Sample records for henri-michaelis-menten enzyme kinetics

  1. Practical steady-state enzyme kinetics. (United States)

    Lorsch, Jon R


    Enzymes are key components of most biological processes. Characterization of enzymes is therefore frequently required during the study of biological systems. Steady-state kinetics provides a simple and rapid means of assessing the substrate specificity of an enzyme. When combined with site-directed mutagenesis (see Site-Directed Mutagenesis), it can be used to probe the roles of particular amino acids in the enzyme in substrate recognition and catalysis. Effects of interaction partners and posttranslational modifications can also be assessed using steady-state kinetics. This overview explains the general principles of steady-state enzyme kinetics experiments in a practical, rather than theoretical, way. Any biochemistry textbook will have a section on the theory of Michaelis-Menten kinetics, including derivations of the relevant equations. No specific enzymatic assay is described here, although a method for monitoring product formation or substrate consumption over time (an assay) is required to perform the experiments described. © 2014 Elsevier Inc. All rights reserved.

  2. A Comprehensive Enzyme Kinetic Exercise for Biochemistry (United States)

    Barton, Janice S.


    This article describes a comprehensive treatment of experimental enzyme kinetics strongly coupled to electronic data acquisition and use of spreadsheets to organize data and perform linear and nonlinear least-squares analyses, all in a manner that promotes development of important reasoning skills. Kinetic parameters are obtained for the stable…

  3. A stochastic model of enzyme kinetics (United States)

    Stefanini, Marianne; Newman, Timothy; McKane, Alan


    Enzyme kinetics is generally modeled by deterministic rate equations, and in the simplest case leads to the well-known Michaelis-Menten equation. It is plausible that stochastic effects will play an important role at low enzyme concentrations. We have addressed this by constructing a simple stochastic model which can be exactly solved in the steady-state. Throughout a wide range of parameter values Michaelis-Menten dynamics is replaced by a new and simple theoretical result.

  4. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.


    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  5. Extracellular enzyme kinetics scale with resource availability (United States)

    Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.


    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

  6. Kinetics of enzyme action: essential principles for drug hunters

    National Research Council Canada - National Science Library

    Stein, Ross L


    ... field. Beginning with the most basic principles pertaining to simple, one-substrate enzyme reactions and their inhibitors, and progressing to a thorough treatment of two-substrate enzymes, Kinetics of Enzyme Action...

  7. Thermodynamic Activity-Based Progress Curve Analysis in Enzyme Kinetics. (United States)

    Pleiss, Jürgen


    Macrokinetic Michaelis-Menten models based on thermodynamic activity provide insights into enzyme kinetics because they separate substrate-enzyme from substrate-solvent interactions. Kinetic parameters are estimated from experimental progress curves of enzyme-catalyzed reactions. Three pitfalls are discussed: deviations between thermodynamic and concentration-based models, product effects on the substrate activity coefficient, and product inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Internal Diffusion-Controlled Enzyme Reaction: The Acetylcholinesterase Kinetics. (United States)

    Lee, Sangyun; Kim, Ji-Hyun; Lee, Sangyoub


    Acetylcholinesterase is an enzyme with a very high turnover rate; it quenches the neurotransmitter, acetylcholine, at the synapse. We have investigated the kinetics of the enzyme reaction by calculating the diffusion rate of the substrate molecule along an active site channel inside the enzyme from atomic-level molecular dynamics simulations. In contrast to the previous works, we have found that the internal substrate diffusion is the determinant of the acetylcholinesterase kinetics in the low substrate concentration limit. Our estimate of the overall bimolecular reaction rate constant for the enzyme is in good agreement with the experimental data. In addition, the present calculation provides a reasonable explanation for the effects of the ionic strength of solution and the mutation of surface residues of the enzyme. The study suggests that internal diffusion of the substrate could be a key factor in understanding the kinetics of enzymes of similar characteristics.

  9. Stochastic theory of interfacial enzyme kinetics: A kinetic Monte Carlo study

    International Nuclear Information System (INIS)

    Das, Biswajit; Gangopadhyay, Gautam


    Graphical abstract: Stochastic theory of interfacial enzyme kinetics is formulated. Numerical results of macroscopic phenomenon of lag-burst kinetics is obtained by using a kinetic Monte Carlo approach to single enzyme activity. Highlights: ► An enzyme is attached with the fluid state phospholipid molecules on the Langmuir monolayer. ► Through the diffusion, the enzyme molecule reaches the gel–fluid interface. ► After hydrolysing a phospholipid molecule it predominantly leaves the surface in the lag phase. ► The enzyme is strictly attached to the surface with scooting mode of motion and the burst phase appears. - Abstract: In the spirit of Gillespie’s stochastic approach we have formulated a theory to explore the advancement of the interfacial enzyme kinetics at the single enzyme level which is ultimately utilized to obtain the ensemble average macroscopic feature, lag-burst kinetics. We have provided a theory of the transition from the lag phase to the burst phase kinetics by considering the gradual development of electrostatic interaction among the positively charged enzyme and negatively charged product molecules deposited on the phospholipid surface. It is shown that the different diffusion time scales of the enzyme over the fluid and product regions are responsible for the memory effect in the correlation of successive turnover events of the hopping mode in the single trajectory analysis which again is reflected on the non-Gaussian distribution of turnover times on the macroscopic kinetics in the lag phase unlike the burst phase kinetics.

  10. Current IUBMB recommendations on enzyme nomenclature and kinetics

    Directory of Open Access Journals (Sweden)

    Athel Cornish-Bowden


    Full Text Available The International Union of Biochemistry (IUB, now IUBMB prepared recommendations for describing the kinetic behaviour of enzymes in 1981. Despite the more than 30 years that have passed since these have not subsequently been revised, though in various respects they do not adequately cover current needs. The IUBMB is also responsible for recommendations on the naming and classification of enzymes. In contrast to the case of kinetics, these recommendations are kept continuously up to date.

  11. Reexamining Michaelis-Menten Enzyme Kinetics for Xanthine Oxidase (United States)

    Bassingthwaighte, James B.; Chinn, Tamara M.


    Abbreviated expressions for enzyme kinetic expressions, such as the Michaelis-Menten (M-M) equations, are based on the premise that enzyme concentrations are low compared with those of the substrate and product. When one does progress experiments, where the solute is consumed during conversion to form a series of products, the idealized conditions…

  12. An Inverse Michaelis–Menten Approach for Interfacial Enzyme Kinetics

    DEFF Research Database (Denmark)

    Kari, Jeppe; Andersen, Morten; Borch, Kim


    Interfacial enzyme reactions are ubiquitous both in vivo and in technical applications, but analysis of their kinetics remains controversial. In particular, it is unclear whether conventional Michaelis–Menten theory, which requires a large excess of substrate, can be applied. Here, an extensive...... experimental study of the enzymatic hydrolysis of insoluble cellulose indeed showed that the conventional approach had a limited applicability. Instead we argue that, unlike bulk reactions, interfacial enzyme catalysis may reach a steady-state condition in the opposite experimental limit, where...... for kinetic analyses of interfacial enzyme reactions and that its analogy to established theory provides a bridge to the accumulated understanding of steady-state enzyme kinetics. Finally, we show that the ratio of parameters from conventional and inverted Michaelis–Menten analysis reveals the density...

  13. A century of enzyme kinetic analysis, 1913 to 2013. (United States)

    Johnson, Kenneth A


    This review traces the history and logical progression of methods for quantitative analysis of enzyme kinetics from the 1913 Michaelis and Menten paper to the application of modern computational methods today. Following a brief review of methods for fitting steady state kinetic data, modern methods are highlighted for fitting full progress curve kinetics based upon numerical integration of rate equations, including a re-analysis of the original Michaelis-Menten full time course kinetic data. Finally, several illustrations of modern transient state kinetic methods of analysis are shown which enable the elucidation of reactions occurring at the active sites of enzymes in order to relate structure and function. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  14. Enzyme Kinetics? Elementary, my dear 3 -8 ...

    Indian Academy of Sciences (India)

    research interests are in the areas of protein- ... rate constant for the formation of products, k3 is significantly of some enzymes. ... tissue at different stages of development. .... represent the only values of Km and V max that satisfy all of the sets.

  15. Transition state theory for enzyme kinetics (United States)

    Truhlar, Donald G.


    This article is an essay that discusses the concepts underlying the application of modern transition state theory to reactions in enzymes. Issues covered include the potential of mean force, the quantization of vibrations, the free energy of activation, and transmission coefficients to account for nonequilibrium effect, recrossing, and tunneling. PMID:26008760

  16. Bioethanol from lignocellulose - pretreatment, enzyme immobilization and hydrolysis kinetics

    DEFF Research Database (Denmark)

    Tsai, Chien Tai

    , the cost of enzyme is still the bottle neck, re-using the enzyme is apossible way to reduce the input of enzyme in the process. In the point view of engineering, the prediction of enzymatic hydrolysis kinetics under different substrate loading, enzyme combination is usful for process design. Therefore...... lignocellulose is the required high cellulase enzyme dosages that increase the processing costs. One method to decrease the enzyme dosage is to re-use BG, which hydrolyze the soluble substrate cellobiose. Based on the hypothesis that immobilized BG can be re-used, how many times the enzyme could be recycled...... liquid and pretreatment time can be reduced, the influence of substrate concentration, pretreatment time and temperature were investigated and optimized. Pretreatment of barley straw by [EMIM]Ac, correlative models were constructed using 3 different pretreatment parameters (temperature, time...

  17. Mesoscopic dynamics of diffusion-influenced enzyme kinetics. (United States)

    Chen, Jiang-Xing; Kapral, Raymond


    A particle-based mesoscopic model for enzyme kinetics is constructed and used to investigate the influence of diffusion on the reactive dynamics. Enzymes and enzyme-substrate complexes are modeled as finite-size soft spherical particles, while substrate, product, and solvent molecules are point particles. The system is evolved using a hybrid molecular dynamics-multiparticle collision dynamics scheme. Both the nonreactive and reactive dynamics are constructed to satisfy mass, momentum, and energy conservation laws, and reversible reaction steps satisfy detailed balance. Hydrodynamic interactions among the enzymes and complexes are automatically accounted for in the dynamics. Diffusion manifests itself in various ways, notably in power-law behavior in the evolution of the species concentrations. In accord with earlier investigations, regimes where the product production rate exhibits either monotonic or nonmonotonic behavior as a function of time are found. In addition, the species concentrations display both t(-1/2) and t(-3/2) power-law behavior, depending on the dynamical regime under investigation. For high enzyme volume fractions, cooperative effects influence the enzyme kinetics. The time dependent rate coefficient determined from the mass action rate law is computed and shown to depend on the enzyme concentration. Lifetime distributions of substrate molecules newly released in complex dissociation events are determined and shown to have either a power-law form for rebinding to the same enzyme from which they were released or an exponential form for rebinding to different enzymes. The model can be used and extended to explore a variety of issues related concentration effects and diffusion on enzyme kinetics.

  18. Mesoscopic dynamics of diffusion-influenced enzyme kinetics (United States)

    Chen, Jiang-Xing; Kapral, Raymond


    A particle-based mesoscopic model for enzyme kinetics is constructed and used to investigate the influence of diffusion on the reactive dynamics. Enzymes and enzyme-substrate complexes are modeled as finite-size soft spherical particles, while substrate, product, and solvent molecules are point particles. The system is evolved using a hybrid molecular dynamics-multiparticle collision dynamics scheme. Both the nonreactive and reactive dynamics are constructed to satisfy mass, momentum, and energy conservation laws, and reversible reaction steps satisfy detailed balance. Hydrodynamic interactions among the enzymes and complexes are automatically accounted for in the dynamics. Diffusion manifests itself in various ways, notably in power-law behavior in the evolution of the species concentrations. In accord with earlier investigations, regimes where the product production rate exhibits either monotonic or nonmonotonic behavior as a function of time are found. In addition, the species concentrations display both t^{-1/2} and t^{-3/2} power-law behavior, depending on the dynamical regime under investigation. For high enzyme volume fractions, cooperative effects influence the enzyme kinetics. The time dependent rate coefficient determined from the mass action rate law is computed and shown to depend on the enzyme concentration. Lifetime distributions of substrate molecules newly released in complex dissociation events are determined and shown to have either a power-law form for rebinding to the same enzyme from which they were released or an exponential form for rebinding to different enzymes. The model can be used and extended to explore a variety of issues related concentration effects and diffusion on enzyme kinetics.

  19. Thermodynamic activity-based intrinsic enzyme kinetic sheds light on enzyme-solvent interactions. (United States)

    Grosch, Jan-Hendrik; Wagner, David; Nistelkas, Vasilios; Spieß, Antje C


    The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme-solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert-butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity-based kinetic modeling might be a suitable tool to initially curtail the type of enzyme-solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:96-103, 2017. © 2016 American Institute of Chemical Engineers.

  20. Enzyme activity and kinetics in substrate-amended river sediments

    Energy Technology Data Exchange (ETDEWEB)

    Duddridge, J E; Wainwright, M


    In determining the effects of heavy metals in microbial activity and litter degradation in river sediments, one approach is to determine the effects of these pollutants on sediment enzyme activity and synthesis. Methods to assay amylase, cellulase and urease activity in diverse river sediments are reported. Enzyme activity was low in non-amended sediments, but increased markedly when the appropriate substrate was added, paralleling both athropogenic and natural amendment. Linear relationships between enzyme activity, length of incubation, sample size and substrate concentration were established. Sediment enzyme activity generally obeyed Michaelis-Menton kinetics, but of the three enzymes, urease gave least significant correlation coefficients when the data for substrate concentration versus activity was applied to the Eadie-Hofstee transformation of the Michaelis-Menten equation. K/sub m/ and V/sub max/ for amylase, cellulase and urease in sediments are reported. (JMT)

  1. [Enzyme kinetic glucose determination by the glucose dehydrogenase method. Enzyme kinetic substrate determination using competitive inhibitors, II (author's transl)]. (United States)

    Müller-Matthesius, R


    The sensitivity of enzyme kinetic substrate determinations can be improved with the aid of competitive inhibitors. As an example, the determination of glucose dehydrogenase in the presence of potassium thiocyanate is described. The method has the advantage of rapid operation with satisfactory precision.

  2. Modeling physiological processes in plankton on enzyme kinetic principles

    Directory of Open Access Journals (Sweden)

    Ted Packard


    Full Text Available Many ecologically important chemical transformations in the ocean are controlled by biochemical enzyme reactions in plankton. Nitrogenase regulates the transformation of N2 to ammonium in some cyanobacteria and serves as the entryway for N2 into the ocean biosphere. Nitrate reductase controls the reduction of NO3 to NO2 and hence new production in phytoplankton. The respiratory electron transfer system in all organisms links the carbon oxidation reactions of intermediary metabolism with the reduction of oxygen in respiration. Rubisco controls the fixation of CO2 into organic matter in phytoplankton and thus is the major entry point of carbon into the oceanic biosphere. In addition to these, there are the enzymes that control CO2 production, NH4 excretion and the fluxes of phosphate. Some of these enzymes have been recognized and researched by marine scientists in the last thirty years. However, until recently the kinetic principles of enzyme control have not been exploited to formulate accurate mathematical equations of the controlling physiological expressions. Were such expressions available they would increase our power to predict the rates of chemical transformations in the extracellular environment of microbial populations whether this extracellular environment is culture media or the ocean. Here we formulate from the principles of bisubstrate enzyme kinetics, mathematical expressions for the processes of NO3 reduction, O2 consumption, N2 fixation, total nitrogen uptake.

  3. A Sensitive and Robust Enzyme Kinetic Experiment Using Microplates and Fluorogenic Ester Substrates (United States)

    Johnson, R. Jeremy; Hoops, Geoffrey C.; Savas, Christopher J.; Kartje, Zachary; Lavis, Luke D.


    Enzyme kinetics measurements are a standard component of undergraduate biochemistry laboratories. The combination of serine hydrolases and fluorogenic enzyme substrates provides a rapid, sensitive, and general method for measuring enzyme kinetics in an undergraduate biochemistry laboratory. In this method, the kinetic activity of multiple protein…

  4. On enzyme kinetic parameters modification of gamma irradiation

    International Nuclear Information System (INIS)

    Ferdes, O.S.; Ferdes, M.; Turcu, G.R.


    To elucidate the molecular mechanisms of gamma-ray action on biomolecules there were investigated the modifications in activity and other kinetic parameters for some enzymes irradiated in pure dry state at relative high doses. There were considered bacterial and fungal α-amylases, glucoamylase and Mucor sp. protease irradiated by a 60 Co gamma-ray source in the dose range 1.0-30.0 kGy, at different dose-rates between 0.5-2.0 kGy/h, at room temperature. Considering the enzyme inactivation in this dose range, the dose-effect relationships have an expected form and depend on the irradiation conditions but not significantly on the dose rate. The catalytic properties of enzymes were modified by irradiation. By usual methods it is evidenced a direct correlation between the enzymatic activities, Michaelis-Menten constant, K m , reaction velocities, v, and the irradiation dose. These experimental findings can support a self-consistent theoretical approach on biophysical radiation action on biological active molecules like enzymes. At the same time, some enzyme behaviour to irradiation could be considered like a good biological indicator of radiation response. (Author) 4 Figs., 19 Refs

  5. Kinetic memory based on the enzyme-limited competition. (United States)

    Hatakeyama, Tetsuhiro S; Kaneko, Kunihiko


    Cellular memory, which allows cells to retain information from their environment, is important for a variety of cellular functions, such as adaptation to external stimuli, cell differentiation, and synaptic plasticity. Although posttranslational modifications have received much attention as a source of cellular memory, the mechanisms directing such alterations have not been fully uncovered. It may be possible to embed memory in multiple stable states in dynamical systems governing modifications. However, several experiments on modifications of proteins suggest long-term relaxation depending on experienced external conditions, without explicit switches over multi-stable states. As an alternative to a multistability memory scheme, we propose "kinetic memory" for epigenetic cellular memory, in which memory is stored as a slow-relaxation process far from a stable fixed state. Information from previous environmental exposure is retained as the long-term maintenance of a cellular state, rather than switches over fixed states. To demonstrate this kinetic memory, we study several models in which multimeric proteins undergo catalytic modifications (e.g., phosphorylation and methylation), and find that a slow relaxation process of the modification state, logarithmic in time, appears when the concentration of a catalyst (enzyme) involved in the modification reactions is lower than that of the substrates. Sharp transitions from a normal fast-relaxation phase into this slow-relaxation phase are revealed, and explained by enzyme-limited competition among modification reactions. The slow-relaxation process is confirmed by simulations of several models of catalytic reactions of protein modifications, and it enables the memorization of external stimuli, as its time course depends crucially on the history of the stimuli. This kinetic memory provides novel insight into a broad class of cellular memory and functions. In particular, applications for long-term potentiation are discussed

  6. Measurement of Enzyme Kinetics by Use of a Blood Glucometer: Hydrolysis of Sucrose and Lactose (United States)

    Heinzerling, Peter; Schrader, Frank; Schanze, Sascha


    An alternative analytical method for measuring the kinetic parameters of the enzymes invertase and lactase is described. Invertase hydrolyzes sucrose to glucose and fructose and lactase hydrolyzes lactose to glucose and galactose. In most enzyme kinetics studies, photometric methods or test strips are used to quantify the derivates of the…

  7. Use of Mushroom Tyrosinase to Introduce Michaelis-Menten Enzyme Kinetics to Biochemistry Students (United States)

    Flurkey, William H.; Inlow, Jennifer K.


    An inexpensive enzyme kinetics laboratory exercise for undergraduate biochemistry students is described utilizing tyrosinase from white button mushrooms. The exercise can be completed in one or two three-hour lab sessions. The optimal amounts of enzyme, substrate (catechol), and inhibitor (kojic acid) are first determined, and then kinetic data is…

  8. A Hands-On Classroom Simulation to Demonstrate Concepts in Enzyme Kinetics (United States)

    Junker, Matthew


    A classroom exercise is described to introduce enzyme kinetics in an undergraduate biochemistry or chemistry course. The exercise is a simulation in which a student acts as an enzyme that "catalyzes" the unscrewing of a nut from a bolt. With other students assisting, the student enzyme carries out reactions with bolt-nut substrates under different…

  9. Enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, Evert; Barends, Thomas; Terwisscha van Scheltinga, Anke C.; Dijkstra, Bauke W.; Beintema, Jaap J.


    The enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis, were studied in detail with a new enzyme assay. In this assay, the enzyme reaction products were derivatized by reductive coupling to a chromophore, Products mere separated by HPLC and the amount of product was

  10. Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes

    International Nuclear Information System (INIS)

    Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu; Dua, Arti


    Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kinetics resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.

  11. Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu; Dua, Arti, E-mail: [Department of Chemistry, Indian Institute of Technology, Madras, Chennai 600036 (India)


    Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kinetics resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.

  12. Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes (United States)

    Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu; Dua, Arti


    Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kinetics resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.

  13. Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

    Directory of Open Access Journals (Sweden)

    Supaporn Kradtap Hartwell


    Full Text Available Enzyme kinetics studies normally focus on the initial rate of enzymatic reaction. However, the manual operation of steps of the conventional enzyme kinetics method has some drawbacks. Errors can result from the imprecise time control and time necessary for manual changing the reaction cuvettes into and out of the detector. By using the automatic flow-based analytical systems, enzyme kinetics studies can be carried out at real-time initial rate avoiding the potential errors inherent in manual operation. Flow-based systems have been developed to provide rapid, low-volume, and high-precision analyses that effectively replace the many tedious and high volume requirements of conventional wet chemistry analyses. This article presents various arrangements of flow-based techniques and their potential use in future enzyme kinetics applications.

  14. Automated Determination of Oxygen-Dependent Enzyme Kinetics in a Tube-in-Tube Flow Reactor. (United States)

    Ringborg, Rolf H; Toftgaard Pedersen, Asbjørn; Woodley, John M


    Enzyme-mediated oxidation is of particular interest to synthetic organic chemists. However, the implementation of such systems demands knowledge of enzyme kinetics. Conventionally collecting kinetic data for biocatalytic oxidations is fraught with difficulties such as low oxygen solubility in water and limited oxygen supply. Here, we present a novel method for the collection of such kinetic data using a pressurized tube-in-tube reactor, operated in the low-dispersed flow regime to generate time-series data, with minimal material consumption. Experimental development and validation of the instrument revealed not only the high degree of accuracy of the kinetic data obtained, but also the necessity of making measurements in this way to enable the accurate evaluation of high K MO enzyme systems. For the first time, this paves the way to integrate kinetic data into the protein engineering cycle.

