate for the hydride transfer reaction in FDHs. The chosen mutations were selected so that enzyme activity could remain at an adequate level. In CtFDH, the mutation Asn120Cys in the active site inactivated the enzyme for formate (oxidation) but increased the specific activity for hydrogen carbonate (reduction) as a function of substrate concentration. The mutation Asn120Cys in CtFDH increased 6.5-fold the KM, indicating that substrate binding was weakened. A 6.5-fold increase of kcat compensated the lower affinity suggesting that product release was improved. The corresponding mutation Asn119Cys in CmFDH inactivated the enzyme for both substrates. Molecular dynamics simulations indicated that the active site dimensions change differently with different substrates after mutations, and in the mutant Asn120Cys of CtFDH, hydrogen carbonate adopted better reactive position than formate. With hydrogen carbonate, the active site enlarged enough for two hydrogen carbonate molecules to be placed there. The change of Asn119 to bulky Tyr or His in CmFDH requires changes in the active site to accommodate the substrate; activity with formate was retained but not with hydrogen carbonate. This study showed that the active site of FDHs can be modified radically, which gives possibilities for further enzyme engineering to improve the reaction with hydrogen carbonate or carbon dioxide for enzymatic fixing of carbon dioxide.

Generic Schemes for Single-Molecule Kinetics. 2: Information Content of the Poisson Indicator.

Recently, we described a pathway analysis technique (paper 1) for analyzing generic schemes for single-molecule kinetics based upon the first-passage time distribution. Here, we employ this method to derive expressions for the Poisson indicator, a normalized measure of stochastic variation (essentially equivalent to the Fano factor and Mandel's Q parameter), for various renewal (i.e., memoryless) enzymatic reactions. We examine its dependence on substrate concentration, without assuming all steps follow Poissonian kinetics. Based upon fitting to the functional forms of the first two waiting time moments, we show that, to second order, the non-Poissonian kinetics are generally underdetermined but can be specified in certain scenarios. For an enzymatic reaction with an arbitrary intermediate topology, we identify a generic minimum of the Poisson indicator as a function of substrate concentration, which can be used to tune substrate concentration to the stochastic fluctuations and to estimate the largest number of underlying consecutive links in a turnover cycle. We identify a local maximum of the Poisson indicator (with respect to substrate concentration) for a renewal process as a signature of competitive binding, either between a substrate and an inhibitor or between multiple substrates. Our analysis explores the rich connections between Poisson indicator measurements and microscopic kinetic mechanisms.

An eco-friendly approach to control Oxya velox infestation: Mangifera indica exoglucanase and endoglucanase cellulose ingestion inhibition activity

Abstract Oxya velox (Grasshopper ) is considered to be one of the damaging pests. The Mangifera indica ( M. indica ) crude enzyme extracts (5 g powder/20 mL of water, ethyl acetate, butanol and chloroform separately) were evaluated for their cellulose ingestion inhibition activity in O. velox . The extracts activities were studied as a function of substrate concentration, pH and temperature. Dinitrosalisalic acid (DNSA) method was adopted to study the kinetic of both endoglucanase and exoglucanase enzymes. The K m values of both enzymes were recorded to be 2 mg/mL and 0.92 mg/mL, respectively, which revealed greater affinity of enzymes for the substrate. All solvents showed considerable enzymes inhibition activity and variation among solvents was insignificant. Results revealed that the cellulose digestion by O. velox enzymes could possibly be inhibited using M. indica extracts.