  15. Modeling nitrous oxide production and reduction in soil through explicit representation of denitrification enzyme kinetics. (United States)

    Zheng, Jianqiu; Doskey, Paul V


    An enzyme-explicit denitrification model with representations for pre- and de novo synthesized enzymes was developed to improve predictions of nitrous oxide (N2O) accumulations in soil and emissions from the surface. The metabolic model of denitrification is based on dual-substrate utilization and Monod growth kinetics. Enzyme synthesis/activation was incorporated into each sequential reduction step of denitrification to regulate dynamics of the denitrifier population and the active enzyme pool, which controlled the rate function. Parameterizations were developed from observations of the dynamics of N2O production and reduction in soil incubation experiments. The model successfully reproduced the dynamics of N2O and N2 accumulation in the incubations and revealed an important regulatory effect of denitrification enzyme kinetics on the accumulation of denitrification products. Pre-synthesized denitrification enzymes contributed 20, 13, 43, and 62% of N2O that accumulated in 48 h incubations of soil collected from depths of 0-5, 5-10, 10-15, and 15-25 cm, respectively. An enzyme activity function (E) was defined to estimate the relative concentration of active enzymes and variation in response to environmental conditions. The value of E allows for activities of pre-synthesized denitrification enzymes to be differentiated from de novo synthesized enzymes. Incorporating explicit representations of denitrification enzyme kinetics into biogeochemical models is a promising approach for accurately simulating dynamics of the production and reduction of N2O in soils.

  16. Automated Determination of Oxygen-Dependent Enzyme Kinetics in a Tube-in-Tube Flow Reactor

    DEFF Research Database (Denmark)

    Ringborg, Rolf Hoffmeyer; Pedersen, Asbjørn Toftgaard; Woodley, John


    revealed not only the high degree of accuracy of the kinetic data obtained, but also the necessity of making measurements in this way to enable the accurate evaluation of high KMO enzyme systems. For the first time, this paves the way to integrate kinetic data into the protein engineering cycle....

  17. SigrafW: An Easy-to-Use Program for Fitting Enzyme Kinetic Data (United States)

    Leone, Francisco Assis; Baranauskas, Jose Augusto; Furriel, Rosa Prazeres Melo; Borin, Ivana Aparecida


    SigrafW is Windows-compatible software developed using the Microsoft[R] Visual Basic Studio program that uses the simplified Hill equation for fitting kinetic data from allosteric and Michaelian enzymes. SigrafW uses a modified Fibonacci search to calculate maximal velocity (V), the Hill coefficient (n), and the enzyme-substrate apparent…

  18. More Nuts and Bolts of Michaelis-Menten Enzyme Kinetics (United States)

    Lechner, Joseph H.


    Several additions to a classroom activity are proposed in which an "enzyme" (the student) converts "substrates" (nut-bolt assemblies) into "products" (separated nuts and bolts) by unscrewing them. (Contains 1 table.)

  19. An Enzyme Kinetics Experiment for the Undergraduate Organic Chemistry Laboratory (United States)

    Olsen, Robert J.; Olsen, Julie A.; Giles, Greta A.


    An experiment using [superscript 1]H NMR spectroscopy to observe the kinetics of the acylase 1-catalyzed hydrolysis of "N"-acetyl-DL-methionine has been developed for the organic laboratory. The L-enantiomer of the reactant is hydrolyzed completely in less than 2 h, and [superscript 1]H NMR spectroscopic data from a single sample can be worked up…

  20. Enzyme-catalyzed and binding reaction kinetics determined by titration calorimetry. (United States)

    Hansen, Lee D; Transtrum, Mark K; Quinn, Colette; Demarse, Neil


    Isothermal calorimetry allows monitoring of reaction rates via direct measurement of the rate of heat produced by the reaction. Calorimetry is one of very few techniques that can be used to measure rates without taking a derivative of the primary data. Because heat is a universal indicator of chemical reactions, calorimetry can be used to measure kinetics in opaque solutions, suspensions, and multiple phase systems and does not require chemical labeling. The only significant limitation of calorimetry for kinetic measurements is that the time constant of the reaction must be greater than the time constant of the calorimeter which can range from a few seconds to a few minutes. Calorimetry has the unique ability to provide both kinetic and thermodynamic data. This article describes the calorimetric methodology for determining reaction kinetics and reviews examples from recent literature that demonstrate applications of titration calorimetry to determine kinetics of enzyme-catalyzed and ligand binding reactions. A complete model for the temperature dependence of enzyme activity is presented. A previous method commonly used for blank corrections in determinations of equilibrium constants and enthalpy changes for binding reactions is shown to be subject to significant systematic error. Methods for determination of the kinetics of enzyme-catalyzed reactions and for simultaneous determination of thermodynamics and kinetics of ligand binding reactions are reviewed. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Kinetics based reaction optimization of enzyme catalysed reduction of formaldehyde to methanol with synchronous cofactor regeneration

    DEFF Research Database (Denmark)

    Marpani, Fauziah Binti; Sárossy, Zsuzsa; Pinelo, Manuel


    regeneration of the reducing equivalents during reaction is required. Herein, we report the optimization of the enzymatic conversion of formaldehyde (CHOH) to CH3 OH by alcohol dehydrogenase, the final step of the enzymatic redox reaction of CO2 to CH3 OH, with kinetically synchronous enzymatic cofactor...... regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). A mathematical model of the enzyme kinetics was employed to identify the best reaction set-up for attaining optimal cofactor recycling rate and enzyme utilization efficiency. Targeted process optimization...... experiments were conducted to verify the kinetically modelled results. Repetitive reaction cycles were shown to enhance the yield of CH3 OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes...

  2. Diameter dependent electron transfer kinetics in semiconductor-enzyme complexes. (United States)

    Brown, Katherine A; Song, Qing; Mulder, David W; King, Paul W


    Excited state electron transfer (ET) is a fundamental step for the catalytic conversion of solar energy into chemical energy. To understand the properties controlling ET between photoexcited nanoparticles and catalysts, the ET kinetics were measured for solution-phase complexes of CdTe quantum dots and Clostridium acetobutylicum [FeFe]-hydrogenase I (CaI) using time-resolved photoluminescence spectroscopy. Over a 2.0-3.5 nm diameter range of CdTe nanoparticles, the observed ET rate (kET) was sensitive to CaI concentration. To account for diameter effects on CaI binding, a Langmuir isotherm and two geometric binding models were created to estimate maximal CaI affinities and coverages at saturating concentrations. Normalizing the ET kinetics to CaI surface coverage for each CdTe diameter led to k(ET) values that were insensitive to diameter, despite a decrease in the free energy for photoexcited ET (ΔGET) with increasing diameter. The turnover frequency (TOF) of CaI in CdTe-CaI complexes was measured at several molar ratios. Normalization for diameter-dependent changes in CaI coverage showed an increase in TOF with diameter. These results suggest that k(ET) and H2 production for CdTe-CaI complexes are not strictly controlled by ΔG(ET) and that other factors must be considered.

  3. New types of experimental data shape the use of enzyme kinetics for dynamic network modeling. (United States)

    Tummler, Katja; Lubitz, Timo; Schelker, Max; Klipp, Edda


    Since the publication of Leonor Michaelis and Maude Menten's paper on the reaction kinetics of the enzyme invertase in 1913, molecular biology has evolved tremendously. New measurement techniques allow in vivo characterization of the whole genome, proteome or transcriptome of cells, whereas the classical enzyme essay only allows determination of the two Michaelis-Menten parameters V and K(m). Nevertheless, Michaelis-Menten kinetics are still commonly used, not only in the in vitro context of enzyme characterization but also as a rate law for enzymatic reactions in larger biochemical reaction networks. In this review, we give an overview of the historical development of kinetic rate laws originating from Michaelis-Menten kinetics over the past 100 years. Furthermore, we briefly summarize the experimental techniques used for the characterization of enzymes, and discuss web resources that systematically store kinetic parameters and related information. Finally, describe the novel opportunities that arise from using these data in dynamic mathematical modeling. In this framework, traditional in vitro approaches may be combined with modern genome-scale measurements to foster thorough understanding of the underlying complex mechanisms. © 2013 FEBS.

  4. Explaining the atypical reaction profiles of heme enzymes with a novel mechanistic hypothesis and kinetic treatment.

    Directory of Open Access Journals (Sweden)

    Kelath Murali Manoj

    Full Text Available Many heme enzymes show remarkable versatility and atypical kinetics. The fungal extracellular enzyme chloroperoxidase (CPO characterizes a variety of one and two electron redox reactions in the presence of hydroperoxides. A structural counterpart, found in mammalian microsomal cytochrome P450 (CYP, uses molecular oxygen plus NADPH for the oxidative metabolism (predominantly hydroxylation of substrate in conjunction with a redox partner enzyme, cytochrome P450 reductase. In this study, we employ the two above-mentioned heme-thiolate proteins to probe the reaction kinetics and mechanism of heme enzymes. Hitherto, a substrate inhibition model based upon non-productive binding of substrate (two-site model was used to account for the inhibition of reaction at higher substrate concentrations for the CYP reaction systems. Herein, the observation of substrate inhibition is shown for both peroxide and final substrate in CPO catalyzed peroxidations. Further, analogy is drawn in the "steady state kinetics" of CPO and CYP reaction systems. New experimental observations and analyses indicate that a scheme of competing reactions (involving primary product with enzyme or other reaction components/intermediates is relevant in such complex reaction mixtures. The presence of non-selective reactive intermediate(s affords alternate reaction routes at various substrate/product concentrations, thereby leading to a lowered detectable concentration of "the product of interest" in the reaction milieu. Occam's razor favors the new hypothesis. With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth. We also introduce a key concept of "substrate concentration at maximum observed rate". The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes.

  5. Simulation of the kinetics of enzymic hydrolysis of starch in standard apparatus used in alcohol production

    Energy Technology Data Exchange (ETDEWEB)

    Rovinskii, L A; Yarovenko, V L


    A mathematical model is described for kinetics of enzymic hydrolysis of starch in standard apparatus used in alcohol fermentation. The apparatus with uniform mixing and displacement was highly superior compared with other apparatuses. Differences of temperature with the apparatus significantly affects the rate of starch hydrolysis compared with constant temperature.

  6. A quenched-flow system for measuring heterogeneous enzyme kinetics with sub-second time resolution

    DEFF Research Database (Denmark)

    Olsen, Johan Pelck; Kari, Jeppe; Borch, Kim


    of insoluble substrate. Perhaps for this reason, transient kinetics has rarely been reported for heterogeneous enzyme reactions. Here, we describe a quenched-flow system using peristaltic pumps and stirred substrate suspensions with a dead time below 100 ms. The general performance was verified by alkali...

  7. The renneting of milk : a kinetic study of the enzymic and aggregation reactions

    NARCIS (Netherlands)

    Hooydonk, van A.C.M.


    The rennet-induced clotting of milk was studied under various conditions. The kinetics of the enzymic and aggregation reactions was analysed separately and, where possible, related to the physico-chemical properties of the casein micelle and its environment.

    The effects of important

  8. Enzyme catalyzed oxidative gelation of sugar beet pectin: Kinetics and rheology

    DEFF Research Database (Denmark)

    Abang Zaidel, Dayang Norulfairuz; Chronakis, Ioannis S.; Meyer, Anne S.


    Sugar beet pectin (SBP) is a marginally utilized co-processing product from sugar production from sugar beets. In this study, the kinetics of oxidative gelation of SBP, taking place via enzyme catalyzed cross-linking of ferulic acid moieties (FA), was studied using small angle oscillatory...

  9. Enzyme allocation problems in kinetic metabolic networks: Optimal solutions are elementary flux modes

    Czech Academy of Sciences Publication Activity Database

    Müller, Stefan; Regensburger, G.; Steuer, Ralf


    Roč. 347, APR 2014 (2014), s. 182-190 ISSN 0022-5193 R&D Projects: GA MŠk(CZ) EE2.3.20.0256 Institutional support: RVO:67179843 Keywords : metabolic optimization * enzyme kinetics * oriented matroid * elementary vector * conformal sum Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.116, year: 2014

  10. Kinetics of Single-Enzyme Reactions on Vesicles: Role of Substrate Aggregation (United States)

    Zhdanov, Vladimir P.


    Enzymatic reactions occurring in vivo on lipid membranes can be influenced by various factors including macromolecular crowding in general and substrate aggregation in particular. In academic studies, the role of these factors can experimentally be clarified by tracking single-enzyme kinetics occurring on individual lipid vesicles. To extend the conceptual basis for such experiments, we analyze herein the corresponding kinetics mathematically with emphasis on the role of substrate aggregation. In general, the aggregation may occur on different length scales. Small aggregates may e.g. contain a few proteins or peptides while large aggregates may be mesoscopic as in the case of lipid domains which can be formed in the membranes composed of different lipids. We present a kinetic model describing comprehensively the effect of aggregation of the former type on the dependence of the reaction rate on substrate membrane concentration. The results obtained with physically reasonable parameters indicate that the aggregation-related deviations from the conventional Michaelis-Menten kinetics may be appreciable. Special Issue Comments: This theoretical article is focused on single-enzyme reactions occurring in parallel with substrate aggregation on individual vesicles. This subject is related to a few Special Issue articles concerning enzyme dynamics6,7 and function8 and mathematical aspects of stochastic kinetics.9

  11. Functional Enzyme-Based Approach for Linking Microbial Community Functions with Biogeochemical Process Kinetics

    Energy Technology Data Exchange (ETDEWEB)

    Li, Minjing [School; Qian, Wei-jun [Pacific Northwest National Laboratory, Richland, Washington 99354, United States; Gao, Yuqian [Pacific Northwest National Laboratory, Richland, Washington 99354, United States; Shi, Liang [School; Liu, Chongxuan [Pacific Northwest National Laboratory, Richland, Washington 99354, United States; School


    The kinetics of biogeochemical processes in natural and engineered environmental systems are typically described using Monod-type or modified Monod-type models. These models rely on biomass as surrogates for functional enzymes in microbial community that catalyze biogeochemical reactions. A major challenge to apply such models is the difficulty to quantitatively measure functional biomass for constraining and validating the models. On the other hand, omics-based approaches have been increasingly used to characterize microbial community structure, functions, and metabolites. Here we proposed an enzyme-based model that can incorporate omics-data to link microbial community functions with biogeochemical process kinetics. The model treats enzymes as time-variable catalysts for biogeochemical reactions and applies biogeochemical reaction network to incorporate intermediate metabolites. The sequences of genes and proteins from metagenomes, as well as those from the UniProt database, were used for targeted enzyme quantification and to provide insights into the dynamic linkage among functional genes, enzymes, and metabolites that are necessary to be incorporated in the model. The application of the model was demonstrated using denitrification as an example by comparing model-simulated with measured functional enzymes, genes, denitrification substrates and intermediates

  12. Kinetics and spatial distribution of enzymes of carbon, nitrogen and phosphorus cycles in earthworm biopores (United States)

    Hoang Thi Thu, Duyen; Razavi, Bahar S.


    Earthworms boost microbial activities and consequently form hotspots in soil. The distribution of enzyme activities inside the earthworm biopores is completely unknown. For the first time, we analyzed enzyme kinetics and visualized enzyme distribution inside and outside biopores by in situ soil zymography. Kinetic parameters (Vmax and Km) of 6 enzymes β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) were determined in biopores formed by Lumbricus terrestris L.. The spatial distributions of GLU, NAG and APT become visible via zymograms in comparison between earthworm-inhabited and earthworm-free soil. Zymography showed heterogeneous distribution of hotspots in the rhizosphere and biopores. The hotspot areas were 2.4 to 14 times larger in the biopores than in soil without earthworms. The significantly higher Vmax values for GLU, CBH, XYL, NAG and APT in biopores confirmed the stimulation of enzyme activities by earthworms. For CBH, XYL and NAG, the 2- to 3-fold higher Km values in biopores indicated different enzyme systems with lower substrate affinity compared to control soil. The positive effects of earthworms on Vmax were cancelled by the Km increase for CBH, XYL and NAG at a substrate concentration below 20 μmol g-1 soil. The change of enzyme systems reflected a shift in dominant microbial populations toward species with lower affinity to holo-celluloses and to N-acetylglucosamine, and with higher affinity to proteins as compared to the biopores-free soil. We conclude that earthworm biopores are microbial hotspots with much higher and dense distribution of enzyme activities compared to bulk soil. References Spohn M, Kuzyakov Y. (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots - a soil zymography analysis, Plant Soil 379: 67-77. Blagodatskaya, E., Kuzyakov, Y., 2013. Review paper: Active microorganisms in soil

  13. Kinetics based reaction optimization of enzyme catalyzed reduction of formaldehyde to methanol with synchronous cofactor regeneration. (United States)

    Marpani, Fauziah; Sárossy, Zsuzsa; Pinelo, Manuel; Meyer, Anne S


    Enzymatic reduction of carbon dioxide (CO 2 ) to methanol (CH 3 OH) can be accomplished using a designed set-up of three oxidoreductases utilizing reduced pyridine nucleotide (NADH) as cofactor for the reducing equivalents electron supply. For this enzyme system to function efficiently a balanced regeneration of the reducing equivalents during reaction is required. Herein, we report the optimization of the enzymatic conversion of formaldehyde (CHOH) to CH 3 OH by alcohol dehydrogenase, the final step of the enzymatic redox reaction of CO 2 to CH 3 OH, with kinetically synchronous enzymatic cofactor regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). A mathematical model of the enzyme kinetics was employed to identify the best reaction set-up for attaining optimal cofactor recycling rate and enzyme utilization efficiency. Targeted process optimization experiments were conducted to verify the kinetically modeled results. Repetitive reaction cycles were shown to enhance the yield of CH 3 OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes to high concentrations of CHOH. System II was found to be superior to System I with a yield of 8 mM CH 3 OH, a TTN of 160 and BPR of 24 μmol CH 3 OH/U · h during 6 hr of reaction. The study demonstrates that an optimal reaction set-up could be designed from rational kinetics modeling to maximize the yield of CH 3 OH, whilst simultaneously optimizing cofactor recycling and enzyme utilization efficiency. © 2017 Wiley Periodicals, Inc.

  14. Kinetics of leather dyeing pretreated with enzymes: role of acid protease. (United States)

    Kanth, Swarna Vinodh; Venba, Rajangam; Jayakumar, Gladstone Christopher; Chandrababu, Narasimhan Kannan


    In the present investigation, kinetics of dyeing involving pretreatment with acid protease has been presented. Application of acid protease in dyeing process resulted in increased absorption and diffusion of dye into the leather matrix. Enzyme treatment at 1% concentration, 60 min duration and 50 degrees C resulted in maximum of 98% dye exhaustion and increased absorption rate constants. The final exhaustion (C(infinity)) for the best fit of CI Acid Black 194 dye has been 98.5% with K and r2 values from the modified Cegarra-Puente isotherm as 0.1033 and 0.0631. CI Acid Black 194 being a 2:1 metal complex acid dye exhibited higher absorption rate than the acid dye CI Acid Black 210. A reduction in 50% activation energy calculated from Arrhenius equation has been observed in enzyme assisted dyeing process of both the dyes that substantiates enhanced dye absorption. The absorption rate constant calculated with modified Cegarra-Puente equation confirm higher rate constants and faster kinetics for enzyme assisted dyeing process. Enzyme treated leather exhibited richness of color and shade when compared with control. The present study substantiates the essential role of enzyme pretreatment as an eco-friendly leather dyeing process.

  15. Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation (United States)

    Yunus, Ali A.; Lima, Christopher D.


    SUMO conjugation to protein substrates requires the concerted action of a dedicated E2 ubiquitin conjugation enzyme (Ubc9) and associated E3 ligases. Although Ubc9 can directly recognize and modify substrate lysine residues that occur within a consensus site for SUMO modification, E3 ligases can redirect specificity and enhance conjugation rates during SUMO conjugation in vitro and in vivo. In this chapter, we will describe methods utilized to purify SUMO conjugating enzymes and model substrates which can be used for analysis of SUMO conjugation in vitro. We will also describe methods to extract kinetic parameters during E3-dependent or E3-independent substrate conjugation. PMID:19107417

  16. A Numerical Procedure for Model Identifiability Analysis Applied to Enzyme Kinetics

    DEFF Research Database (Denmark)

    Daele, Timothy, Van; Van Hoey, Stijn; Gernaey, Krist


    The proper calibration of models describing enzyme kinetics can be quite challenging. In the literature, different procedures are available to calibrate these enzymatic models in an efficient way. However, in most cases the model structure is already decided on prior to the actual calibration...... and Pronzato (1997) and which can be easily set up for any type of model. In this paper the proposed approach is applied to the forward reaction rate of the enzyme kinetics proposed by Shin and Kim(1998). Structural identifiability analysis showed that no local structural model problems were occurring......) identifiability problems. By using the presented approach it is possible to detect potential identifiability problems and avoid pointless calibration (and experimental!) effort....