Application of the median method to estimate the kinetic constants of the substrate uncompetitive inhibition equation

In 1974, Eisenthal and Cornish-Bowden published the direct linear plot method, which used the median to estimate the Vmax and Km from a set of initial rates as a function of substrate concentrations. The robustness of this non-parametric method was clearly demonstrated by comparing it with the least-squares method. The authors commented that the method cannot readily be generalized to equations of more than two parameters. Unfortunately, this comment has been misread by other authors. Comments such as “this method cannot be extended directly to equations with more than two parameters” were found in some publications. In addition, recently, the most drastic comment was published: “this method cannot be applied for the analysis of substrate inhibition.” Given all of these presumptions, we have been motivated to publish a demonstration of the contrary: the median method can be applied to more than two-parameter equations, using as an example, the substrate uncompetitive inhibition equation. A computer algorithm was written to evaluate the effect of simulated experimental error of the initial rates on the estimation of Vmax, Km and KS. The error was assigned to different points of the experimental design. Four different KS/Km ratios were analyzed with the values 10, 100, 1000 and 10,000. The results indicated that the least-squares method was slightly better than the median method in terms of accuracy and variance. However, the presence of outliers affected the estimation of kinetic constants using the least-squares method more severely than the median method. The estimation of KS using the median method to estimate 1/KS was much better than the direct estimation of KS, causing a negative effect of non-linearity of KS in the kinetic equation. Considering that the median method is free from the assumptions of the least-squares method and the arbitrary assumptions implicit in the linearization methods to estimate the kinetic constants Vmax, Km and KS from the substrate uncompetitive inhibition equation, the median method is highly superior to all published methods, including the non-linear regression by least squares. We concluded that the median method can be applied to the substrate uncompetitive inhibition equation and other equations with more than two parameters. In addition, as we can project, the median method is the most reliable and robust method for the estimation of kinetic parameters from enzyme kinetic models.

Granular biomass floatation: A simple kinetic/stoichiometric explanation

Floatation events are commonly observed in anammox, denitrifying and anaerobic granular systems mostly subjected to overloading conditions. Although several operational strategies have been proposed to avoid floatation of granular biomass, until now, there is no consensus about the conditions responsible for this phenomenon. In the present study, a simple explanation based on kinetic and stoichiometric principles defining the aforementioned processes is provided. The operational zones corresponding to evaluated parameters where risk of floatation exists are defined as a function of substrate concentration in the bulk liquid and the radius of the granule. Moreover, the possible control of biomass floatation by changing the operating temperature was analyzed. Defined operational zones and profiles fit data reported in literature for granular biomass floatation events. From the study the most influencing parameter on floatation occurrence has been identified as the substrate concentration in the bulk media.

Mitochondrial substrate specificity in beetle flight muscle: assessing respiratory oxygen flux in small samples from Dermestes maculatus and Tenebrio molitor

AbstractIn the present study, the permeabilized fibre approach is adapted to investigate substrate utilization patterns in the flight muscle mitochondria of Dermestes maculatus (Coleoptera: Dermestidae; a carrion scavenger beetle) and Tenebrio molitor (Coleoptera: Tenebrionidae; a phytophagous scavenger beetle). Respiration in saponin‐permeabilized fibres is measured during titration of palmitoyl‐carnitine (Palm‐C), pyruvate (Pyr) or l‐glycerol 3‐phosphate (G3‐P). Michaelis–Menten‐type enzyme kinetics for oxygen consumption are observed as a function of substrate concentration in Pyr and G3‐P, from which substrate‐specific apparent Km (sensitivity) and Vmax (capacity) are determined. Compared with D. maculatus, the apparent Km in T. molitor is lower (P < 0.001) for Pyr, and Vmax is greater for G3‐P (P < 0.001). In D. maculatus, the apparent Km for G3‐P is greater (P < 0.001), and respiratory Vmax is lower (P < 0.001), than kinetics for Pyr. Robust respiration with l‐proline (Pro) is also observed in both beetle species tested; however, it is over 2.5‐fold greater in D. maculatus than T. molitor (P < 0.05). These results demonstrate that respiration in beetle flight muscle mitochondria can be assessed in small samples (i.e. at the individual beetle level) using the approach adapted for the present study. The results of the present study also highlight the substrate oxidative capacity patterns in both D. maculatus and T. molitor, which rank Palm‐C < G3‐P < Pyr < Pro.