  17. Proteomic analyses for profiling regulated proteins/enzymes by Fucus vesiculosus fucoidan in B16 melanoma cells: A combination of enzyme kinetics functional study. (United States)

    Wang, Zhi-Jiang; Zheng, Li; Yang, Jun-Mo; Kang, Yani; Park, Yong-Doo


    Fucoidans are complex sulfated polysaccharides that have a wide range of biological activities. Previously, we reported the various effects of Fucus vesiculosus fucoidan on tyrosinase and B16 melanoma cells. In this study, to identify fucoidan-targeted proteins in B16 melanoma cells, we performed a proteomics study and integrated enzyme kinetics. We detected 19 candidate proteins dysregulated by fucoidan treatment. Among the probed proteins, the enzyme kinetics of two candidate enzymes, namely lactate dehydrogenase (LDH) as an upregulated protein and superoxide dismutase (SOD) as a downregulated enzyme, were determined. The enzyme kinetics results showed that Fucus vesiculosus fucoidan significantly inhibited LDH catalytic function while it did not affect SOD activity even at a high dose, while only slightly decreased activity (up to 10%) at a low dose. Based on our previous and present observations, fucoidan could inhibit B16 melanoma cells growth via regulating proteins/enzymes expression levels such as LDH and SOD known as cell survival biomarkers. Interestingly, both expression level and enzyme catalytic activity of LDH were regulated by fucoidan, which could directly induce the apoptotic effect on B16 melanoma cells along with SOD downregulation. This study highlights how combining proteomics with enzyme kinetics can yield valuable insights into fucoidan targets. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Empirical evaluation of a virtual laboratory approach to teach lactate dehydrogenase enzyme kinetics. (United States)

    Booth, Christine; Cheluvappa, Rajkumar; Bellinson, Zack; Maguire, Danni; Zimitat, Craig; Abraham, Joyce; Eri, Rajaraman


    Personalised instruction is increasingly recognised as crucial for efficacious learning today. Our seminal work delineates and elaborates on the principles, development and implementation of a specially-designed adaptive, virtual laboratory. We strived to teach laboratory skills associated with lactate dehydrogenase (LDH) enzyme kinetics to 2nd-year biochemistry students using our adaptive learning platform. Pertinent specific aims were to:(1)design/implement a web-based lesson to teach lactate dehydrogenase(LDH) enzyme kinetics to 2nd-year biochemistry students(2)determine its efficacious in improving students' comprehension of enzyme kinetics(3)assess their perception of its usefulness/manageability(vLab versus Conventional Tutorial). Our tools were designed using HTML5 technology. We hosted the program on an adaptive e-learning platform (AeLP). Provisions were made to interactively impart informed laboratory skills associated with measuring LDH enzyme kinetics. A series of e-learning methods were created. Tutorials were generated for interactive teaching and assessment. The learning outcomes herein were on par with that from a conventional classroom tutorial. Student feedback showed that the majority of students found the vLab learning experience "valuable"; and the vLab format/interface "well-designed". However, there were a few technical issues with the 1st roll-out of the platform. Our pioneering effort resulted in productive learning with the vLab, with parity with that from a conventional tutorial. Our contingent discussion emphasises not only the cornerstone advantages, but also the shortcomings of the AeLP method utilised. We conclude with an astute analysis of possible extensions and applications of our methodology.

  19. Enzyme-catalyzed synthesis and kinetics of ultrasonic-assisted biodiesel production from waste tallow. (United States)

    Adewale, Peter; Dumont, Marie-Josée; Ngadi, Michael


    The use of ultrasonic processing was evaluated for its ability to achieve adequate mixing while providing sufficient activation energy for the enzymatic transesterification of waste tallow. The effects of ultrasonic parameters (amplitude, cycle and pulse) and major reaction factors (molar ratio and enzyme concentration) on the reaction kinetics of biodiesel generation from waste tallow bio-catalyzed by immobilized lipase [Candida antarctica lipase B (CALB)] were investigated. Three sets of experiments namely A, B, and C were conducted. In experiment set A, two factors (ultrasonic amplitude and cycle) were investigated at three levels; in experiment set B, two factors (molar ratio and enzyme concentration) were examined at three levels; and in experiment set C, two factors (ultrasonic amplitude and reaction time) were investigated at five levels. A Ping Pong Bi Bi kinetic model approach was employed to study the effect of ultrasonic amplitude on the enzymatic transesterification. Kinetic constants of transesterification reaction were determined at different ultrasonic amplitudes (30%, 35%, 40%, 45%, and 50%) and enzyme concentrations (4, 6, and 8 wt.% of fat) at constant molar ratio (fat:methanol); 1:6, and ultrasonic cycle; 5 Hz. Optimal conditions for ultrasound-assisted biodiesel production from waste tallow were fat:methanol molar ratio, 1:4; catalyst level 6% (w/w of fat); reaction time, 20 min (30 times less than conventional batch processes); ultrasonic amplitude 40% at 5 Hz. The kinetic model results revealed interesting features of ultrasound assisted enzyme-catalyzed transesterification (as compared to conventional system): at ultrasonic amplitude 40%, the reaction activities within the system seemed to be steady after 20 min which means the reaction could proceed with or without ultrasonic mixing. Reversed phase high performance liquid chromatography indicated the biodiesel yield to be 85.6±0.08%. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays. (United States)

    Jung, Se-Hui; Kong, Deok-Hoon; Park, Seoung-Woo; Kim, Young-Myeong; Ha, Kwon-Soo


    Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

  1. A Kinetic Modelling of Enzyme Inhibitions in the Central Metabolism of Yeast Cells (United States)

    Kasbawati; Kalondeng, A.; Aris, N.; Erawaty, N.; Azis, M. I.


    Metabolic regulation plays an important role in the metabolic engineering of a cellular process. It is conducted to improve the productivity of a microbial process by identifying the important regulatory nodes of a metabolic pathway such as fermentation pathway. Regulation of enzymes involved in a particular pathway can be held to improve the productivity of the system. In the central metabolism of yeast cell, some enzymes are known as regulating enzymes that can be inhibited to increase the production of ethanol. In this research we study the kinetic modelling of the enzymes in the central pathway of yeast metabolism by taking into consideration the enzyme inhibition effects to the ethanol production. The existence of positive steady state solution and the stability of the system are also analysed to study the property and dynamical behaviour of the system. One stable steady state of the system is produced if some conditions are fulfilled. The conditions concern to the restriction of the maximum reactions of the enzymes in the pyruvate and acetaldehyde branch points. There exists a certain time of fermentation reaction at which a maximum and a minimum ethanol productions are attained after regulating the system. Optimal ethanol concentration is also produced for a certain initial concentration of inhibitor.

  2. Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene. (United States)

    Zuccaro, Laura; Tesauro, Cinzia; Kurkina, Tetiana; Fiorani, Paola; Yu, Hak Ki; Knudsen, Birgitta R; Kern, Klaus; Desideri, Alessandro; Balasubramanian, Kannan


    Monolayer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner. Moreover, an appropriate design of the capture probes allows us to distinctly follow the cleavage step of topoisomerase I functioning in real-time down to picomolar concentrations. The presented results are promising for future rapid screening of drugs that are being evaluated for regulating enzyme activity.

  3. Thymidine kinase 2 enzyme kinetics elucidate the mechanism of thymidine-induced mitochondrial DNA depletion. (United States)

    Sun, Ren; Wang, Liya


    Mitochondrial thymidine kinase 2 (TK2) is a nuclear gene-encoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis-Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in late-onset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.

  4. An Integrated Circuit for Chip-Based Analysis of Enzyme Kinetics and Metabolite Quantification. (United States)

    Cheah, Boon Chong; Macdonald, Alasdair Iain; Martin, Christopher; Streklas, Angelos J; Campbell, Gordon; Al-Rawhani, Mohammed A; Nemeth, Balazs; Grant, James P; Barrett, Michael P; Cumming, David R S


    We have created a novel chip-based diagnostic tools based upon quantification of metabolites using enzymes specific for their chemical conversion. Using this device we show for the first time that a solid-state circuit can be used to measure enzyme kinetics and calculate the Michaelis-Menten constant. Substrate concentration dependency of enzyme reaction rates is central to this aim. Ion-sensitive field effect transistors (ISFET) are excellent transducers for biosensing applications that are reliant upon enzyme assays, especially since they can be fabricated using mainstream microelectronics technology to ensure low unit cost, mass-manufacture, scaling to make many sensors and straightforward miniaturisation for use in point-of-care devices. Here, we describe an integrated ISFET array comprising 2(16) sensors. The device was fabricated with a complementary metal oxide semiconductor (CMOS) process. Unlike traditional CMOS ISFET sensors that use the Si3N4 passivation of the foundry for ion detection, the device reported here was processed with a layer of Ta2O5 that increased the detection sensitivity to 45 mV/pH unit at the sensor readout. The drift was reduced to 0.8 mV/hour with a linear pH response between pH 2-12. A high-speed instrumentation system capable of acquiring nearly 500 fps was developed to stream out the data. The device was then used to measure glucose concentration through the activity of hexokinase in the range of 0.05 mM-231 mM, encompassing glucose's physiological range in blood. Localised and temporal enzyme kinetics of hexokinase was studied in detail. These results present a roadmap towards a viable personal metabolome machine.

  5. Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria. (United States)

    Moreadith, R W; Lehninger, A L


    The purification and kinetic characterization of an NAD(P)+-malic enzyme from 22aH mouse hepatoma mitochondria are described. The enzyme was purified 328-fold with a final yield of 51% and specific activity of 38.1 units/mg of protein by employing DEAE-cellulose chromatography and an ATP affinity column. Sephadex G-200 chromatography yielded a native Mr = 240,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major subunit with Mr = 61,000, suggesting a tetrameric structure, and also showed that the preparation contained less than 10% polypeptide impurities. Use of the ATP affinity column required the presence of MnCl2 and fumarate (an allosteric activator) in the elution buffers. In the absence of fumarate, the Michaelis constants for malate, NAD+, and NADP+ were 3.6 mM, 55 microM, and 72 microM, respectively; in the presence of fumarate (2 mM), the constants were 0.34 mM, 9 microM, and 13 microM, respectively. ATP was shown to be an allosteric inhibitor, competitive with malate. However, the inhibition by ATP displayed hyperbolic competitive kinetics with a KI (ATP) of 80 microM (minus fumarate) and 0.5 mM (plus 2 mM fumarate). The allosteric properties of the enzyme are integrated into a rationale for its specific role in the pathways of malate and glutamate oxidation in tumor mitochondria.

  6. Enzyme Kinetics Experiment with the Multienzyme Complex Viscozyme L and Two Substrates for the Accurate Determination of Michaelian Parameters (United States)

    Guerra, Nelson Pérez


    A laboratory experiment in which students study the kinetics of the Viscozyme-L-catalyzed hydrolysis of cellulose and starch comparatively was designed for an upper-division biochemistry laboratory. The main objective of this experiment was to provide an opportunity to perform enhanced enzyme kinetics data analysis using appropriate informatics…

  7. Enzyme Kinetics By Directly Imaging A Porous Silicon Microfluidic Reactor Using Desorption/Ionization on Silicon Mass Spectrometry

    NARCIS (Netherlands)

    Nichols, K.P.F.; Azoz, Seyla; Gardeniers, Johannes G.E.


    Enzyme kinetics were obtained in a porous silicon microfluidic channel by combining an enzyme and substrate droplet, allowing them to react and deposit a small amount of residue on the channel walls, and then analyzing this residue by directly ionizing the channel walls using a matrix assisted laser

  8. Determination Of Enzyme Kinetic Parameters on Sago Starch Hydrolysis By Linearized Graphical Methods

    International Nuclear Information System (INIS)

    Lai, L.W.; Teo, C.L.; Suzana Wahidin; Mohamad Suffian Mohamad Annuar


    Amyloglucosidase (E.C. from Aspergillus niger was used to hydrolyze the sago (Metro xylon sagu) starch into reducing sugars. The experiment was conducted at constant temperature, 55 degree Celsius; pH, 4.5 and enzyme amount, 0.2 U/ ml, respectively. In this investigation, the substrate concentration was varied ranging from 1.0 - 7.0 g/ L. The obtained data were then fixed into linearized plots namely Lineweaver-Burk and Langmuir models to calculate enzyme kinetic parameters, K m and V max . Both of the K m and V max (mM, mol/min) values from each plot were: Lineweaver-Burk (26.53, 3.31) and Langmuir (13.52, 2.35). Among the linearized models, K m and V max values acquired from Langmuir plot was chosen. (author)

  9. Large-scale ruthenium- and enzyme-catalyzed dynamic kinetic resolution of (rac-1-phenylethanol

    Directory of Open Access Journals (Sweden)

    Bäckvall Jan-E


    Full Text Available Abstract The scale-up of the ruthenium- and enzyme-catalyzed dynamic kinetic resolution (DKR of (rac-1-phenylethanol (2 is addressed. The immobilized lipase Candida antarctica lipase B (CALB was employed for the resolution, which shows high enantioselectivity in the transesterification. The ruthenium catalyst used, (η 5-C5Ph5RuCl(CO2 1, was shown to possess very high reactivity in the "in situ" redox racemization of 1-phenylethanol (2 in the presence of the immobilized enzyme, and could be used in 0.05 mol% with high efficiency. Commercially available isopropenyl acetate was employed as acylating agent in the lipase-catalyzed transesterifications, which makes the purification of the product very easy. In a successful large-scale DKR of 2, with 0.05 mol% of 1, (R-1-phenylethanol acetate (3 was obtained in 159 g (97% yield in excellent enantiomeric excess (99.8% ee.

  10. Enzyme kinetics and identification of the rate-limiting step of enzymatic arabinoxylan degradation

    DEFF Research Database (Denmark)

    Rasmussen, Louise Enggaard; Xu, Cheng; Sørensen, Jens


    This study investigated the kinetics of multi-enzymatic degradation of soluble wheat arabinoxylan by monitoring the release of xylose and arabinose during designed treatments with mono-component enzymes at different substrate concentrations. The results of different combinations of α...... α-l-arabinofuranosidases catalyze liberation of arabinose residues linked 1→3 to singly (AFAn) or doubly (AFBa) substituted xyloses in arabinoxylan, respectively. When added to arabinoxylan at equimolar levels, the AFBa enzyme catalyzed the release of more arabinose, i.e. had a higher rate constant...... than AFAn, but with respect to the xylose release, AFAn – as expected – exhibited a better synergistic effect than AFBa with β-xylosidase. This synergistic effect with AFAn was estimated to increase the number of β-xylosidase catalyzed cuts from ∼3 (with β-xylosidase alone) to ∼7 in each arabinoxylan...

  11. [Enzyme kinetic analysis of Oncomelania hupensis exposed to active ingredient of Buddleja lindleyana (AIBL)]. (United States)

    Bang-Xing, Han; Jun, Chen


    To analyze the enzyme kinetics of active ingredient of Buddleja lindleyana (AIBL) against Oncomelania hupensis , the intermediate host of Schistosoma japonicum . O . hupensis snails were placed in 1 000 ml of 3.55 mg/L AIBL solution for 24, 48 h and 72 h, respectively, and the enzyme kinetics of alanine aminotransferase (GPT) was determined by Reitman-Frankel assay, lactate dehydrogenase (LDH) by the chemical inhibition lactic acid substrate method, alkaline phosphatase (AKP) by the disodium phenyl phosphate colorimetric method, acetylcholine esterase (AChE) and malate dehydrogenas (MDH) by ELISA, and succinate dehydrogenase (SDH) by the phenazine methyl sulfate reaction method (PMS) in the soft tissues of O. hupensis before and after AIBL treatment. Following exposure to 3.55 mg/L AIBL solution for 24 h, the GPT, LDH, and AKP activities significantly improved in the soft tissues of O. hupensis , while the SDH and MDH activities were significantly lowered in the head-foot and liver. However, AIBL treatment did not cause significant effect on AChE activity in O. hupensis . AIBL causes significant damages to O. hupensis liver and can efficiently act on anaerobic and aerobic respiration loci, which will hinder energy metabolism, and cause inadequate energy supply in cells used for normal secretion, eventually leading to O. hupensis death.

  12. Microbial respiration and kinetics of extracellular enzymes activities through rhizosphere and detritusphere at agricultural site (United States)

    Löppmann, Sebastian; Blagodatskaya, Evgenia; Kuzyakov, Yakov


    Rhizosphere and detritusphere are soil microsites with very high resource availability for microorganisms affecting their biomass, composition and functions. In the rhizosphere low molecular compounds occur with root exudates and low available polymeric compounds, as belowground plant senescence. In detritusphere the substrate for decomposition is mainly a polymeric material of low availability. We hypothesized that microorganisms adapted to contrasting quality and availability of substrates in the rhizosphere and detritusphere are strongly different in affinity of hydrolytic enzymes responsible for decomposition of organic compounds. According to common ecological principles easily available substrates are quickly consumed by microorganisms with enzymes of low substrate affinity (i.e. r-strategists). The slow-growing K-strategists with enzymes of high substrate affinity are better adapted for growth on substrates of low availability. Estimation of affinity of enzyme systems to the substrate is based on Michaelis-Menten kinetics, reflecting the dependency of decomposition rates on substrate amount. As enzymes-mediated reactions are substrate-dependent, we further hypothesized that the largest differences in hydrolytic activity between the rhizosphere and detritusphere occur at substrate saturation and that these differences are smoothed with increasing limitation of substrate. Affected by substrate limitation, microbial species follow a certain adaptation strategy. To achieve different depth gradients of substrate availability 12 plots on an agricultural field were established in the north-west of Göttingen, Germany: 1) 4 plots planted with maize, reflecting lower substrate availability with depth; 2) 4 unplanted plots with maize litter input (0.8 kg m-2 dry maize residues), corresponding to detritusphere; 3) 4 bare fallow plots as control. Maize litter was grubbed homogenously into the soil at the first 5 cm to ensure comparable conditions for the herbivore and

  13. Some Investigations on Protease Enzyme Production Kinetics Using Bacillus licheniformis BBRC 100053 and Effects of Inhibitors on Protease Activity

    Directory of Open Access Journals (Sweden)

    Zahra Ghobadi Nejad


    Full Text Available Due to great commercial application of protease, it is necessary to study kinetic characterization of this enzyme in order to improve design of enzymatic reactors. In this study, mathematical modeling of protease enzyme production kinetics which is derived from Bacillus licheniformis BBRC 100053 was studied (at 37°C, pH 10 after 73 h in stationary phase, and 150 rpm. The aim of the present paper was to determine the best kinetic model and kinetic parameters for production of protease and calculating Ki (inhibition constant of different inhibitors to find the most effective one. The kinetic parameters Km (Michaelis-Menten constant and Vm (maximum rate were calculated 0.626 mM and 0.0523 mM/min. According to the experimental results, using DFP (diisopropyl fluorophosphate and PMSF (phenylmethanesulfonyl fluoride as inhibitors almost 50% of the enzyme activity could be inhibited when their concentrations were 0.525 and 0.541 mM, respectively. Ki for DFP and PMSF were 0.46 and 0.56 mM, respectively. Kinetic analysis showed that the Lineweaver-Burk model was the best fitting model for protease production kinetics DFP was more effective than PMSF and both of them should be covered in the group of noncompetitive inhibitors.

  14. Deduction of kinetic mechanism in multisubstrate enzyme reactions from tritium isotope effects. Application to dopamine beta-hydroxylase

    International Nuclear Information System (INIS)

    Klinman, J.P.; Humphries, H.; Voet, J.G.


    Primary tritium isotope effects have been measured for the hydroxylation of [2-3H] dopamine catalyzed by dopamine beta-hydroxylase. Experimental values vary from 8.8 +/- 1.4 at 0.02 mM oxygen to 4.1 +/- 0.6 at 1.0 mM oxygen. It is shown that the observed dependence of the isotope effect on oxygen concentration provides unequivocal evidence for a kinetically significant dissociation of both dopamine and oxygen from enzyme, ternary complex. This approach, which is applicable to any multisubstrate enzyme characterized by detectable kinetic isotope effects, provides an alternate to classical methods for the elucidation of kinetic order in enzyme-catalyzed reactions

  15. Thermodynamics of information processing based on enzyme kinetics: An exactly solvable model of an information pump. (United States)

    Cao, Yuansheng; Gong, Zongping; Quan, H T


    Motivated by the recent proposed models of the information engine [Proc. Natl. Acad. Sci. USA 109, 11641 (2012)] and the information refrigerator [Phys. Rev. Lett. 111, 030602 (2013)], we propose a minimal model of the information pump and the information eraser based on enzyme kinetics. This device can either pump molecules against the chemical potential gradient by consuming the information to be encoded in the bit stream or (partially) erase the information initially encoded in the bit stream by consuming the Gibbs free energy. The dynamics of this model is solved exactly, and the "phase diagram" of the operation regimes is determined. The efficiency and the power of the information machine is analyzed. The validity of the second law of thermodynamics within our model is clarified. Our model offers a simple paradigm for the investigating of the thermodynamics of information processing involving the chemical potential in small systems.

  16. Thermodynamics of information processing based on enzyme kinetics: An exactly solvable model of an information pump (United States)

    Cao, Yuansheng; Gong, Zongping; Quan, H. T.


    Motivated by the recent proposed models of the information engine [Proc. Natl. Acad. Sci. USA 109, 11641 (2012), 10.1073/pnas.1204263109] and the information refrigerator [Phys. Rev. Lett. 111, 030602 (2013), 10.1103/PhysRevLett.111.030602], we propose a minimal model of the information pump and the information eraser based on enzyme kinetics. This device can either pump molecules against the chemical potential gradient by consuming the information to be encoded in the bit stream or (partially) erase the information initially encoded in the bit stream by consuming the Gibbs free energy. The dynamics of this model is solved exactly, and the "phase diagram" of the operation regimes is determined. The efficiency and the power of the information machine is analyzed. The validity of the second law of thermodynamics within our model is clarified. Our model offers a simple paradigm for the investigating of the thermodynamics of information processing involving the chemical potential in small systems.

  17. Enzyme (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  18. Interactions of nitrite with catalase: Enzyme activity and reaction kinetics studies. (United States)

    Krych-Madej, Justyna; Gebicka, Lidia


    Catalase, a heme enzyme, which catalyzes decomposition of hydrogen peroxide to water and molecular oxygen, is one of the main enzymes of the antioxidant defense system of the cell. Nitrite, used as a food preservative has long been regarded as a harmful compound due to its ability to form carcinogenic nitrosamines. Recently, much evidence has been presented that nitrite plays a protective role as a nitric oxide donor under hypoxic conditions. In this work the effect of nitrite on the catalytic reactions of catalase was studied. Catalase was inhibited by nitrite, and this process was pH-dependent. IC 50 values varied from about 1μM at pH5.0 to about 150μM of nitrite at pH7.4. The presence of chloride significantly enhanced nitrite-induced catalase inhibition, in agreement with earlier observations. The kinetics of the reactions of nitrite with ferric catalase, its redox intermediate, Compound I, and catalase inactive form, Compound II, was also studied. Possible mechanisms of nitrite-induced catalase inhibition are analyzed and the biological consequences of the reactions of catalase with nitrite are discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Rapid Determination of Enzyme Kinetics from Fluorescence: Overcoming the Inner Filter Effect (United States)

    Palmier, Mark O.; Van Doren, Steven R.


    Fluorescence change is convenient for monitoring enzyme kinetics. Unfortunately, it looses linearity as the absorbance of the fluorescent substrate increases with concentration. When the sum of absorbance at excitation and emission wavelengths exceeds 0.08, this inner filtering effect (IFE) alters apparent initial velocities, Km, and kcat. The IFE distortion of apparent initial velocities can be corrected without doing fluorophore dilution assays. Using the substrate’s extinction coefficients at excitation and emission wavelengths, the inner filter effect can be modeled during curve fitting for more accurate Michaelis-Menten parameters. A faster and simpler approach is to derive kcat and Km from progress curves. Strategies to obtain reliable and reproducible estimates of kcat and Km from only two or three progress curves are illustrated using matrix metalloproteinase-12 and alkaline phosphatase. Accurate estimates of concentration of enzyme active sites and specificity constant kcat/Km (from one progress curve with [S] ≪ Km) confer accuracy, freedom of choices of [S], and robustness to kcat and Km globally fitted to a few progress curves. The economies of the progress curve approach make accurate kcat and Km more accessible from fluorescence measurements. PMID:17706587

  20. Photoperiodism and enzyme rhythms: Kinetic characteristics of the photoperiodic induction of Crassulacean acid metabolism. (United States)

    Brulfert, J; Guerrier, D; Queiroz, O


    The effect of photoperiod on Crassulacean acid metabolism (CAM) in Kalanchoe blossfeldiana Poellniz, cv. Tom Thumb, has characteristics similar to its effect on flowering in this plant (although these two phenomena are not causally related). The photoperiodic control of CAM is based on (a) dependance on phytochrome, (b) an endogenous circadian rhythm of sensitivity to photoperiodic signals, (c) a balance between specific positive (increase in enzyme capacity) and negative (inhibitory substances) effects of the photoperiod. Variations in malate content, capacity of phosphoenolpyruvate (PEP) carboxylase, and capacity of CAM inhibitors in young leaves were measured under photoperiodic conditions noninductive for CAM and after transfer into photoperiodic conditions inductive for CAM. Essential characteristics of the photoperiodic induction of CAM are: 1) lag time for malate accumulation; 2) after-effect of the inductive photoperiod on the malate accumulation, on the increase in PEP carboxylase capacity, and on the decrease in the level of long-day produced inhibitors; final levels of malate, enzyme capacity and inhibitor are proportional to the number of inductive day-night cycles; 3) cireadian rhythm in PEP carboxylase capacity with a fixed phase under noninductive photoperiods and a continuously shifting phase under inductive photoperiods, after complex advancing and delaying transients. Kinetic similarities indicate that photoperiodic control of different physiological functions, namely, CAM and flowering, may be achieved through similar mechanisms. Preliminary results with species of Bryophyllum and Sedum support this hypothesis. Phase relationships suggest different degrees of coupling between endogenous enzymic rhythm and photoperiod, depending on whether the plants are under long days or short days.

  1. The alteration of intracellular enzymes. III. The effect of temperature on the kinetics of altered and unaltered yeast catalase. (United States)



    1. The very large increase in catalase activity (Euler effect) which follows treatment of yeast cells with CHCl(3), UV and n-propanol is accompanied by highly significant changes in kinetic properties. With respect to the enzymatic decomposition of H(2)O(2), the thermodynamic constants of the activation process micro, DeltaHdouble dagger, DeltaSdouble dagger, DeltaFdouble dagger, decrease, following treatment of the intracellular enzyme, by 4.5 kcal., 4.5 kcal., 10.1 e.u. and 1.7 kcal., respectively, all these differences being significant at the 1 per cent level. 2. Similar differences exist between the untreated, intracellular enzyme on the one hand, and the extracted yeast and crystalline beef liver catalases on the other. Significant differences in these thermodynamic constants do not exist among the treated intracellular, extracted yeast, and crystalline liver catalases. 3. These data provide unequivocal confirmation of the phenomenon of enzyme alteration reported previously, and confirm previous evidence that the extracted and crystalline enzymes have also undergone enzyme alteration and have properties which are identical with, or very similar to, those of the catalase altered in situ. 4. With respect to the process of heat destruction of catalase, the greatly diminished stability to heat of the altered enzymes, previously reported, has been confirmed. The thermodynamic constants of activation of this process have likewise changed following alteration, in the case of micro, DeltaHdouble dagger, and DeltaSdouble dagger an increase of 20.6 kcal., 20.6 kcal., and 70 e.u., respectively, and of DeltaFdouble dagger a decrease of 2.8 kcal. 5. All these data have been shown to be consistent with, and in some cases predictable from, the interfacial hypothesis, which states that the unaltered catalase exists within the cell adsorbed to some interface, in a partially, but reversibly, unfolded configuration of relatively low specificity; enzyme alteration consists, in

  2. Drying characteristic, enzyme inactivation and browning pigmentation kinetics of controlled humidity-convective drying of banana slices (United States)

    Sarpong, Frederick; Yu, Xiaojie; Zhou, Cunshan; Oteng-Darko, Patricia; Amenorfe, Leticia Peace; Wu, Bengang; Bai, Junwen; Ma, Haile


    Investigating the kinetics of enzyme activities and browning indexes in food are very essential in understanding the enzyme inactivation and browning pigmentation reaction during drying processing. In order to understand and predict accurately the enzyme inactivation and browning pigmentation of banana slices using Relative Humidity (RH)-convective hot air dryer aided by ultrasound (US) pretreatment, this study was conducted. Drying was carried out with 20 kHz frequency of US-pretreatment using three durations (10 20 and 30 min) and RH (10 20 and 30%) conditions at 70 °C and 2.0 m/s air velocity. The kinetic study of both enzyme inactivation and browning pigmentation results were compared to their relevance of fit in terms of coefficient of correlation (R2), the root mean square error (RMSE) and the reduced chi-square (χ 2). First order and second-order polynomial kinetic model fitted well for enzyme inactivation and browning indexes respectively. Both enzymes inactivation kinetics and enzymatic browning index (EBI) declined significantly (p drying time in all drying conditions and rate of decrease intensified in longer US-pretreatment duration and lower RH conditions. However, shorter US-pretreatment duration and higher RH conditions reduced the non- enzymatic browning index (NBI) significantly. Again, longer US-pretreatment duration and lower RH shortened the drying time but adversely created more microspores from the micrograph study. Longer US pretreatment and lower RH decrease significantly (p < 0.05) the L* and b* values whereas the a* values was increased.

  3. Computer controlled automated assay for comprehensive studies of enzyme kinetic parameters.

    Directory of Open Access Journals (Sweden)

    Felix Bonowski

    Full Text Available Stability and biological activity of proteins is highly dependent on their physicochemical environment. The development of realistic models of biological systems necessitates quantitative information on the response to changes of external conditions like pH, salinity and concentrations of substrates and allosteric modulators. Changes in just a few variable parameters rapidly lead to large numbers of experimental conditions, which go beyond the experimental capacity of most research groups. We implemented a computer-aided experimenting framework ("robot lab assistant" that allows us to parameterize abstract, human-readable descriptions of micro-plate based experiments with variable parameters and execute them on a conventional 8 channel liquid handling robot fitted with a sensitive plate reader. A set of newly developed R-packages translates the instructions into machine commands, executes them, collects the data and processes it without user-interaction. By combining script-driven experimental planning, execution and data-analysis, our system can react to experimental outcomes autonomously, allowing outcome-based iterative experimental strategies. The framework was applied in a response-surface model based iterative optimization of buffer conditions and investigation of substrate, allosteric effector, pH and salt dependent activity profiles of pyruvate kinase (PYK. A diprotic model of enzyme kinetics was used to model the combined effects of changing pH and substrate concentrations. The 8 parameters of the model could be estimated from a single two-hour experiment using nonlinear least-squares regression. The model with the estimated parameters successfully predicted pH and PEP dependence of initial reaction rates, while the PEP concentration dependent shift of optimal pH could only be reproduced with a set of manually tweaked parameters. Differences between model-predictions and experimental observations at low pH suggest additional protonation

  4. Solution of non-steady-state substrate concentration in the action of biosensor response at mixed enzyme kinetics (United States)

    Senthamarai, R.; Jana Ranjani, R.


    In this paper, a mathematical model of an amperometric biosensor at mixed enzyme kinetics and diffusion limitation in the case of substrate inhibition has been developed. The model is based on time dependent reaction diffusion equation containing a non -linear term related to non -Michaelis - Menten kinetics of the enzymatic reaction. Solution for the concentration of the substrate has been derived for all values of parameters using the homotopy perturbation method. All the approximate analytic expressions of substrate concentration are compared with simulation results using Scilab/Matlab program. Finally, we have given a satisfactory agreement between them.

  5. Evaluation of reaction mechanisms and the kinetic parameters for the transesterification of castor oil by liquid enzymes

    DEFF Research Database (Denmark)

    Andrade, Thalles Allan; Errico, Massimiliano; Christensen, Knud Villy


    of the transesterification of castor oil with methanol using the enzyme Eversa® Transform as catalyst were investigated. Reactions were carried out for 8 hours at 35 °C with: an alcohol-to-oil molar ratio equal to 6:1, a 5 wt% of liquid enzyme solution and addition of 5 wt% of water by weight of castor oil. From...... methanolysis rates of glycerides obtained, indicated that transesterification dominates over hydrolysis. The mechanism among the four models proposed that gave the best fit could be simplified, eliminating the kinetic parameters with negligible effects on the reaction rates. This model was able to fit...

  6. Inhibition and kinetic studies of cellulose- and hemicellulose-degrading enzymes of Ganoderma boninense by naturally occurring phenolic compounds. (United States)

    Surendran, A; Siddiqui, Y; Ali, N S; Manickam, S


    Ganoderma sp, the causal pathogen of the basal stem rot (BSR) disease of oil palm, secretes extracellular hydrolytic enzymes. These play an important role in the pathogenesis of BSR by nourishing the pathogen through the digestion of cellulose and hemicellulose of the host tissue. Active suppression of hydrolytic enzymes secreted by Ganoderma boninense by various naturally occurring phenolic compounds and estimation of their efficacy on pathogen suppression is focused in this study. Ten naturally occurring phenolic compounds were assessed for their inhibitory effect on the hydrolytic enzymes of G. boninense. The enzyme kinetics (V max and K m ) and the stability of the hydrolytic enzymes were also characterized. The selected compounds had shown inhibitory effect at various concentrations. Two types of inhibitions namely uncompetitive and noncompetitive were observed in the presence of phenolic compounds. Among all the phenolic compounds tested, benzoic acid was the most effective compound suppressive to the growth and production of hydrolytic enzymes secreted by G. boninense. The phenolic compounds as inhibitory agents can be a better replacement for the metal ions which are known as conventional inhibitors till date. The three hydrolytic enzymes were stable in a wide range of pH and temperature. These findings highlight the efficacy of the applications of phenolic compounds to control Ganoderma. The study has proved a replacement for chemical controls of G. boninense with naturally occurring phenolic compounds. © 2018 The Society for Applied Microbiology.

  7. Kinetic and structural characterization of amyloid-β peptide hydrolysis by human angiotensin-1-converting enzyme. (United States)

    Larmuth, Kate M; Masuyer, Geoffrey; Douglas, Ross G; Schwager, Sylva L; Acharya, K Ravi; Sturrock, Edward D


    Angiotensin-1-converting enzyme (ACE), a zinc metallopeptidase, consists of two homologous catalytic domains (N and C) with different substrate specificities. Here we report kinetic parameters of five different forms of human ACE with various amyloid beta (Aβ) substrates together with high resolution crystal structures of the N-domain in complex with Aβ fragments. For the physiological Aβ(1-16) peptide, a novel ACE cleavage site was found at His14-Gln15. Furthermore, Aβ(1-16) was preferentially cleaved by the individual N-domain; however, the presence of an inactive C-domain in full-length somatic ACE (sACE) greatly reduced enzyme activity and affected apparent selectivity. Two fluorogenic substrates, Aβ(4-10)Q and Aβ(4-10)Y, underwent endoproteolytic cleavage at the Asp7-Ser8 bond with all ACE constructs showing greater catalytic efficiency for Aβ(4-10)Y. Surprisingly, in contrast to Aβ(1-16) and Aβ(4-10)Q, sACE showed positive domain cooperativity and the double C-domain (CC-sACE) construct no cooperativity towards Aβ(4-10)Y. The structures of the Aβ peptide-ACE complexes revealed a common mode of peptide binding for both domains which principally targets the C-terminal P2' position to the S2' pocket and recognizes the main chain of the P1' peptide. It is likely that N-domain selectivity for the amyloid peptide is conferred through the N-domain specific S2' residue Thr358. Additionally, the N-domain can accommodate larger substrates through movement of the N-terminal helices, as suggested by the disorder of the hinge region in the crystal structures. Our findings are important for the design of domain selective inhibitors as the differences in domain selectivity are more pronounced with the truncated domains compared to the more physiological full-length forms. The atomic coordinates and structure factors for N-domain ACE with Aβ peptides 4-10 (5AM8), 10-16 (5AM9), 1-16 (5AMA), 35-42 (5AMB) and (4-10)Y (5AMC) complexes have been deposited in the

  8. Hybrid quantum and classical methods for computing kinetic isotope effects of chemical reactions in solutions and in enzymes. (United States)

    Gao, Jiali; Major, Dan T; Fan, Yao; Lin, Yen-Lin; Ma, Shuhua; Wong, Kin-Yiu


    A method for incorporating quantum mechanics into enzyme kinetics modeling is presented. Three aspects are emphasized: 1) combined quantum mechanical and molecular mechanical methods are used to represent the potential energy surface for modeling bond forming and breaking processes, 2) instantaneous normal mode analyses are used to incorporate quantum vibrational free energies to the classical potential of mean force, and 3) multidimensional tunneling methods are used to estimate quantum effects on the reaction coordinate motion. Centroid path integral simulations are described to make quantum corrections to the classical potential of mean force. In this method, the nuclear quantum vibrational and tunneling contributions are not separable. An integrated centroid path integral-free energy perturbation and umbrella sampling (PI-FEP/UM) method along with a bisection sampling procedure was summarized, which provides an accurate, easily convergent method for computing kinetic isotope effects for chemical reactions in solution and in enzymes. In the ensemble-averaged variational transition state theory with multidimensional tunneling (EA-VTST/MT), these three aspects of quantum mechanical effects can be individually treated, providing useful insights into the mechanism of enzymatic reactions. These methods are illustrated by applications to a model process in the gas phase, the decarboxylation reaction of N-methyl picolinate in water, and the proton abstraction and reprotonation process catalyzed by alanine racemase. These examples show that the incorporation of quantum mechanical effects is essential for enzyme kinetics simulations.

  9. The maximum entropy production and maximum Shannon information entropy in enzyme kinetics (United States)

    Dobovišek, Andrej; Markovič, Rene; Brumen, Milan; Fajmut, Aleš


    We demonstrate that the maximum entropy production principle (MEPP) serves as a physical selection principle for the description of the most probable non-equilibrium steady states in simple enzymatic reactions. A theoretical approach is developed, which enables maximization of the density of entropy production with respect to the enzyme rate constants for the enzyme reaction in a steady state. Mass and Gibbs free energy conservations are considered as optimization constraints. In such a way computed optimal enzyme rate constants in a steady state yield also the most uniform probability distribution of the enzyme states. This accounts for the maximal Shannon information entropy. By means of the stability analysis it is also demonstrated that maximal density of entropy production in that enzyme reaction requires flexible enzyme structure, which enables rapid transitions between different enzyme states. These results are supported by an example, in which density of entropy production and Shannon information entropy are numerically maximized for the enzyme Glucose Isomerase.

  10. Sugarcane bagasse pretreatment using three imidazolium-based ionic liquids; mass balances and enzyme kinetics

    Directory of Open Access Journals (Sweden)

    Karatzos Sergios


    Full Text Available Abstract Background Effective pretreatment is key to achieving high enzymatic saccharification efficiency in processing lignocellulosic biomass to fermentable sugars, biofuels and value-added products. Ionic liquids (ILs, still relatively new class of solvents, are attractive for biomass pretreatment because some demonstrate the rare ability to dissolve all components of lignocellulosic biomass including highly ordered (crystalline cellulose. In the present study, three ILs, 1-butyl-3-methylimidazolium chloride ([C4mim]Cl, 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl, 1-ethyl-3-methylimidazolium acetate ([C2mim]OAc are used to dissolve/pretreat and fractionate sugarcane bagasse. In these IL-based pretreatments the biomass is completely or partially dissolved in ILs at temperatures greater than 130°C and then precipitated by the addition of an antisolvent to the IL biomass mixture. For the first time mass balances of IL-based pretreatments are reported. Such mass balances, along with kinetics data, can be used in process modelling and design. Results Lignin removals of 10% mass of lignin in bagasse with [C4mim]Cl, 50% mass with [C2mim]Cl and 60% mass with [C2mim]OAc, are achieved by limiting the amount of water added as antisolvent to 0.5 water:IL mass ratio thus minimising lignin precipitation. Enzyme saccharification (24 h, 15FPU yields (% cellulose mass in starting bagasse from the recovered solids rank as: [C2mim]OAc(83% > >[C2mim]Cl(53% = [C4mim]Cl(53%. Composition of [C2mim]OAc-treated solids such as low lignin, low acetyl group content and preservation of arabinosyl groups are characteristic of aqueous alkali pretreatments while those of chloride IL-treated solids resemble aqueous acid pretreatments. All ILs are fully recovered after use (100% mass as determined by ion chromatography. Conclusions In all three ILs regulated addition of water as an antisolvent effected a polysaccharide enriched precipitate since some of the lignin

  11. Temperature sensitivity of extracellular enzyme kinetics in subtropical wetland soils under different nutrient and water level conditions (United States)

    Goswami, S.; Inglett, K.; Inglett, P.


    Microbial extracellular enzymes play an important role in the initial steps of soil organic matter decomposition and are involved in regulating nutrient cycle processes. Moreover, with the recent concern of climate change, microbial extracellular enzymes may affect the functioning (C losses, C sequestration, greenhouse gas emissions, vegetation changes) of different ecosystems. Hence, it is imperative to understand the biogeochemical processes that may be climate change sensitive. Here, we have measured the Michaelis Menten Kinetics [maximal rate of velocity (Vmax) and half-saturation constant (Km)] of 6 enzymes involved in soil organic matter decomposition (phosphatase, phosphodiesterase, β-D-glucosidase, cellobiohydrolase, leucine aminopeptidase, N-Acetyl-β-D glucosaminidase) in different nutrient(P) concentration both aerobically and anaerobically in Everglade water conservation area 2A (F1, F4-slough and U3-slough). Temperature sensitivity of different enzymes is assessed within soil of different P concentrations. We hypothesized that the temperature sensitivity of the enzyme changes with the biogeochemical conditions including water level and nutrient condition. Furthermore, we have tested specific hypothesis that higher P concentration will initiate more C demand for microbes leading to higher Vmax value for carbon processing enzymes in high P site. We found temperature sensitivity of all enzymes for Vmax and Km under both aerobic and anaerobic condition ranges from 0.6 to 3.2 for Vmax and 0.5 to 2.5 for Km. Q10 values of Km for glucosidase indicate more temperature sensitivity under anaerobic condition. Under aerobic condition higher temperature showed significant effect on Vmax for bisphosphatase between high P and low P site. Decreasing P concentration from F1 site to U3-S site had showed significant effect in all temperature on carbon processing enzyme. This suggests that in high P site, microbes will use more carbon-processing enzyme to get more carbon

  12. [Treatment of surface burns with proteolytic enzymes: mathematic description of lysis kinetics]. (United States)

    Domogatskaia, A S; Domogatskiĭ, S P; Ruuge, E K


    The lysis of necrotic tissue by a proteolytic enzyme applied to the surface of a burn wound was studied. A mathematical model was proposed, which describes changes in the thickness of necrotic tissue as a function of the proteolytic activity of the enzyme. The model takes into account the inward-directed diffusion of the enzyme, the counterflow of interstitial fluid (exudates) containing specific inhibitors, and the extracellular matrix proteolysis. It was shown in terms of the quasi-stationary approach that the thickness of the necrotic tissue layer decreases exponentially with time; i.e., the lysis slows down as the thickness of the necrotic tissue layer decreases. The dependence of the characteristic time of this decrease on enzyme concentration was obtained. It was shown that, at high enzyme concentrations (more than 5 mg/ml), the entire time of lysis (after the establishment of quasi-stationary equilibrium) is inversely proportional to the concentration of the enzyme.

  13. Inhibition and kinetic studies of lignin degrading enzymes of Ganoderma boninense by naturally occurring phenolic compounds. (United States)

    Surendran, Arthy; Siddiqui, Yasmeen; Saud, Halimi Mohd; Ali, Nusaibah Syd; Manickam, Sivakumar


    Lignolytic (Lignin degrading) enzyme, from oil palm pathogen Ganoderma boninense Pat. (Syn G. orbiforme (Ryvarden), is involved in the detoxification and the degradation of lignin in the oil palm and is the rate-limiting step in the infection process of this fungus. Active inhibition of lignin degrading enzymes secreted by G. boninense by various naturally occurring phenolic compounds and estimation of efficiency on pathogen suppression was aimed at. In our work, ten naturally occurring phenolic compounds were evaluated for their inhibitory potential towards the lignolytic enzymes of G.boninense. Additionally, the lignin degrading enzymes were characterised. Most of the peholic compounds exhibited an uncompetitive inhibition towards the lignin degrading enzymes. Benzoic acid was the superior inhibitor to the production of lignin degrading enzymes, when compared between the ten phenolic compounds. The inhibitory potential of the phenolic compounds toward the lignin degrading enzymes are higher than that of the conventional metal ion inhibitor. The lignin degrading enzymes were stable in a wide range of pH but were sensitive to higher to temperature. The study demonstrated the inhibitor potential of ten naturally occurring phenolic compounds toward the lignin degrading enzymes of G. boninense with different efficacies. The study has shed a light towards a new management strategy to control BSR in oil palm. It serves as replacement for the existing chemical control. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  14. [Molecular-kinetic parameters of thiamine enzymes and the mechanism of antivitamin action of hydroxythiamine in animal organisms]. (United States)

    Ostrovskiĭ KuM; Voskoboev, A I; Gorenshtenĭn, B I; Dosta, G A


    The molecula-kinetic parameters (Km, Ki) of three thiamine enzymes, e. g. thiamine pyrophosphokinase (EC, pyruvate dehydrogenase (EC and transketolase (EC with respect to the effects of the thiamine antimetabolite hydroxythiamine in the whole animal organism have been compared. It has been shown that only the first two enzymes, which interact competitively with the vitamin, antivitamin or their pyrophosphate ethers, obey the kinetic parameters obtained for the purified enzymes in vitro. The anticoenzymic effect of hydroxythiamine pyrophosphate with respect to transketolase is not observed in vivo at maximal concentration of the anticoenzyme in tissues due to the absence of competitive interactions with thiamine pyrophosphate. The incorporation of the true and false coenzymes into transketolase occurs only during de novo transketolase synthesis (the apoform is absent in tissues, with the exception of erythrocytes) and proceeds slowly with a half-life time equal to 24--30 hrs. After a single injection of hydroxythiamine at a large dose (70--400 mg/kg) the maximal inhibition of the transketolase activity in tissues (liver, heart, kidney, muscle, spleen, lungs adrenal grands) manifests itself by the 48th--72nd hour, when the concentration of free hydroxythiamine and its pyrophosphate is minimal and the whole anticoenzyme is tightly bound to the protein, forming the false holoenzyme. The use of hydroxythiamine for inhibition of pyruvate dehydrogenase or transketolase in animal organism is discussed.