Using simple models to describe the kinetics of growth, glucose consumption, and monoclonal antibody formation in naive and infliximab producer CHO cells.

Despite their practical and commercial relevance, there are few reports on the kinetics of growth and production of Chinese hamster ovary (CHO) cells—the most frequently used host for the industrial production of therapeutic proteins. We characterize the kinetics of cell growth, substrate consumption, and product formation in naive and monoclonal antibody (mAb) producing recombinant CHO cells. Culture experiments were performed in 125 mL shake flasks on commercial culture medium (CD Opti CHO™ Invitrogen, Carlsbad, CA, USA) diluted to different glucose concentrations (1.2–4.8 g/L). The time evolution of cell, glucose, lactic acid concentration and monoclonal antibody concentrations was monitored on a daily basis for mAb-producing cultures and their naive counterparts. The time series were differentiated to calculate the corresponding kinetic rates (rx = d[X]/dt; rs = d[S]/dt; rp = d[mAb]/dt). Results showed that these cell lines could be modeled by Monod-like kinetics if a threshold substrate concentration value of [S]t = 0.58 g/L (for recombinant cells) and [S]t = 0.96 g/L (for naïve cells), below which growth is not observed, was considered. A set of values for μmax, and Ks was determined for naive and recombinant cell cultures cultured at 33 and 37 °C. The yield coefficient (Yx/s) was observed to be a function of substrate concentration, with values in the range of 0.27–1.08 × 107 cell/mL and 0.72–2.79 × 106 cells/mL for naive and recombinant cultures, respectively. The kinetics of mAb production can be described by a Luedeking–Piret model (d[mAb]/dt = αd[X]/dt + β[X]) with values of α = 7.65 × 10−7 µg/cell and β = 7.68 × 10−8 µg/cell/h for cultures conducted in batch-agitated flasks and batch and instrumented bioreactors operated in batch and fed-batch mode.

Functional Investigations of Thromboxane Synthase (CYP5A1) in Lipid Bilayers of Nanodiscs

CYP5A1 is a membrane-associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH2 ) into thromboxane A2 (TXA2 ), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER-bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug-metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small-molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.

Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4 reconstituted in Nanodiscs. We discovered that the “oxidase” uncoupling pathway is also operating in the substrate free form of the enzyme with rate of this pathway substantially increasing with the first substrate binding event. Surprisingly, a large fraction of the reducing equivalents used by the P450 system is wasted in this oxidase pathway. In addition, the overall coupling with testosterone and bromocryptine as substrates is significantly higher in the presence of anionic lipids, which is attributed to the changes in the redox potential of CYP3A4 and reductase.

A novel amperometric phenol biosensor based on immobilized HRP on poly(glycidylmethacrylate)-grafted iron oxide nanoparticles for the determination of phenol derivatives

Abstract A new type of amperometric phenol biosensor was fabricated for the determination of phenol and its derivatives such as, phenol, catechol, p -cresol, 2-aminophenol and pyrogallol. The telomere of poly(glycidylmethacrylate) (PGMA) with a trimethoxysilyl terminal group was synthesized by telomerization of glycidylmethacrylate. Iron oxide nanoparticles were coated with telomere of poly(glycidylmethacrylate) in order to obtain good enzyme immobilization platform. PGMA covered Fe 3 O 4 was covalently attached Au electrode surface and horseradish peroxidase (HRP) was immobilized on nanoparticles covered surface, respectively. The telomere, nanoparticles and modified electrodes were characterized by Fourier transform infrared (FT-IR), X-ray diffraction (XRD), transmission electron microscopy (TEM) and cyclic voltammetry (CV) before and after immobilization of HRP. Amperometric response was measured as a function of substrate concentration, at a fixed potential of −0.50 V vs. Ag/AgCl in phosphate-buffered saline (pH 7.5). Analytical parameters for the fabricated phenol biosensor were obtained from the calibration curves. The basic features (optimum pH, optimum temperature, reusability, and storage stability) of the enzyme electrode were determined.