  15. Kinetic characteristics of polygalacturonase enzymes hydrolyzing galacturonic acid oligomers using isothermal titration calorimetry (United States)

    Polygalacturonase enzymes hydrolyze the polygalacturonic acid chains found in pectin. Interest in polygalacturonase enzymes continues as they are useful in a number of industrial processes and conversely, detrimental, as they are involved in maceration of economically important crops. While a good...

  16. Enzymes or redox couples? The kinetics of thioredoxin and glutaredoxin reactions in a systems biology context

    NARCIS (Netherlands)

    Pillay, Ché S.; Hofmeyr, Jan Hendrik S; Olivier, Brett G.; Snoep, Jacky L.; Rohwer, Johann M.


    Systems biology approaches, such as kinetic modelling, could provide valuable insights into how thioredoxins, glutaredoxins and peroxiredoxins (here collectively called redoxins), and the systems that reduce these molecules are regulated. However, it is not clear whether redoxins should be described

  17. Quantitative production of compound I from a cytochrome P450 enzyme at low temperatures. Kinetics, activation parameters, and kinetic isotope effects for oxidation of benzyl alcohol. (United States)

    Wang, Qin; Sheng, Xin; Horner, John H; Newcomb, Martin


    Cytochrome P450 enzymes are commonly thought to oxidize substrates via an iron(IV)-oxo porphyrin radical cation transient termed Compound I, but kinetic studies of P450 Compounds I are essentially nonexistent. We report production of Compound I from cytochrome P450 119 (CYP119) in high conversion from the corresponding Compound II species at low temperatures in buffer mixtures containing 50% glycerol by photolysis with 365 nm light from a pulsed lamp. Compound I was studied as a reagent in oxidations of benzyl alcohol and its benzylic mono- and dideuterio isotopomers. Pseudo-first-order rate constants obtained at -50 degrees C with concentrations of substrates between 1.0 and 6.0 mM displayed saturation kinetics that gave binding constants for the substrate in the Compound I species (K(bind)) and first-order rate constants for the oxidation reactions (k(ox)). Representative results are K(bind) = 214 M(-1) and k(ox) = 0.48 s(-1) for oxidation of benzyl alcohol. For the dideuterated substrate C(6)H(5)CD(2)OH, kinetics were studied between -50 and -25 degrees C, and a van't Hoff plot for complexation and an Arrhenius plot for the oxidation reaction were constructed. The H/D kinetic isotope effects (KIEs) at -50 degrees C were resolved into a large primary KIE (P = 11.9) and a small, inverse secondary KIE (S = 0.96). Comparison of values extrapolated to 22 degrees C of both the rate constant for oxidation of C(6)H(5)CD(2)OH and the KIE for the nondeuterated and dideuterated substrates to values obtained previously in laser flash photolysis experiments suggested that tunneling could be a significant component of the total rate constant at -50 degrees C.

  18. Approaching a Conceptual Understanding of Enzyme Kinetics and Inhibition: Development of an Active Learning Inquiry Activity for Prehealth and Nonscience Majors (United States)

    House, Chloe; Meades, Glen; Linenberger, Kimberly J.


    Presented is a guided inquiry activity designed to be conducted with prenursing students using an analogous system to help develop a conceptual understanding of factors impacting enzyme kinetics and the various types of enzyme inhibition. Pre- and postconceptual understanding evaluations and effectiveness of implementation surveys were given to…

  19. Purification of PON1 from human serum and assessment of enzyme kinetics against metal toxicity. (United States)

    Ekinci, Deniz; Beydemir, Sükrü


    Paraoxonase-1 (PON1) is an organophosphate hydrolyser enzyme which has also antioxidant properties in metabolism. Due to its crucial functions, inhibition of the enzyme is undesirable and very dangerous. PON1 enzyme activity should not be altered in any case. Inhibitory investigations of this enzyme are therefore important and useful. Metal toxicology of enzymes has become popular in the recent years. Here, we report the in vitro inhibitory effects of some metal ions, including Pb(+2), Cr(+2), Fe(+2), and Zn(+2), on the activity of human serum PON1 (hPON1; EC For this purpose, we purified the enzyme from human serum and analyzed the alterations in the enzyme activity in the presence of metal ions. The results show that metal ions exhibit inhibitory effects on hPON1 at low concentrations with IC (50) values ranging from 0.838 to 7.410 mM. Metal ions showed different inhibition mechanisms: lead and iron were competitive, chrome was noncompetitive, and zinc was uncompetitive. Lead was determined to be the most effective inhibitor.

  20. Plackett-Burman Design for rGILCC1 Laccase Activity Enhancement in Pichia pastoris: Concentrated Enzyme Kinetic Characterization

    Directory of Open Access Journals (Sweden)

    Edwin D. Morales-Álvarez


    Full Text Available Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1 laccase production at low scale from Ganoderma lucidum containing the construct pGAPZαA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations. Plackett-Burman Design allowed improving enzyme activity from previous work 36.08-fold, with a laccase activity of 4.69 ± 0.39 UL−1 at 168 h of culture in a 500 mL shake-flask. Concentrated rGILCC1 remained stable between 10 and 50°C and retained a residual enzymatic activity greater than 70% at 60°C and 50% at 70°C. In regard to pH stability, concentrated enzyme was more stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity greater than 55% was obtained at pH 10.0 ± 0.2. Furthermore, calculated apparent enzyme kinetic parameters were a Vmax of 6.87 × 10−5 mM s−1, with an apparent Km of 5.36 × 10−2 mM. Collectively, these important stability findings open possibilities for applications involving a wide pH and temperature ranges.

  1. Fibrolytic enzyme and ammonia application effects on the nutritive value, intake, and digestion kinetics of bermudagrass hay in beef cattle. (United States)

    Romero, J J; Zarate, M A; Queiroz, O C M; Han, J H; Shin, J H; Staples, C R; Brown, W F; Adesogan, A T


    The objectives were to compare the effect of exogenous fibrolytic enzyme (Biocellulase A20) or anhydrous ammonia (4% DM) treatment on the nutritive value, voluntary intake, and digestion kinetics of bermudagrass (Cynodon dactylon cultivar Coastal) hay harvested after 2 maturities (5- and 13-wk regrowths). Six individually housed, ruminally cannulated Brangus steers (BW 325 ± 10 kg) were used in an experiment with a 6 × 6 Latin square design with a 3 (additives) × 2 (maturities) factorial arrangement of treatments. Each period consisted of 14 d of adaptation and 7, 4, 1, 1, and 4 d for measuring in vivo digestibility, in situ degradability, no measurements, rumen liquid fermentation and passage indices, and rate of solid passage, respectively. Steers were fed hay for ad libitum intake and supplemented with sugarcane molasses and distillers grain (supplement total of 2.88 kg DM/d). Enzyme did not affect the nutritional composition of hay but ammonia treatment decreased hay NDF, hemicellulose, and ADL concentrations and increased the CP concentration particularly for the mature lignified 13-wk hay. The enzyme increased NDF and hemicellulose digestibility of the 5-wk hay but decreased those of the 13-wk hay. Ammoniation decreased intake of hay but increased digestibility of DM, OM, NDF, hemicellulose, ADF, and cellulose and increased the ruminal in situ soluble and potentially digestible fractions and the rate of DM degradation of the 13-wk hay. Also, ammoniation increased the concentrations of ruminal NH3, total VFA, acetate, and butyrate but enzyme treatment did not. Neither enzyme addition nor ammoniation affected rate of liquid and solid passage. In conclusion, ammoniation decreased the concentration of most fiber fractions, decreased the intake of hays, and increased their CP concentration, in vivo digestibility, and in situ degradability at both maturities whereas enzyme application increased fiber digestibility of the 5-wk hay but decreased it in the case of

  2. Single lipid vesicle assay for characterizing single-enzyme kinetics of phospholipid hydrolysis in a complex biological fluid. (United States)

    Tabaei, Seyed R; Rabe, Michael; Zetterberg, Henrik; Zhdanov, Vladimir P; Höök, Fredrik


    Imaging of individual lipid vesicles is used to track single-enzyme kinetics of phospholipid hydrolysis. The method is employed to quantify the catalytic activity of phospholipase A2 (PLA2) in both pure and complex biological fluids. The measurements are demonstrated to offer a subpicomolar limit of detection (LOD) of human secretory PLA2 (sPLA2) in up to 1000-fold-diluted cerebrospinal fluid (CSF). An additional new feature provided by the single-enzyme sensitivity is that information about both relative concentration variations of active sPLA2 in CSF and the specific enzymatic activity can be simultaneously obtained. When CSF samples from healthy controls and individuals diagnosed with Alzheimer's disease (AD) are analyzed, the specific enzymatic activity is found to be preserved within 7% in the different CSF samples whereas the enzyme concentration differs by up to 56%. This suggests that the previously reported difference in PLA2 activity in CSF samples from healthy and AD individuals originates from differences in the PLA2 expression level rather than from the enzyme activity. Conventional ensemble averaging methods used to probe sPLA2 activity do not allow one to obtain such information. Together with an improvement in the LOD of at least 1 order of magnitude compared to that of conventional assays, this suggests that the method will become useful in furthering our understanding of the role of PLA2 in health and disease and in detecting the pharmacodynamic effects of PLA2-targeting drug candidates.

  3. Kinetic properties of a sex pheromone-degrading enzyme: the sensillar esterase of Antheraea polyphemus.


    Vogt, R G; Riddiford, L M; Prestwich, G D


    Behavioral and electrophysiological evidence has suggested that sex pheromone is rapidly inactivated within the sensory hairs soon after initiation of the action-potential spike. We report the isolation and characterization of a sex-pheromone-degrading enzyme from the sensory hairs of the silkmoth Antheraea polyphemus. In the presence of this enzyme at physiological concentration, the pheromone [(6E,11Z)-hexadecadienyl acetate] has an estimated half-life of 15 msec. Our findings suggest a mol...

  4. Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible competitive inhibitor, tested by simulated progress curves. (United States)

    Moruno-Dávila, M A; Garrido-del Solo, C; García-Moreno, M; Havsteen, B H; Garcia-Sevilla, F; Garcia-Cánovas, F; Varón, R


    The use of suicide substrates remains a very important and useful method in enzymology for studying enzyme mechanisms and designing potential drugs. Suicide substrates act as modified substrates for the target enzymes and bind to the active site. Therefore the presence of a competitive reversible inhibitor decreases the rate of substrate-induced inactivation and protects the enzyme from this inactivation. This lowering on the inactivation rate has evident physiological advantages, since it allows the easy acquisition of experimental data and facilitates kinetic data analysis by providing another variable (inhibitor concentration). However despite the importance of the simultaneous action of a suicide substrate and a competitive reversible inhibition, to date no corresponding kinetic analysis has been carried out. Therefore we present a general kinetic analysis of a Michaelis-Menten reaction mechanism with double inhibition caused by both, a suicide substrate and a competitive reversible inhibitor. We assume rapid equilibrium of the reversible reaction steps involved, while the time course equations for the reaction product have been derived with the assumption of a limiting enzyme. The goodness of the analytical solutions has been tested by comparison with the simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested. In conclusion, we present a complete kinetic analysis of an enzyme reaction mechanism as described above in an attempt to fill a gap in the theoretical treatment of this type of system.

  5. Kinetics of enzyme-catalyzed cross-linking of feruloylated arabinan from sugar beet

    DEFF Research Database (Denmark)

    Abang Zaidel, Dayang Norulfairuz; Arnous, Anis; Holck, Jesper


    the kinetics of HRP catalyzed cross-linking of FA esterified to α-(1,5)-linked arabinans are affected by the length of the arabinan chains carrying the feruloyl substitutions. The kinetics of the HRP-catalyzed cross-linking of four sets of arabinan samples from sugar beet pulp, having different molecular...... weights and hence different degrees of polymerization, were monitored by the disappearance of FA absorbance at 316 nm. MALDI-TOF/TOF-MS analysis confirmed that the sugar beet arabinans were feruloyl-substituted, and HPLC analysis verified that the amounts of diFAs increased when FA levels decreased...

  6. Kinetic and structural evidences on human prolidase pathological mutants suggest strategies for enzyme functional rescue.

    Directory of Open Access Journals (Sweden)

    Roberta Besio

    Full Text Available Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients' fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.

  7. Application of A Microfluidic Tool for the Determination of Enzyme Kinetics

    DEFF Research Database (Denmark)

    Ringborg, Rolf H.

    thermodynamics, inhibition and stability. It is a difficult task to assess where the current bottle neck is for a desired process. Moreover, it cannot be expected that a single solution to the limitations can be found and rather an integrated solution of all of the problems should be the future aim. All...... alreadyexist and can be found in common text books. These models do however require mutant specific data and must be collected with the target reaction. In this thesis a novel way of collecting kinetic data is created, this is carried out by combining existing technology and enables the analysis of aqueous...

  8. Understanding system dynamics of an adaptive enzyme network from globally profiled kinetic parameters. (United States)

    Chiang, Austin W T; Liu, Wei-Chung; Charusanti, Pep; Hwang, Ming-Jing


    A major challenge in mathematical modeling of biological systems is to determine how model parameters contribute to systems dynamics. As biological processes are often complex in nature, it is desirable to address this issue using a systematic approach. Here, we propose a simple methodology that first performs an enrichment test to find patterns in the values of globally profiled kinetic parameters with which a model can produce the required system dynamics; this is then followed by a statistical test to elucidate the association between individual parameters and different parts of the system's dynamics. We demonstrate our methodology on a prototype biological system of perfect adaptation dynamics, namely the chemotaxis model for Escherichia coli. Our results agreed well with those derived from experimental data and theoretical studies in the literature. Using this model system, we showed that there are motifs in kinetic parameters and that these motifs are governed by constraints of the specified system dynamics. A systematic approach based on enrichment statistical tests has been developed to elucidate the relationships between model parameters and the roles they play in affecting system dynamics of a prototype biological network. The proposed approach is generally applicable and therefore can find wide use in systems biology modeling research.

  9. Optimizing electrode-attached redox-peptide systems for kinetic characterization of protease action on immobilized substrates. Observation of dissimilar behavior of trypsin and thrombin enzymes. (United States)

    Anne, Agnès; Chovin, Arnaud; Demaille, Christophe


    In this work, we experimentally address the issue of optimizing gold electrode attached ferrocene (Fc)-peptide systems for kinetic measurements of protease action. Considering human α-thrombin and bovine trypsin as proteases of interest, we show that the recurring problem of incomplete cleavage of the peptide layer by these enzymes can be solved by using ultraflat template-stripped gold, instead of polished polycrystalline gold, as the Fc-peptide bearing electrode material. We describe how these fragile surfaces can be mounted in a rotating disk configuration so that enzyme mass transfer no longer limits the overall measured cleavage kinetics. Finally, we demonstrate that, once the system has been optimized, in situ real-time cyclic voltammetry monitoring of the protease action can yield high-quality kinetic data, showing no sign of interfering effects. The cleavage progress curves then closely match the Langmuirian variation expected for a kinetically controlled surface process. Global fit of the progress curves yield accurate values of the peptide cleavage rate for both trypsin and thrombin. It is shown that, whereas trypsin action on the surface-attached peptide closely follows Michaelis-Menten kinetics, thrombin displays a specific and unexpected behavior characterized by a nearly enzyme-concentration-independent cleavage rate in the subnanomolar enzyme concentration range. The reason for this behavior has still to be clarified, but its occurrence may limit the sensitivity of thrombin sensors based on Fc-peptide layers.

  10. Enzyme kinetics in acoustically levitated droplets of supercooled water: a novel approach to cryoenzymology. (United States)

    Weis, David D; Nardozzi, Jonathan D


    The rate of the alkaline phosphatase-catalyzed hydrolysis of 4-methylumbelliferone phosphate was measured in acoustically levitated droplets of aqueous tris (50 mM) at pH 8.5 at 22 +/- 2 degrees C and in supercooled solution at -6 +/- 2 degrees C. At 22 degrees C, the rate of product formation was in excellent agreement with the rate observed in bulk solution in a cuvette, indicating that the acoustic levitation process does not alter the enzyme activity. The rate of the reaction decreased 6-fold in supercooled solution at -6 +/- 2 degrees C. The acoustic levitator apparatus is described in detail.

  11. Utilization of integrated Michaelis-Menten equations for enzyme inhibition diagnosis and determination of kinetic constants using Solver supplement of Microsoft Office Excel. (United States)

    Bezerra, Rui M F; Fraga, Irene; Dias, Albino A


    Enzyme kinetic parameters are usually determined from initial rates nevertheless, laboratory instruments only measure substrate or product concentration versus reaction time (progress curves). To overcome this problem we present a methodology which uses integrated models based on Michaelis-Menten equation. The most severe practical limitation of progress curve analysis occurs when the enzyme shows a loss of activity under the chosen assay conditions. To avoid this problem it is possible to work with the same experimental points utilized for initial rates determination. This methodology is illustrated by the use of integrated kinetic equations with the well-known reaction catalyzed by alkaline phosphatase enzyme. In this work nonlinear regression was performed with the Solver supplement (Microsoft Office Excel). It is easy to work with and track graphically the convergence of SSE (sum of square errors). The diagnosis of enzyme inhibition was performed according to Akaike information criterion. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  12. Brownian dynamic study of an enzyme metabolon in the TCA cycle: Substrate kinetics and channeling. (United States)

    Huang, Yu-Ming M; Huber, Gary A; Wang, Nuo; Minteer, Shelley D; McCammon, J Andrew


    Malate dehydrogenase (MDH) and citrate synthase (CS) are two pacemaking enzymes involved in the tricarboxylic acid (TCA) cycle. Oxaloacetate (OAA) molecules are the intermediate substrates that are transferred from the MDH to CS to carry out sequential catalysis. It is known that, to achieve a high flux of intermediate transport and reduce the probability of substrate leaking, a MDH-CS metabolon forms to enhance the OAA substrate channeling. In this study, we aim to understand the OAA channeling within possible MDH-CS metabolons that have different structural orientations in their complexes. Three MDH-CS metabolons from native bovine, wild-type porcine, and recombinant sources, published in recent work, were selected to calculate OAA transfer efficiency by Brownian dynamics (BD) simulations and to study, through electrostatic potential calculations, a possible role of charges that drive the substrate channeling. Our results show that an electrostatic channel is formed in the metabolons of native bovine and recombinant porcine enzymes, which guides the oppositely charged OAA molecules passing through the channel and enhances the transfer efficiency. However, the channeling probability in a suggested wild-type porcine metabolon conformation is reduced due to an extended diffusion length between the MDH and CS active sites, implying that the corresponding arrangements of MDH and CS result in the decrease of electrostatic steering between substrates and protein surface and then reduce the substrate transfer efficiency from one active site to another. © 2017 The Protein Society.

  13. Analysis of residuals from enzyme kinetic and protein folding experiments in the presence of correlated experimental noise. (United States)

    Kuzmic, Petr; Lorenz, Thorsten; Reinstein, Jochen


    Experimental data from continuous enzyme assays or protein folding experiments often contain hundreds, or even thousands, of densely spaced data points. When the sampling interval is extremely short, the experimental data points might not be statistically independent. The resulting neighborhood correlation invalidates important theoretical assumptions of nonlinear regression analysis. As a consequence, certain goodness-of-fit criteria, such as the runs-of-signs test and the autocorrelation function, might indicate a systematic lack of fit even if the experiment does agree very well with the underlying theoretical model. A solution to this problem is to analyze only a subset of the residuals of fit, such that any excessive neighborhood correlation is eliminated. Substrate kinetics of the HIV protease and the unfolding kinetics of UMP/CMP kinase, a globular protein from Dictyostelium discoideum, serve as two illustrative examples. A suitable data-reduction algorithm has been incorporated into software DYNAFIT [P. Kuzmic, Anal. Biochem. 237 (1996) 260-273], freely available to all academic researchers from

  14. Acetazolamide Inhibits the Level of Tyrosinase and Melanin: An Enzyme Kinetic, In Vitro, In Vivo, and In Silico Studies. (United States)

    Abbas, Qamar; Raza, Hussain; Hassan, Mubashir; Phull, Abdul Rehman; Kim, Song Ja; Seo, Sung-Yum


    Melanin is the major factor that determines skin color and protects from ultraviolet radiation. In present study we evaluated the anti-melanogenesis effect of acetazolamide (ACZ) using four different approaches: enzyme kinetic, in vitro, in vivo and in silico. ACZ demonstrated significant inhibitory activity (IC 50 7.895 ± 0.24 μm) against tyrosinase as compared to the standard drug kojic acid (IC 50 16.84 ± 0.64 μm) and kinetic analyses showed that ACZ is a non-competitive inhibitor without cytotoxic effect. In in vitro experiments, A375 human melanoma cells were treated with 20 or 40 μm of ACZ with or without 50 μm of l-DOPA. Western blot results showed that ACZ significantly (P melanin and it could be used as a lead for developing the drugs for hyperpigmentary disorders and skin whitening. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

  15. Bilirubin glucuronidation revisited: proper assay conditions to estimate enzyme kinetics with recombinant UGT1A1. (United States)

    Zhou, Jin; Tracy, Timothy S; Remmel, Rory P


    Bilirubin, an end product of heme catabolism, is primarily eliminated via glucuronic acid conjugation by UGT1A1. Impaired bilirubin conjugation, caused by inhibition of UGT1A1, can result in clinical consequences, including jaundice and kernicterus. Thus, evaluation of the ability of new drug candidates to inhibit UGT1A1-catalyzed bilirubin glucuronidation in vitro has become common practice. However, the instability of bilirubin and its glucuronides presents substantial technical challenges to conduct in vitro bilirubin glucuronidation assays. Furthermore, because bilirubin can be diglucuronidated through a sequential reaction, establishment of initial rate conditions can be problematic. To address these issues, a robust high-performance liquid chromatography assay to measure both bilirubin mono- and diglucuronide conjugates was developed, and the incubation conditions for bilirubin glucuronidation by human embryonic kidney 293-expressed UGT1A1 were carefully characterized. Our results indicated that bilirubin glucuronidation should be assessed at very low protein concentrations (0.05 mg/ml protein) and over a short incubation time (5 min) to assure initial rate conditions. Under these conditions, bilirubin total glucuronide formation exhibited a hyperbolic (Michaelis-Menten) kinetic profile with a K(m) of ∼0.2 μM. In addition, under these initial rate conditions, the relative proportions between the total monoglucuronide and the diglucuronide product were constant across the range of bilirubin concentration evaluated (0.05-2 μM), with the monoglucuronide being the predominant species (∼70%). In conclusion, establishment of appropriate incubation conditions (i.e., very low protein concentrations and short incubation times) is necessary to properly characterize the kinetics of bilirubin glucuronidation in a recombinant UGT1A1 system.

  16. The mechanism distinguishability problem in biochemical kinetics: the single-enzyme, single-substrate reaction as a case study. (United States)

    Schnell, Santiago; Chappell, Michael J; Evans, Neil D; Roussel, Marc R


    A theoretical analysis of the distinguishability problem of two rival models of the single enzyme-single substrate reaction, the Michaelis-Menten and Henri mechanisms, is presented. We also outline a general approach for analysing the structural indistinguishability between two mechanisms. The approach involves constructing, if possible, a smooth mapping between the two candidate models. Evans et al. [N.D. Evans, M.J. Chappell, M.J. Chapman, K.R. Godfrey, Structural indistinguishability between uncontrolled (autonomous) nonlinear analytic systems, Automatica 40 (2004) 1947-1953] have shown that if, in addition, either of the mechanisms satisfies a particular criterion then such a transformation always exists when the models are indistinguishable from their experimentally observable outputs. The approach is applied to the single enzyme-single substrate reaction mechanism. In principle, mechanisms can be distinguished using this analysis, but we show that our ability to distinguish mechanistic models depends both on the precise measurements made, and on our knowledge of the system prior to performing the kinetics experiments.

  17. Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes. (United States)

    Tóth, Júlia; Bollins, Jack; Szczelkun, Mark D


    DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This 'DNA sliding' is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. The relative importance of kinetic mechanisms and variable enzyme abundances for the regulation of hepatic glucose metabolism--insights from mathematical modeling. (United States)

    Bulik, Sascha; Holzhütter, Hermann-Georg; Berndt, Nikolaus


    Adaptation of the cellular metabolism to varying external conditions is brought about by regulated changes in the activity of enzymes and transporters. Hormone-dependent reversible enzyme phosphorylation and concentration changes of reactants and allosteric effectors are the major types of rapid kinetic enzyme regulation, whereas on longer time scales changes in protein abundance may also become operative. Here, we used a comprehensive mathematical model of the hepatic glucose metabolism of rat hepatocytes to decipher the relative importance of different regulatory modes and their mutual interdependencies in the hepatic control of plasma glucose homeostasis. Model simulations reveal significant differences in the capability of liver metabolism to counteract variations of plasma glucose in different physiological settings (starvation, ad libitum nutrient supply, diabetes). Changes in enzyme abundances adjust the metabolic output to the anticipated physiological demand but may turn into a regulatory disadvantage if sudden unexpected changes of the external conditions occur. Allosteric and hormonal control of enzyme activities allow the liver to assume a broad range of metabolic states and may even fully reverse flux changes resulting from changes of enzyme abundances alone. Metabolic control analysis reveals that control of the hepatic glucose metabolism is mainly exerted by enzymes alone, which are differently controlled by alterations in enzyme abundance, reversible phosphorylation, and allosteric effects. In hepatic glucose metabolism, regulation of enzyme activities by changes of reactants, allosteric effects, and reversible phosphorylation is equally important as changes in protein abundance of key regulatory enzymes.

  19. KID - an algorithm for fast and efficient text mining used to automatically generate a database containing kinetic information of enzymes

    Directory of Open Access Journals (Sweden)

    Schomburg Dietmar


    Full Text Available Abstract Background The amount of available biological information is rapidly increasing and the focus of biological research has moved from single components to networks and even larger projects aiming at the analysis, modelling and simulation of biological networks as well as large scale comparison of cellular properties. It is therefore essential that biological knowledge is easily accessible. However, most information is contained in the written literature in an unstructured way, so that methods for the systematic extraction of knowledge directly from the primary literature have to be deployed. Description Here we present a text mining algorithm for the extraction of kinetic information such as KM, Ki, kcat etc. as well as associated information such as enzyme names, EC numbers, ligands, organisms, localisations, pH and temperatures. Using this rule- and dictionary-based approach, it was possible to extract 514,394 kinetic parameters of 13 categories (KM, Ki, kcat, kcat/KM, Vmax, IC50, S0.5, Kd, Ka, t1/2, pI, nH, specific activity, Vmax/KM from about 17 million PubMed abstracts and combine them with other data in the abstract. A manual verification of approx. 1,000 randomly chosen results yielded a recall between 51% and 84% and a precision ranging from 55% to 96%, depending of the category searched. The results were stored in a database and are available as "KID the KInetic Database" via the internet. Conclusions The presented algorithm delivers a considerable amount of information and therefore may aid to accelerate the research and the automated analysis required for today's systems biology approaches. The database obtained by analysing PubMed abstracts may be a valuable help in the field of chemical and biological kinetics. It is completely based upon text mining and therefore complements manually curated databases. The database is available at The source code of the algorithm is provided under the GNU General Public

  20. Enzyme kinetics, inhibitors, mutagenesis and electron paramagnetic resonance analysis of dual-affinity nitrate reductase in unicellular N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801. (United States)

    Wang, Tung-Hei; Chen, Yung-Han; Huang, Jine-Yung; Liu, Kang-Cheng; Ke, Shyue-Chu; Chu, Hsiu-An


    The assimilatory nitrate reductase (NarB) of N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801 is a monomeric enzyme with dual affinity for substrate nitrate. We purified the recombinant NarB of Cyanothece sp. PCC 8801 and further investigated it by enzyme kinetics analysis, site-directed mutagenesis, inhibitor kinetics analysis, and electron paramagnetic resonance (EPR) spectroscopy. The NarB showed 2 kinetic regimes at pH 10.5 or 8 and electron-donor conditions methyl viologen or ferredoxin (Fd). Fd-dependent NR assay revealed NarB with very high affinity for nitrate (K(m)1, ∼1μM; K(m)2, ∼270μM). Metal analysis and EPR results showed that NarB contains a Mo cofactor and a [4Fe-4S] cluster. In addition, the R352A mutation on the proposed nitrate-binding site of NarB greatly altered both high- and low-affinity kinetic components. Furthermore, the effect of azide on the NarB of Cyanothece sp. PCC 8801 was more complex than that on the NarB of Synechococcus sp. PCC 7942 with its single kinetic regime. With 1mM azide, the kinetics of the wild-type NarB was transformed from 2 kinetic regimes to hyperbolic kinetics, and its activity was enhanced significantly under medium nitrate concentrations. Moreover, EPR results also suggested a structural difference between the two NarBs. Taken together, our results show that the NarB of Cyanothece sp. PCC 8801 contains only a single Mo-catalytic center, and we rule out that the enzyme has 2 independent, distinct catalytic sites. In addition, the NarB of Cyanothece sp. PCC 8801 may have a regulatory nitrate-binding site. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  1. Polyol specificity of recombinant Arabidopsis thaliana sorbitol dehydrogenase studied by enzyme kinetics and in silico modeling

    Directory of Open Access Journals (Sweden)

    María Francisca eAguayo


    Full Text Available Polyols are enzymatically-produced plant compounds which can act as compatible solutes during periods of abiotic stress. NAD+-dependent SORBITOL DEHYDROGENASE (SDH, E.C. from Arabidopsis thaliana L. (AtSDH is capable of oxidizing several polyols including sorbitol, ribitol and xylitol. In the present study, enzymatic assays using recombinant AtSDH demonstrated a higher specificity constant for xylitol compared to sorbitol and ribitol, all of which are C2 (S and C4 (R polyols. Enzyme activity was reduced by preincubation with ethylenediaminetetraacetic acid (EDTA, indicating a requirement for zinc ions. In humans, it has been proposed that sorbitol becomes part of a pentahedric coordination sphere of the catalytic zinc during the reaction mechanism. In order to determine the validity of this pentahedric coordination model in a plant SDH, homology modeling and Molecular Dynamics simulations of AtSDH ternary complexes with the three polyols were performed using crystal structures of human and Bemisia argentifolii (Genn. (Hemiptera: Aleyrodidae SDHs as scaffolds. The results indicate that the differences in interaction with structural water molecules correlate very well with the observed enzymatic parameters, validate the proposed pentahedric coordination of the catalytic zinc ion in a plant SDH, and provide an explanation for why AtSDH shows a preference for polyols with a chirality of C2 (S and C4 (R.

  2. Real-Time Enzyme Kinetics by Quantitative NMR Spectroscopy and Determination of the Michaelis-Menten Constant Using the Lambert-W Function (United States)

    Her, Cheenou; Alonzo, Aaron P.; Vang, Justin Y.; Torres, Ernesto; Krishnan, V. V.


    Enzyme kinetics is an essential part of a chemistry curriculum, especially for students interested in biomedical research or in health care fields. Though the concept is routinely performed in undergraduate chemistry/biochemistry classrooms using other spectroscopic methods, we provide an optimized approach that uses a real-time monitoring of the…

  3. Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation. (United States)

    Coitinho, Juliana B; Pereira, Mozart S; Costa, Débora M A; Guimarães, Samuel L; Araújo, Simara S; Hengge, Alvan C; Brandão, Tiago A S; Nagem, Ronaldo A P


    The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

  4. Large deviation theory for the kinetics and energetics of turnover of enzyme catalysis in a chemiostatic flow (United States)

    Das, Biswajit; Gangopadhyay, Gautam


    In the framework of large deviation theory, we have characterized nonequilibrium turnover statistics of enzyme catalysis in a chemiostatic flow with externally controllable parameters, like substrate injection rate and mechanical force. In the kinetics of the process, we have shown the fluctuation theorems in terms of the symmetry of the scaled cumulant generating function (SCGF) in the transient and steady state regime and a similar symmetry rule is reflected in a large deviation rate function (LDRF) as a property of the dissipation rate through boundaries. Large deviation theory also gives the thermodynamic force of a nonequilibrium steady state, as is usually recorded experimentally by a single molecule technique, which plays a key role responsible for the dynamical symmetry of the SCGF and LDRF. Using some special properties of the Legendre transformation, here, we have provided a relation between the fluctuations of fluxes and dissipation rates, and among them, the fluctuation of the turnover rate is routinely estimated but the fluctuation in the dissipation rate is yet to be characterized for small systems. Such an enzymatic reaction flow system can be a very good testing ground to systematically understand the rare events from the large deviation theory which is beyond fluctuation theorem and central limit theorem.

  5. The Non-Linear Child: Ontogeny, Isoniazid Concentration, and NAT2 Genotype Modulate Enzyme Reaction Kinetics and Metabolism. (United States)

    Rogers, Zoe; Hiruy, Hiwot; Pasipanodya, Jotam G; Mbowane, Chris; Adamson, John; Ngotho, Lihle; Karim, Farina; Jeena, Prakash; Bishai, William; Gumbo, Tawanda


    N-acetyltransferase 2 (NAT2) catalyzes the acetylation of isoniazid to N-acetylisoniazid. NAT2 polymorphism explains 88% of isoniazid clearance variability in adults. We examined the effects of clinical and genetic factors on Michaelis-Menten reaction kinetic constants of maximum velocity (V max ) and affinity (K m ) in children 0-10years old. We measured the rates of isoniazid elimination and N-acetylisoniazid production in the blood of 30 children. Since maturation effects could be non-linear, we utilized a pharmacometric approach and the artificial intelligence method, multivariate adaptive regression splines (MARS), to identify factors predicting NAT2 V max and K m by examining clinical, genetic, and laboratory factors in toto. Isoniazid concentration predicted both V max and K m and superseded the contribution of NAT2 genotype. Age non-linearly modified the NAT2 genotype contribution until maturation at ≥5.3years. Thus, enzyme efficiency was constrained by substrate concentration, genes, and age. Since MARS output is in the form of basis functions and equations, it allows multiscale systems modeling from the level of cellular chemical reactions to whole body physiological parameters, by automatic selection of significant predictors by the algorithm. Copyright © 2016 Forschungsgesellschaft für Arbeitsphysiologie und Arbeitschutz e.V. Published by Elsevier B.V. All rights reserved.

  6. The Non-Linear Child: Ontogeny, Isoniazid Concentration, and NAT2 Genotype Modulate Enzyme Reaction Kinetics and Metabolism

    Directory of Open Access Journals (Sweden)

    Zoe Rogers


    Full Text Available N-acetyltransferase 2 (NAT2 catalyzes the acetylation of isoniazid to N-acetylisoniazid. NAT2 polymorphism explains 88% of isoniazid clearance variability in adults. We examined the effects of clinical and genetic factors on Michaelis-Menten reaction kinetic constants of maximum velocity (Vmax and affinity (Km in children 0–10 years old. We measured the rates of isoniazid elimination and N-acetylisoniazid production in the blood of 30 children. Since maturation effects could be non-linear, we utilized a pharmacometric approach and the artificial intelligence method, multivariate adaptive regression splines (MARS, to identify factors predicting NAT2 Vmax and Km by examining clinical, genetic, and laboratory factors in toto. Isoniazid concentration predicted both Vmax and Km and superseded the contribution of NAT2 genotype. Age non-linearly modified the NAT2 genotype contribution until maturation at ≥5.3 years. Thus, enzyme efficiency was constrained by substrate concentration, genes, and age. Since MARS output is in the form of basis functions and equations, it allows multiscale systems modeling from the level of cellular chemical reactions to whole body physiological parameters, by automatic selection of significant predictors by the algorithm.

  7. A Chimeric LysK-Lysostaphin Fusion Enzyme Lysing Staphylococcus aureus Cells: a Study of Both Kinetics of Inactivation and Specifics of Interaction with Anionic Polymers. (United States)

    Filatova, Lyubov Y; Donovan, David M; Ishnazarova, Nadiya T; Foster-Frey, Juli A; Becker, Stephen C; Pugachev, Vladimir G; Balabushevich, Nadezda G; Dmitrieva, Natalia F; Klyachko, Natalia L


    A staphylolytic fusion protein (chimeric enzyme K-L) was created, harboring three unique lytic activities composed of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of chimeric enzyme K-L was investigated. As a protein antimicrobial, with potential antigenic properties, the biophysical effect of including chimeric enzyme K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. Chimeric enzyme K-L reveals a high lytic activity under the following optimal ( opt ) conditions: pH opt 6.0-10.0, t opt 20-30 °C, NaCl opt 400-800 mM. At the working temperature of 37 °C, chimeric enzyme K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7 ± 3.0 h. At storage temperatures of 22 and 4 °C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the chimeric enzyme K-L inactivation. The optimal storage conditions under which the enzyme retains 100 % activity after 140 days of incubation (4 °C, the enzyme concentration of 0.8 mg/mL, pH 6.0 or 7.5) were established. Chimeric enzyme K-L is included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol, while the enzyme activity and stability are retained, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent.

  8. Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002. (United States)

    Goltsov, Alexey; Tashkandi, Ghassan; Langdon, Simon P; Harrison, David J; Bown, James L


    The phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin-1 (mTOR1) are two key targets for anti-cancer therapy. Predicting the response of the PI3K/AKT/mTOR1 signalling pathway to targeted therapy is made difficult because of network complexities. Systems biology models can help explore those complexities but the value of such models is dependent on accurate parameterisation. Motivated by a need to increase accuracy in kinetic parameter estimation, and therefore the predictive power of the model, we present a framework to integrate kinetic data from enzyme assays into a unified enzyme kinetic model. We present exemplar kinetic models of PI3K and mTOR1, calibrated on in vitro enzyme data and founded on Michaelis-Menten (MM) approximation. We describe the effects of an allosteric mTOR1 inhibitor (Rapamycin) and ATP-competitive inhibitors (BEZ235 and LY294002) that show dual inhibition of mTOR1 and PI3K. We also model the kinetics of phosphatase and tensin homolog (PTEN), which modulates sensitivity of the PI3K/AKT/mTOR1 pathway to these drugs. Model validation with independent data sets allows investigation of enzyme function and drug dose dependencies in a wide range of experimental conditions. Modelling of the mTOR1 kinetics showed that Rapamycin has an IC 50 independent of ATP concentration and that it is a selective inhibitor of mTOR1 substrates S6K1 and 4EBP1: it retains 40% of mTOR1 activity relative to 4EBP1 phosphorylation and inhibits completely S6K1 activity. For the dual ATP-competitive inhibitors of mTOR1 and PI3K, LY294002 and BEZ235, we derived the dependence of the IC 50 on ATP concentration that allows prediction of the IC 50 at different ATP concentrations in enzyme and cellular assays. Comparison of drug effectiveness in enzyme and cellular assays showed that some features of these drugs arise from signalling modulation beyond the on-target action and MM approximation and require a systems-level consideration of the whole PI3K

  9. Impact of pH and Total Soluble Solids on Enzyme Inactivation Kinetics during High Pressure Processing of Mango (Mangifera indica) Pulp. (United States)

    Kaushik, Neelima; Nadella, Tejaswi; Rao, P Srinivasa


    This study was undertaken with an aim to enhance the enzyme inactivation during high pressure processing (HPP) with pH and total soluble solids (TSS) as additional hurdles. Impact of mango pulp pH (3.5, 4.0, 4.5) and TSS (15, 20, 25 °Brix) variations on the inactivation of pectin methylesterase (PME), polyphenol oxidase (PPO), and peroxidase (POD) enzymes were studied during HPP at 400 to 600 MPa pressure (P), 40 to 70 °C temperature (T), and 6- to 20-min pressure-hold time (t). The enzyme inactivation (%) was modeled using second order polynomial equations with a good fit that revealed that all the enzymes were significantly affected by HPP. Response surface and contour models predicted the kinetic behavior of mango pulp enzymes adequately as indicated by the small error between predicted and experimental data. The predicted kinetics indicated that for a fixed P and T, higher pulse pressure effect and increased isobaric inactivation rates were possible at lower levels of pH and TSS. In contrast, at a fixed pH or TSS level, an increase in P or T led to enhanced inactivation rates, irrespective of the type of enzyme. PPO and POD were found to have similar barosensitivity, whereas PME was found to be most resistant to HPP. Furthermore, simultaneous variation in pH and TSS levels of mango pulp resulted in higher enzyme inactivation at lower pH and TSS during HPP, where the effect of pH was found to be predominant than TSS within the experimental domain. Exploration of additional hurdles such as pH, TSS, and temperature for enzyme inactivation during high pressure processing of fruits is useful from industrial point of view, as these parameters play key role in preservation process design. © 2015 Institute of Food Technologists®

  10. Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis. (United States)

    Deng, Gejing; Gu, Rong-Fang; Marmor, Stephen; Fisher, Stewart L; Jahic, Haris; Sanyal, Gautam


    An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated. Copyright 2004 Elsevier B.V.

  11. Spectroscopic and Kinetic Characterization of Peroxidase-Like π-Cation Radical Pinch-Porphyrin-Iron(III Reaction Intermediate Models of Peroxidase Enzymes

    Directory of Open Access Journals (Sweden)

    Samuel Hernández Anzaldo


    Full Text Available The spectroscopic and kinetic characterization of two intermediates from the H2O2 oxidation of three dimethyl ester [(proto, (meso, (deuteroporphyrinato (picdien]Fe(III complexes ([FePPPic], [FeMPPic] and [FeDPPic], respectively pinch-porphyrin peroxidase enzyme models, with s = 5/2 and 3/2 Fe(III quantum mixed spin (qms ground states is described herein. The kinetic study by UV/Vis at λmax = 465 nm showed two different types of kinetics during the oxidation process in the guaiacol test for peroxidases (1–3 + guaiacol + H2O2 → oxidation guaiacol products. The first intermediate was observed during the first 24 s of the reaction. When the reaction conditions were changed to higher concentration of pinch-porphyrins and hydrogen peroxide only one type of kinetics was observed. Next, the reaction was performed only between pinch-porphyrins-Fe(III and H2O2, resulting in only two types of kinetics that were developed during the first 0–4 s. After this time a self-oxidation process was observed. Our hypotheses state that the formation of the π-cation radicals, reaction intermediates of the pinch-porphyrin-Fe(III family with the ligand picdien [N,N’-bis-pyridin-2-ylmethyl-propane-1,3-diamine], occurred with unique kinetics that are different from the overall process and was involved in the oxidation pathway. UV-Vis, 1H-NMR and ESR spectra confirmed the formation of such intermediates. The results in this paper highlight the link between different spectroscopic techniques that positively depict the kinetic traits of artificial compounds with enzyme-like activity.

  12. Development of ultrasonically levitated drops as microreactors for study of enzyme kinetics and potential as a universal portable analysis system (United States)

    Scheeline, A.; Pierre, Z.; Field, C. R.; Ginsberg, M. D.


    Development of microfluidics has focused on carrying out chemical synthesis and analysis in ever-smaller volumes of solution. In most cases, flow systems are made of either quartz, glass, or an easily moldable polymer such as polydimethylsiloxane (Whitesides 2006). As the system shrinks, the ratio of surface area to volume increases. For studies of either free radical chemistry or protein chemistry, this is undesirable. Proteins stick to surfaces, biofilms grow on surfaces, and radicals annihilate on walls (Lewis et al. 2006). Thus, under those circumstances where small amounts of reactants must be employed, typical microfluidic systems are incompatible with the chemistry one wishes to study. We have developed an alternative approach. We use ultrasonically levitated microliter drops as well mixed microreactors. Depending on whether capillaries (to form the drop) and electrochemical sensors are in contact with the drop or whether there are no contacting solids, the ratio of solid surface area to volume is low or zero. The only interface seen by reactants is a liquid/air interface (or, more generally, liquid/gas, as any gas may be used to support the drop). While drop levitation has been reported since at least the 1940's, we are the second group to carry out enzyme reactions in levitated drops, (Weis; Nardozzi 2005) and have fabricated the lowest power levitator in the literature (Field; Scheeline 2007). The low consumption aspects of ordinary microfluidics combine with a contact-free determination cell (the levitated drop) that ensures against cross-contamination, minimizes the likelihood of biofilm formation, and is robust to changes in temperature and humidity (Lide 1992). We report kinetics measurements in levitated drops and explain how outgrowths of these accomplishments will lead to portable chemistry/biology laboratories well suited to detection of a wide range of chemical and biological agents in the asymmetric battlefield environment.

  13. Occurrence of dead core in catalytic particles containing immobilized enzymes: analysis for the Michaelis-Menten kinetics and assessment of numerical methods. (United States)

    Pereira, Félix Monteiro; Oliveira, Samuel Conceição


    In this article, the occurrence of dead core in catalytic particles containing immobilized enzymes is analyzed for the Michaelis-Menten kinetics. An assessment of numerical methods is performed to solve the boundary value problem generated by the mathematical modeling of diffusion and reaction processes under steady state and isothermal conditions. Two classes of numerical methods were employed: shooting and collocation. The shooting method used the ode function from Scilab software. The collocation methods included: that implemented by the bvode function of Scilab, the orthogonal collocation, and the orthogonal collocation on finite elements. The methods were validated for simplified forms of the Michaelis-Menten equation (zero-order and first-order kinetics), for which analytical solutions are available. Among the methods covered in this article, the orthogonal collocation on finite elements proved to be the most robust and efficient method to solve the boundary value problem concerning Michaelis-Menten kinetics. For this enzyme kinetics, it was found that the dead core can occur when verified certain conditions of diffusion-reaction within the catalytic particle. The application of the concepts and methods presented in this study will allow for a more generalized analysis and more accurate designs of heterogeneous enzymatic reactors.

  14. The influence of carbon nanotubes on enzyme activity and structure: investigation of different immobilization procedures through enzyme kinetics and circular dichroism studies

    International Nuclear Information System (INIS)

    Cang-Rong, Jason Teng; Pastorin, Giorgia


    In the last decade, many environmental organizations have devoted their efforts to identifying renewable biosystems, which could provide sustainable fuels and thus enhance energy security. Amidst the myriad of possibilities, some biofuels make use of different types of waste biomasses, and enzymes are often employed to hydrolyze these biomasses and produce sugars that will be subsequently converted into ethanol. In this project, we aimed to bridge nanotechnology and biofuel production: here we report on the activity and structure of the enzyme amyloglucosidase (AMG), physically adsorbed or covalently immobilized onto single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). In fact, carbon nanotubes (CNTs) present several properties that render them ideal support systems, without the diffusion limitations displayed by porous material and with the advantage of being further functionalizable at their surface. Chemical ligation was achieved both on oxidized nanotubes (via carbodiimide chemistry), as well as on amino-functionalized nanotubes (via periodate-oxidized AMG). Results showed that AMG retained a certain percentage of its specific activity for all enzyme-carbon nanotubes complexes prepared, with the physically adsorbed samples displaying better catalytic efficiency than the covalently immobilized samples. Analysis of the enzyme's structure through circular dichroism (CD) spectroscopy revealed significant structural changes in all samples, the degree of change being consistent with the activity profiles. This study proves that AMG interacts differently with carbon nanotubes depending on the method employed. Due to the higher activity reported by the enzyme physically adsorbed onto CNTs, these samples demonstrated a vast potential for further development. At the same time, the possibility of inducing magnetic properties into CNTs offers the opportunity to easily separate them from the original solution. Hence, substances to which they

  15. Effect of pulsed electric field treatment on enzyme kinetics and thermostability of endogenous ascorbic acid oxidase in carrots (Daucus carota cv. Nantes). (United States)

    Leong, Sze Ying; Oey, Indrawati


    The objective of this research was to study the enzyme kinetics and thermostability of endogenous ascorbic acid oxidase (AAO) in carrot purée (Daucus carota cv. Nantes) after being treated with pulsed electric field (PEF) processing. Various PEF treatments using electric field strength between 0.2 and 1.2kV/cm and pulsed electrical energy between 1 and 520kJ/kg were conducted. The enzyme kinetics and the kinetics of AAO thermal inactivation (55-70°C) were described using Michaelis-Menten model and first order reaction model, respectively. Overall, the estimated Vmax and KM values were situated in the same order of magnitude as the untreated carrot purée after being exposed to pulsed electrical energy between 1 and 400kJ/kg, but slightly changed at pulsed electrical energy above 500kJ/kg. However, AAO presented different thermostability depending on the electric field strength applied. After PEF treatment at the electric field strength between 0.2 and 0.5kV/cm, AAO became thermolabile (i.e. increase in inactivation rate (k value) at reference temperature) but the temperature dependence of k value (Ea value) for AAO inactivation in carrot purée decreased, indicating that the changes in k values were less temperature dependent. It is obvious that PEF treatment affects the temperature stability of endogenous AAO. The changes in enzyme kinetics and thermostability of AAO in carrot purée could be related to the resulting carrot purée composition, alteration in intracellular environment and the effective concentration of AAO released after being subjected to PEF treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Influence of the metal ion on the enzyme activity and kinetics of PepA from Lactobacillus delbrueckii. (United States)

    Ewert, Jacob; Glück, Claudia; Strasdeit, Henry; Fischer, Lutz; Stressler, Timo


    The aminopeptidase A (PepA; EC belongs to the group of metallopeptidases with two bound metal ions per subunit (M1M2(PepA)) and is specific for the cleavage of N-terminal glutamic (Glu) and aspartic acid (Asp) and, in low amounts, serine (Ser) residues. Our group recently characterized the first PepA from a Lactobacillus strain. However, the characterization was performed using synthetic para-nitroaniline substrates and not original peptide substrates, as was done in the current study. Prior to the characterization using original peptide substrates, the PepA purified was converted to its inactive apo-form and eight different metal ions were tested to restore its activity. It was found that five of the metal ions were able to reactivate apo-PepA: Co 2+ , Cu 2+ , Mn 2+ , Ni 2+ and Zn 2+ . Interestingly, depending on the metal ion used for reactivation, the activity and the pH and temperature profile differed. Exemplarily, MnMn(PepA), NiNi(PepA) and ZnZn(PepA) had an activity optimum using MES buffer (50mM, pH 6.0) and 60°C, whereas the activity optimum changed to Na/K-phosphate-buffer (50mM, pH 7.0) and 55°C for CuCu(PepA). However, more important than the changes in optimum pH and temperature, the kinetic properties of PepA were affected by the metal ion used. While all PepA variants could release N-terminal Glu or Asp, only CoCo(PepA), NiNi(PepA) and CuCu(PepA) could release Ser from the particular peptide substrate. In addition, it was found that the enzyme efficiency (V max /K M ) and catalytic mechanism (positive cooperative binding (Hill coefficent; n), substrate inhibition (K IS )) were influenced by the metal ion. Exemplarily, a high cooperativity (n>2),K IS value >20mM and preference for N-terminal Glu were detected for CuCu(PepA). In summary, the results suggested that an exchange of the metal ion can be used for tailoring the properties of PepA for specific hydrolysis requirements. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Kinetic characterization of glucose aerodehydrogenase from Aspergillus niger EMS-150-F after optimizing the dose of mutagen for enhanced production of enzyme

    Directory of Open Access Journals (Sweden)

    Huma Umbreen


    Full Text Available In the present study enhanced production of glucose aerodehydrogenase from Aspergillus niger has been achieved after optimizing the dose of chemical mutagen ethyl methane sulfonate (EMS that has not been reported earlier. Different doses of mutagen were applied and a strain was developed basing upon the best production. The selected strain Aspergillus niger EMS-150-F was optimized for nutrient requirements in order to produce enzyme through fermentation and the results showed the best yield at 2% corn steep liquor (CSL, 36 hours fermentation time, pH 5, 30°C temperature, 0.3% KH2PO4, 0.3% urea and 0.06% CaCO3. The enzyme was then purified and resulted in 57.88 fold purification with 52.12% recovery. On kinetic characterization, the enzyme showed optimum activity at pH 6 and temperature 30°C. The Michaelis-Menton constants (Km, Vmax, Kcat and Kcat/Km were 20 mM, 45.87 U mL-1, 1118.81 s-1 and 55.94 s-1 mM-1, respectively. The enzyme was found to be thermaly stable and the enthalpy and free energy showed an increase with increase in temperature and ΔS* was highly negative proving the enzyme from A. niger EMS-150-F resistant to temperature and showing a very little disorderliness.

  18. An artificial-intelligence technique for qualitatively deriving enzyme kinetic mechanisms from initial-velocity measurements and its application to hexokinase. (United States)

    Garfinkel, L; Cohen, D M; Soo, V W; Garfinkel, D; Kulikowski, C A


    We have developed a computer method based on artificial-intelligence techniques for qualitatively analysing steady-state initial-velocity enzyme kinetic data. We have applied our system to experiments on hexokinase from a variety of sources: yeast, ascites and muscle. Our system accepts qualitative stylized descriptions of experimental data, infers constraints from the observed data behaviour and then compares the experimentally inferred constraints with corresponding theoretical model-based constraints. It is desirable to have large data sets which include the results of a variety of experiments. Human intervention is needed to interpret non-kinetic information, differences in conditions, etc. Different strategies were used by the several experimenters whose data was studied to formulate mechanisms for their enzyme preparations, including different methods (product inhibitors or alternate substrates), different experimental protocols (monitoring enzyme activity differently), or different experimental conditions (temperature, pH or ionic strength). The different ordered and rapid-equilibrium mechanisms proposed by these experimenters were generally consistent with their data. On comparing the constraints derived from the several experimental data sets, they are found to be in much less disagreement than the mechanisms published, and some of the disagreement can be ascribed to different experimental conditions (especially ionic strength). PMID:2690819

  19. Mechanism and activation for allosteric adenosine 5'-monophosphate nucleosidase. Kinetic alpha-deuterium isotope effects for the enzyme-catalyzed hydrolysis of adenosine 5'-monophosphate and nicotinamide mononucleotide

    International Nuclear Information System (INIS)

    Skoog, M.T.


    The kinetic alpha-deuterium isotope effect on Vmax/Km for hydrolysis of NMN catalyzed by AMP nucleosidase at saturating concentrations of the allosteric activator MgATP2- is kH/kD = 1.155 +/- 0.012. This value is close to that reported previously for the nonenzymatic hydrolysis of nucleosides of related structure, suggesting that the full intrinsic isotope effect for enzymatic NMN hydrolysis is expressed under these conditions; that is, bond-changing reactions are largely or completely rate-determining and the transition state has marked oxocarbonium ion character. The kinetic alpha-deuterium isotope effect for this reaction is unchanged when deuterium oxide replaces water as solvent, corroborating this conclusion. Furthermore, this isotope effect is independent of pH over the range 6.95-9.25, for which values of Vmax/Km change by a factor of 90, suggesting that the isotope-sensitive and pH-sensitive steps for AMP-nucleosidase-catalyzed NMN hydrolysis are the same. Values of kH/kD for AMP nucleosidase-catalyzed hydrolysis of NMN decrease with decreasing saturation of enzyme with MgATP2- and reach unity when the enzyme is less than half-saturated with this activator. This requires that the rate-determining step changes from cleavage of the covalent C-N bond to one which is isotope-independent. In contrast to the case for NMN hydrolysis, AMP nucleosidase-catalyzed hydrolysis of AMP at saturating concentrations of MgATP2- shows a kinetic alpha-deuterium isotope effect of unity. Thus, covalent bond-changing reactions are largely or completely rate-determining for hydrolysis of a poor substrate, NMN, but make little or no contribution to rate-determining step for hydrolysis of a good substrate, AMP, by maximally activated enzyme. This behavior has several precedents

  20. In vitro antioxidant activity, enzyme kinetics, biostability and cellular SOD mimicking ability of 1:1 curcumin-copper (II) complex

    International Nuclear Information System (INIS)

    Kunwar, A.; Mishra, B.; Barik, A.; Priyadarsini, K.I.; Narang, H.; Krishna, M.


    In vitro antioxidant activity of 1:1 curcumin copper (II) complex was evaluated by following the inhibition of γ-radiation induced lipid peroxidation and protein oxidation in model systems. The SOD enzyme kinetic parameters K m and V max values and the turn over number of the complex were determined. The complex is stable in bio-fluids and prevents oxidation of lipid and protein solution in presence of H 2 O 2 and showed reduction in MnSOD level in spleen cells without having any effect on cell viability. (author)

  1. In vitro antioxidant activity, enzyme kinetics, biostability and cellular SOD mimicking ability of 1:1 curcumin-copper (II) complex

    Energy Technology Data Exchange (ETDEWEB)

    Kunwar, A; Mishra, B; Barik, A; Priyadarsini, K I [Radiation and Photochemistry Div., Bhabha Atomic Research Centre, Mumbai (India); Narang, H; Krishna, M [Radiation Biology and Health Sciences Div., Bhabha Atomic Research Centre, Mumbai (India)


    In vitro antioxidant activity of 1:1 curcumin copper (II) complex was evaluated by following the inhibition of {gamma}-radiation induced lipid peroxidation and protein oxidation in model systems. The SOD enzyme kinetic parameters K{sub m} and V{sub max} values and the turn over number of the complex were determined. The complex is stable in bio-fluids and prevents oxidation of lipid and protein solution in presence of H{sub 2}O{sub 2} and showed reduction in MnSOD level in spleen cells without having any effect on cell viability. (author)

  2. Kinetic properties of two Rhizopus exo-polygalacturonase enzymes hydrolyzing galacturonic acid oligomers using isothermal titration calorimetry (United States)

    The kinetic characteristics of two Rhizopus oryzae exo-polygalacturonases acting on galacturonic acid oligomers (GalpA) were determined using isothermal titration calorimetry (ITC). RPG15 hydrolyzing (GalpA)2 demonstrated a Km of 55 uM and kcat of 10.3 s^-1^ while RPG16 was shown to have greater af...

  3. Determination of glutamate dehydrogenase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry)

    NARCIS (Netherlands)

    Botman, Dennis; Tigchelaar, Wikky; van Noorden, Cornelis J. F.


    Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)(+) to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we

  4. The purification and steady-state kinetic behaviour of rabbit heart mitochondrial NAD(P)+ malic enzyme.


    Davisson, V J; Schulz, A R


    The mitochondrial NAD(P)+ malic enzyme [EC, L-malate:NAD+ oxidoreductase (decarboxylating)] was purified from rabbit heart to a specific activity of 7 units (mumol/min)/mg at 23 degrees C. A study of the reductive carboxylation reaction indicates that this enzymic reaction is reversible. The rate of the reductive carboxylation reaction appears to be completely inhibited at an NADH concentration of 0.92 mM. A substrate saturation curve of this reaction with NADH as the varied substrat...

  5. Influence of temperature and solvent concentration on the kinetics of the enzyme carbonic anhydrase in carbon capture technology

    DEFF Research Database (Denmark)

    Gladis, Arne; Deslauriers, Maria Gundersen; Fosbøl, Philip Loldrup


    In this study the effect of carbonic anhydrase addition on the absorption of CO2 was investigated in a wetted wall column apparatus. Four different solvents: the primary amine monoethanolamine (MEA), the sterically hindered primary amine 2-amino-2-methyl-1-propanol (AMP), the tertiary amine N......-methyl-diethanolamine (MDEA) and the carbonate salt solution K2CO3 were compared in concentrations from 5 to 50 wt% in a temperature range of 298–328 K with and without enzyme. Necessary mass transfer parameters such as liquid side mass transfer coefficient and solvent and enzyme reaction rates were determined...

  6. Unchanged content of oxidative enzymes in fast-twitch muscle fibers and V˙O2 kinetics after intensified training in trained cyclists

    DEFF Research Database (Denmark)

    Christensen, Peter Møller; Gunnarsson, Thomas Gunnar Petursson; Thomassen, Martin


    perturbation during INT. Pulmonary V˙O2 kinetics was determined in eight trained male cyclists (V˙O2-max: 59 ± 4 (means ± SD) mL min(-1) kg(-1)) during MOD (205 ± 12 W ~65% V˙O2-max) and INT (286 ± 17 W ~85% V˙O2-max) exercise before and after a 7-week HIT period (30-sec sprints and 4-min intervals) with a 50...... DW(-1) min(-1)) of CS (56 ± 8 post-HIT vs. 59 ± 10 pre-HIT), HAD (27 ± 6 vs. 29 ± 3) and PFK (340 ± 69 vs. 318 ± 105) and the capillary to fiber ratio (2.30 ± 0.16 vs. 2.38 ± 0.20) was unaltered following HIT. V˙O2 kinetics was unchanged with HIT and the speed of the primary response did not differ...... of oxidative enzymes in fast-twitch fibers, and did not change V˙O2 kinetics....

  7. Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase. (United States)

    Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen; Christoffersen, Stig; Poulsen, Jens-Christian Navarro; Mølgaard, Anne; Kadziola, Anders


    The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2 was subjected to crystallographic, kinetic, and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product adenosine monophosphate (AMP) or the inhibitor adenosine diphosphate (ADP) in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its evolutionary origin in this family of PRTases. Only the N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded β-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor ADP binds like the product AMP with both the α- and β-phosphates occupying the 5'-phosphoribosyl binding site. The enzyme shows activity over a wide pH range, and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme, may be facilitated by elements in the C-terminal three-helix bundle part of the protein.

  8. Prediction of interindividual variation in drug plasma levels in vivo from individual enzyme kinetic data and physiologically based pharmacokinetic modeling

    NARCIS (Netherlands)

    Bogaards, J.J.P.; Hissink, E.M.; Briggs, M.; Weaver, R.; Jochemsen, R.; Jackson, P.; Bertrand, M.; Bladeren, P. van


    A strategy is presented to predict interindividual variation in drug plasma levels in vivo by the use of physiologically based pharmacokinetic modeling and human in vitro metabolic parameters, obtained through the combined use of microsomes containing single cytochrome P450 enzymes and a human liver

  9. Red Seaweed Enzyme-Catalyzed Bromination of Bromophenol Red: An Inquiry-Based Kinetics Laboratory Experiment for Undergraduates (United States)

    Jittam, Piyachat; Boonsiri, Patcharee; Promptmas, Chamras; Sriwattanarothai, Namkang; Archavarungson, Nattinee; Ruenwongsa, Pintip; Panijpan, Bhinyo


    Haloperoxidase enzymes are of interest for basic and applied bioscientists because of their increasing importance in pharmaceutical industry and environmental cleanups. In a guided inquiry-based laboratory experiment for life-science, agricultural science, and health science undergraduates, the bromoperoxidase from a red seaweed was used to…

  10. Adsorption of Trametes versicolor laccase to soil iron and aluminum minerals: enzyme activity, kinetics and stability studies. (United States)

    Wu, Yue; Jiang, Ying; Jiao, Jiaguo; Liu, Manqiang; Hu, Feng; Griffiths, Bryan S; Li, Huixin


    Laccases play an important role in the degradation of soil phenol or phenol-like substance and can be potentially used in soil remediation through immobilization. Iron and aluminum minerals can adsorb extracellular enzymes in soil environment. In the present study, we investigated the adsorptive interaction of laccase, from the white-rot fungus Trametes versicolor, with soil iron and aluminum minerals and characterized the properties of the enzyme after adsorption to minerals. Results showed that both soil iron and aluminum minerals adsorbed great amount of laccase, independent of the mineral specific surface areas. Adsorbed laccases retained 26-64% of the activity of the free enzyme. Compared to the free laccase, all adsorbed laccases showed higher Km values and lower Vmax values, indicating a reduced enzyme-substrate affinity and a lower rate of substrate conversion in reactions catalyzed by the adsorbed laccase. Adsorbed laccases exhibited increased catalytic activities compared to the free laccase at low pH, implying the suitable application of iron and aluminum mineral-adsorbed T. versicolor laccase in soil bioremediation, especially in acid soils. In terms of the thermal profiles, adsorbed laccases showed decreased thermal stability and higher temperature sensitivity relative to the free laccase. Moreover, adsorption improved the resistance of laccase to proteolysis and extended the lifespan of laccase. Our results implied that adsorbed T. versicolor laccase on soil iron and aluminum minerals had promising potential in soil remediation. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  11. Inhibitory and enzyme-kinetic investigation of chelerythrine and lupeol isolated from Zanthoxylum rhoifolium against krait snake venom acetylcholinesterase

    Energy Technology Data Exchange (ETDEWEB)

    Ahmad, Mustaq, E-mail: [University of Science and Technology, Bannu, (Pakistan). Department of Biotechnology; Weber, Andrea D.; Zanon, Graciane; Tavares, Luciana de C.; Ilha, Vinicius; Dalcol, Ionara I.; Morel, Ademir F., E-mail: [Universidade Federal de Santa Maria, RS (Brazil). Dept. de Quimica


    The in vitro activity of chelerythrine and lupeol, two metabolites isolated from Zanthoxylum rhoifolium were studied against the venom of the snake Bungarus sindanus (Elapidae). The venom, which is highly toxic to humans, consists mainly by the enzyme acetylcholinesterase (AChE). Both compounds showed activity against the venom, and the alkaloid chelerythrine presented higher activity than did triterpene lupeol. (author)

  12. Effect of morin on pharmacokinetics of piracetam in rats, in vitro enzyme kinetics and metabolic stability assay using rapid UPLC method. (United States)

    Sahu, Kapendra; Shaharyar, Mohammad; Siddiqui, Anees A


    The aim of this study was to investigate the effect of Morin on the pharmacokinetics of Piracetam in rats, in vitro enzyme kinetics and metabolic stability (high throughput) studies using human liver microsomes in UPLC. For pharmacokinetics studies, male Wistar rats were pretreated with Morin (10 mg/kg) for one week and on the last day, a single dose of Piracetam (50 mg/kg) was given orally. In another group, both Morin and Piracetam were co-administered to evaluate the acute effect of Morin on Piracetam. The control group received oral distilled water for one week and administered with Piracetam on the last day. As Morin is an inhibitor of P- Glycoprotein (P-gp) and CYP 3A, it was anticipated to improve the bioavailability of Piracetam. Amazingly, relative to control, the areas under the concentration time curve and peak plasma concentration of Piracetam were 1.50- and 1.45-fold, respectively, greater in the Morin-pretreated group. However, co-administration of Morin had no significant effect on these parameters. Apart from the aforementioned merits, the results of this study are further confirmed by clinical trials; Piracetam dosages should be adjusted to avoid potential drug interaction when Piracetam is used clinically in combination with Morin and Morin-containing dietary supplements. The in vitro enzyme kinetics were performed to determined km, Vmax & CLins . The in vitro metabolic stability executed for the estimation of metabolic rate constant and half-life of Piracetam. These studies also extrapolate to in vivo intrinsic hepatic clearance (Clint, in vivo ) from in vitro intrinsic hepatic clearance (CLint, in vitro ). Copyright © 2012 John Wiley & Sons, Ltd.

  13. Synthesis, enzyme inhibitory kinetics mechanism and computational study of N-(4-methoxyphenethyl-N-(substituted-4-methylbenzenesulfonamides as novel therapeutic agents for Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Muhammad Athar Abbasi


    Full Text Available The present study comprises the synthesis of a new series of sulfonamides derived from 4-methoxyphenethylamine (1. The synthesis was initiated by the reaction of 1 with 4-methylbenzenesulfonyl chloride (2 in aqueous sodium carbonate solution at pH 9 to yield N-(4-methoxyphenethyl-4-methylbenzensulfonamide (3.This parent molecule 3 was subsequently treated with various alkyl/aralkyl halides, (4a–j, using N,N-dimethylformamide (DMF as solvent and LiH as activator to produce a series of new N-(4-methoxyphenethyl-N-(substituted-4-methylbenzenesulfonamides (5a–j. The structural characterization of these derivatives was carried out by spectroscopic techniques like IR, 1H-NMR, and 13C-NMR. The elemental analysis data was also coherent with spectral data of these molecules. The inhibitory effects on acetylcholinesterase and DPPH were evaluated and it was observed that N-(4-Methoxyphenethyl-4-methyl-N-(2-propylbenzensulfonamide (5c showed acetylcholinesterase inhibitory activity 0.075 ± 0.001 (IC50 0.075 ± 0.001 µM comparable to Neostigmine methylsulfate (IC50 2.038 ± 0.039 µM.The docking studies of synthesized ligands 5a–j were also carried out against acetylcholinesterase (PDBID 4PQE to compare the binding affinities with IC50 values. The kinetic mechanism analyzed by Lineweaver-Burk plots demonstrated that compound (5c inhibits the acetylcholinesterase competitively to form an enzyme inhibitor complex. The inhibition constants Ki calculated from Dixon plots for compound (5c is 2.5 µM. It was also found from kinetic analysis that derivative 5c irreversible enzyme inhibitor complex. It is proposed on the basis of our investigation that title compound 5c may serve as lead structure for the design of more potent acetylcholinesterase inhibitors.


    Sabiu, Saheed; Ajani, Emmanuel Oladipo; Sunmonu, Taofik Olatunde; Ashafa, Anofi Omotayo Tom


    The continuous search for new lead compounds as viable inhibitors of specific enzymes linked to carbohydrate metabolism has intensified. Cyperus esculentus L. is one of the therapeutically implicated botanicals against several degenerative diseases including diabetes mellitus. This study evaluated the antioxidant and mechanism(s) of inhibitory potential of aqueous extract of C. esculentus on α-amylase and α-glucosidase in vitro . The extract was investigated for its radical scavenging and hypoglycaemic potentials using standard experimental procedures. Lineweaver-Burke plot was used to predict the manner in which the enzymes were inhibited. The data obtained revealed that the extract moderately and potently inhibited the specific activities of α -amylase and α -glucosidase, respectively. The inhibition was concentration-related with respective IC 50 values of 5.19 and 0.78 mg/mL relative to that of the control (3.72 and 3.55 mg/mL). The extract also significantly scavenged free radicals and the effects elicited could be ascribed to its phytoconstituents. The respective competitive and non-competitive mode of action of the extract is due to its inhibitory potentials on the activities of α -amylase and α -glucosidase. Going forward, in addition to completely characterize the exact compound(s) responsible for the elicited activity in this study, pertinent attention will be given to the in vivo evaluation of the identified constituents.

  15. Dose-Dependent Change in Elimination Kinetics of Ethanol due to Shift of Dominant Metabolizing Enzyme from ADH 1 (Class I to ADH 3 (Class III in Mouse

    Directory of Open Access Journals (Sweden)

    Takeshi Haseba


    Full Text Available ADH 1 and ADH 3 are major two ADH isozymes in the liver, which participate in systemic alcohol metabolism, mainly distributing in parenchymal and in sinusoidal endothelial cells of the liver, respectively. We investigated how these two ADHs contribute to the elimination kinetics of blood ethanol by administering ethanol to mice at various doses, and by measuring liver ADH activity and liver contents of both ADHs. The normalized AUC (AUC/dose showed a concave increase with an increase in ethanol dose, inversely correlating with β. CLT (dose/AUC linearly correlated with liver ADH activity and also with both the ADH-1 and -3 contents (mg/kg B.W.. When ADH-1 activity was calculated by multiplying ADH-1 content by its Vmax⁡/mg (4.0 and normalized by the ratio of liver ADH activity of each ethanol dose to that of the control, the theoretical ADH-1 activity decreased dose-dependently, correlating with β. On the other hand, the theoretical ADH-3 activity, which was calculated by subtracting ADH-1 activity from liver ADH activity and normalized, increased dose-dependently, correlating with the normalized AUC. These results suggested that the elimination kinetics of blood ethanol in mice was dose-dependently changed, accompanied by a shift of the dominant metabolizing enzyme from ADH 1 to ADH 3.

  16. Misconceptions regarding basic thermodynamics and enzyme kinetics have led to erroneous conclusions regarding the metabolic importance of lactate dehydrogenase isoenzyme expression. (United States)

    Bak, Lasse K; Schousboe, Arne


    Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate involving the coenzyme NAD + . Part of the foundation for the proposed shuttling of lactate from astrocytes to neurons during brain activation is the differential distribution of LDH isoenzymes between the two cell types. In this short review, we outline the basic kinetic properties of the LDH isoenzymes expressed in neurons and astrocytes, and argue that the distribution of LDH isoenzymes does not in any way govern directional flow of lactate between the two cellular compartments. The two main points are as follows. First, in line with the general concept of chemical catalysis, enzymes do not influence the thermodynamic equilibrium of a chemical reaction but merely the speed at which equilibrium is obtained. Thus, differential distribution of LDH isoenzymes with different kinetic parameters does not predict which cells are producing and which are consuming lactate. Second, the thermodynamic equilibrium of the reaction is toward the reduced substrate (i.e., lactate), which is reflected in the concentrations measured in brain tissue, suggesting that the reaction is at near-equilibrium at steady state. To conclude, the cellular distribution of LDH isoenzymes is of little if any consequence in determining any directional flow of lactate between neurons and astrocytes. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Enzyme Informatics (United States)

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika


    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  18. A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent

    Directory of Open Access Journals (Sweden)

    Lykke Pedersen


    Full Text Available Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

  19. Kinetic Studies on Enzyme-Catalyzed Reactions: Oxidation of Glucose, Decomposition of Hydrogen Peroxide and Their Combination (United States)

    Tao, Zhimin; Raffel, Ryan A.; Souid, Abdul-Kader; Goodisman, Jerry


    The kinetics of the glucose oxidase-catalyzed reaction of glucose with O2, which produces gluconic acid and hydrogen peroxide, and the catalase-assisted breakdown of hydrogen peroxide to generate oxygen, have been measured via the rate of O2 depletion or production. The O2 concentrations in air-saturated phosphate-buffered salt solutions were monitored by measuring the decay of phosphorescence from a Pd phosphor in solution; the decay rate was obtained by fitting the tail of the phosphorescence intensity profile to an exponential. For glucose oxidation in the presence of glucose oxidase, the rate constant determined for the rate-limiting step was k = (3.0 ± 0.7) ×104 M−1s−1 at 37°C. For catalase-catalyzed H2O2 breakdown, the reaction order in [H2O2] was somewhat greater than unity at 37°C and well above unity at 25°C, suggesting different temperature dependences of the rate constants for various steps in the reaction. The two reactions were combined in a single experiment: addition of glucose oxidase to glucose-rich cell-free media caused a rapid drop in [O2], and subsequent addition of catalase caused [O2] to rise and then decrease to zero. The best fit of [O2] to a kinetic model is obtained with the rate constants for glucose oxidation and peroxide decomposition equal to 0.116 s−1 and 0.090 s−1 respectively. Cellular respiration in the presence of glucose was found to be three times as rapid as that in glucose-deprived cells. Added NaCN inhibited O2 consumption completely, confirming that oxidation occurred in the cellular mitochondrial respiratory chain. PMID:19348778

  20. Real-time investigation of human topoisomerase I reaction kinetics using an optical sensor: a fast method for drug screening and determination of active enzyme concentrations (United States)

    Kristoffersen, Emil L.; Jørgensen, Line A.; Franch, Oskar; Etzerodt, Michael; Frøhlich, Rikke; Bjergbæk, Lotte; Stougaard, Magnus; Ho, Yi-Ping; Knudsen, Birgitta R.


    Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment.Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using

  1. ²H kinetic isotope effects and pH dependence of catalysis as mechanistic probes of rat monoamine oxidase A: comparisons with the human enzyme. (United States)

    Wang, Jin; Edmondson, Dale E


    Monoamine oxidase A (MAO A) is a mitochondrial outer membrane-bound flavoenzyme important in the regulation of serotonin and dopamine levels. Because the rat is extensively used as an animal model in drug studies, it is important to understand how rat MAO A behaves in comparison with the more extensively studied human enzyme. For many reversible inhibitors, rat MAO A exhibits K(i) values similar to those of human MAO A. The pH profile of k(cat) for rat MAO A shows a pK(a) of 8.2 ± 0.1 for the benzylamine ES complex and pK(a) values of 7.5 ± 0.1 and 7.6 ± 0.1 for the ES complexes with p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine, respectively. In contrast to the human enzyme, the rat enzyme exhibits a single pK(a) value (8.3 ± 0.1) with k(cat)/K(m) for benzylamine versus pH and pK(a) values of 7.8 ± 0.1 and 8.1 ± 0.2 for the ascending limbs, respectively, of k(cat)/K(m) versus pH profiles for p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine and 9.3 ± 0.1 and 9.1 ± 0.2 for the descending limbs, respectively. The oxidation of para-substituted benzylamine substrate analogues by rat MAO A has large deuterium kinetic isotope effects on k(cat) and on k(cat)/K(m). These effects are pH-independent and range from 7 to 14, demonstrating a rate-limiting α-C-H bond cleavage step in catalysis. Quantitative structure-activity correlations of log k(cat) with the electronic substituent parameter (σ) at pH 7.5 and 9.0 show a dominant contribution with positive ρ values (1.2-1.3) and a pH-independent negative contribution from the steric term. Quantitative structure-activity relationship analysis of the binding affinities of the para-substituted benzylamine analogues for rat MAO A shows an increased van der Waals volume (V(w)) increases the affinity of the deprotonated amine for the enzyme. These results demonstrate that rat MAO A exhibits functional properties similar but not identical with those of the human enzyme and provide additional support for C-H bond cleavage via a polar

  2. Real-time investigation of human topoisomerase I reaction kinetics using an optical sensor: a fast method for drug screening and determination of active enzyme concentrations. (United States)

    Kristoffersen, Emil L; Jørgensen, Line A; Franch, Oskar; Etzerodt, Michael; Frøhlich, Rikke; Bjergbæk, Lotte; Stougaard, Magnus; Ho, Yi-Ping; Knudsen, Birgitta R


    Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment.

  3. Determination of glutamate dehydrogenase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry). (United States)

    Botman, Dennis; Tigchelaar, Wikky; Van Noorden, Cornelis J F


    Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)(+) to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we assessed the effects of various glutamate concentrations in combination with either the coenzyme NAD(+) or NADP(+) on GDH activity in mouse liver cryostat sections using metabolic mapping. NAD(+)-dependent GDH V(max) was 2.5-fold higher than NADP(+)-dependent V(max), whereas the K(m) was similar, 1.92 mM versus 1.66 mM, when NAD(+) or NADP(+) was used, respectively. With either coenzyme, V(max) was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a K(i) of 12.2 and 3.95 for NAD(+) and NADP(+) used as coenzyme, respectively. NAD(+)- and NADP(+)-dependent GDH activities were examined in various mouse tissues. GDH activity was highest in liver and much lower in other tissues. In all tissues, the highest activity was found when NAD(+) was used as a coenzyme. In conclusion, GDH activity in mice is highest in the liver with NAD(+) as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 mM. © The Author(s) 2014.

  4. Isotope partitioning for NAD-malic enzyme from Ascaris suum confirms a steady-state random kinetic mechanism

    International Nuclear Information System (INIS)

    Chen, C.Y.; Harris, B.G.; Cook, P.F.


    Isotope partitioning studies beginning with E-[ 14 C]NAD, E-[ 14 C] malate, E-[ 14 C] NAD-Mg 2+ , and E-Mg-[ 14 C]malate suggest a steady-state random mechanism for the NAD-malic enzyme. Isotope trapping beginning with E-[ 14 C]NAD and with varying concentrations of Mg 2+ and malate in the chase solution indicates that Mg 2+ is added in rapid equilibrium and must be added prior to malate for productive ternary complex formation. Equal percentage trapping from E-[ 14 C]NAD-Mg and E-Mg-[ 14 C] malate indicates the mechanism is steady-state random with equal off-rates for NAD and malate from E-NAD-Mg-malate. The off-rates for both do not change significantly in the ternary E-Mg-malate and E-NAD-Mg complexes, nor does the off-rate change for NAD from E-NAD. No trapping of malate was obtained from E-[ 14 C] malate, suggesting that this complex is nonproductive. A quantitative analysis of the data allows an estimation of values for a number of the rate constants along the reaction pathway

  5. Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen


    The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2, was subjected to crystallographic, kinetic and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5...

  6. Mechanistic studies on β-ketoacyl thiolase from Zoogloea ramigera: Identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes

    International Nuclear Information System (INIS)

    Thompson, S.; Mayerl, F.; Walsh, C.T.; Peoples, O.P.; Masamune, S.; Sinskey, A.J.


    Thiolase proceeds via covalent catalysis involving an acetyl-S-enzyme. The active-site thiol nucleophile is identified as Cys 89 by acetylation with [ 14 C]acetyl-CoA, rapid denaturation, tryptic digestion, and sequencing of the labeled peptide. The native acetyl enzyme is labile to hydrolytic decomposition with t 1/2 of 2 min at pH 7, 25 degree C. Cys 89 has been converted to the alternate nucleophile Ser 89 by mutagenesis and the C89S enzyme overproduced, purified, and assessed for activity. The Ser 89 enzyme retains 1% of the V max of the Cys 89 enzyme in the direction of acetoacetyl-CoA thiolytic cleavage and 0.05% of the V max in the condensation of two acetyl-CoA molecules. A covalent acetyl-O-enzyme intermediate is detected on incubation with [ 14 C]acetyl-CoA and isolation of the labeled Ser 89 -containing tryptic peptide. Comparisons of the Cys 89 and Ser 89 enzymes have been made for kinetic and thermodynamic stability of the acetyl enzyme intermediates both by isolation and by analysis of [ 32 P]CoASH/acetyl-CoA partial reactions and for rate-limiting steps in catalysis with trideuterioacetyl-CoA

  7. Rye Bran Modified with Cell Wall-Degrading Enzymes Influences the Kinetics of Plant Lignans but Not of Enterolignans in Multicatheterized Pigs. (United States)

    Bolvig, Anne K; Nørskov, Natalja P; van Vliet, Sophie; Foldager, Leslie; Curtasu, Mihai V; Hedemann, Mette S; Sørensen, Jens F; Lærke, Helle N; Bach Knudsen, Knud E


    Background: Whole-grain intake is associated with a lower risk of chronic Western-style diseases, possibly brought about by the high concentration of phytochemicals, among them plant lignans (PLs), in the grains. Objective: We studied whether treatment of rye bran with cell wall-degrading enzymes changed the solubility and kinetics of PLs in multicatheterized pigs. Methods: Ten female Duroc × Danish Landrace × Yorkshire pigs (60.3 ± 2.3 kg at surgery) fitted with permanent catheters were included in an incomplete crossover study. The pigs were fed 2 experimental diets for 1-7 d. The diets were rich in PLs and based on nontreated lignan-rich [LR; lignan concentration: 20.2 mg dry matter (DM)/kg] or enzymatically treated lignan-rich (ENZLR; lignan concentration: 27.8 mg DM/kg) rye bran. Plasma concentrations of PLs and enterolignans were quantified with the use of targeted LC-tandem mass spectrometry. Data were log transformed and analyzed with mixed-effects, 1-compartment, and asymptotic regression models. Results: The availability of PLs was 38% greater in ENZLR than in LR, and the soluble fraction of PLs was 49% in ENZLR compared with 35% in LR diets. PLs appeared in the circulation 30 min after intake of both the ENZLR and LR diets. Postprandially, consumption of ENZLR resulted in a 4-times-greater ( P concentration compared with LR. The area under the curve (AUC) measured 0-360 min after ENZLR intake was ∼2 times higher than after LR intake. A 1-compartment model could describe the postprandial increase in plasma concentration after ENZLR intake, whereas an asymptotic regression model described the plasma concentrations after LR intake. Despite increased available and soluble PLs, ENZLR did not increase plasma enterolignans. Conclusion: The modification of rye bran with cell wall-degrading enzymes resulted in significantly greater plasma concentrations of PLs and the 4-h AUC, particularly syringaresinol, in multicatheterized pigs. © 2017 American Society

  8. Enzyme Kinetics and Molecular Docking Studies on Cytochrome 2B6, 2C19, 2E1, and 3A4 Activities by Sauchinone

    Directory of Open Access Journals (Sweden)

    Eun Chae Gong


    Full Text Available Sauchinone, an active lignan isolated from the aerial parts of Saururus chinensis (Saururaceae, exhibits anti-inflammatory, anti-obesity, anti-hyperglycemic, and anti-hepatic steatosis effects. As herb–drug interaction (HDI through cytochrome P450s (CYPs-mediated metabolism limits clinical application of herbs and drugs in combination, this study sought to explore the enzyme kinetics of sauchinone towards CYP inhibition in in vitro human liver microsomes (HLMs and in vivo mice studies and computational molecular docking analysis. In in vitro HLMs, sauchinone reversibly inhibited CYP2B6, 2C19, 2E1, and 3A4 activities in non-competitive modes, showing inhibition constant (Ki values of 14.3, 16.8, 41.7, and 6.84 μM, respectively. Also, sauchinone time-dependently inhibited CYP2B6, 2E1 and 3A4 activities in vitro HLMs. Molecular docking study showed that sauchinone could be bound to a few key amino acid residues in the active site of CYP2B6, 2C19, 2E1, and 3A4. When sibutramine, clopidogrel, or chlorzoxazone was co-administered with sauchinone to mice, the systemic exposure of each drug was increased compared to that without sauchinone, because sauchinone reduced the metabolic clearance of each drug. In conclusion, when sauchinone was co-treated with drugs metabolized via CYP2B6, 2C19, 2E1, or 3A4, sauchinone–drug interactions occurred because sauchinone inhibited the CYP-mediated metabolic activities.

  9. Combining Microbial Enzyme Kinetics Models with Light Use Efficiency Models to Predict CO2 and CH4 Ecosystem Exchange from Flooded and Drained Peatland Systems (United States)

    Oikawa, P. Y.; Jenerette, D.; Knox, S. H.; Sturtevant, C. S.; Verfaillie, J. G.; Baldocchi, D. D.


    Under California's Cap-and-Trade program, companies are looking to invest in land-use practices that will reduce greenhouse gas (GHG) emissions. The Sacramento-San Joaquin River Delta is a drained cultivated peatland system and a large source of CO2. To slow soil subsidence and reduce CO2 emissions, there is growing interest in converting drained peatlands to wetlands. However, wetlands are large sources of CH4 that could offset CO2-based GHG reductions. The goal of our research is to provide accurate measurements and model predictions of the changes in GHG budgets that occur when drained peatlands are restored to wetland conditions. We have installed a network of eddy covariance towers across multiple land use types in the Delta and have been measuring CO2 and CH4 ecosystem exchange for multiple years. In order to upscale these measurements through space and time we are using these data to parameterize and validate a process-based biogeochemical model. To predict gross primary productivity (GPP), we are using a simple light use efficiency (LUE) model which requires estimates of light, leaf area index and air temperature and can explain 90% of the observed variation in GPP in a mature wetland. To predict ecosystem respiration we have adapted the Dual Arrhenius Michaelis-Menten (DAMM) model. The LUE-DAMM model allows accurate simulation of half-hourly net ecosystem exchange (NEE) in a mature wetland (r2=0.85). We are working to expand the model to pasture, rice and alfalfa systems in the Delta. To predict methanogenesis, we again apply a modified DAMM model, using simple enzyme kinetics. However CH4 exchange is complex and we have thus expanded the model to predict not only microbial CH4 production, but also CH4 oxidation, CH4 storage and the physical processes regulating the release of CH4 to the atmosphere. The CH4-DAMM model allows accurate simulation of daily CH4 ecosystem exchange in a mature wetland (r2=0.55) and robust estimates of annual CH4 budgets. The LUE

  10. Kinetic studies of the acylation of pig muscle–d-glyceraldehyde 3-phosphate dehydrogenase by 1,3-diphosphoglycerate and of proton uptake and release in the overall enzyme mechanism (United States)

    Harrigan, P. J.; Trentham, D. R.


    In the presence of NAD+ the acylation by 1,3-diphosphoglycerate of the four active sites of pig muscle d-glyceraldehyde 3-phosphate dehydrogenase can be monitored at 365nm by the disappearance of the absorption band present in the binary complex of NAD+ and the enzyme. A non-specific salt effect decreased the acylation rate 25-fold when the ionic strength was increased from 0.10 to 1.0. This caused acylation to be the rate-limiting process in the enzyme-catalysed reductive dephosphorylation of 1,3-diphosphoglycerate at high ionic strength at pH8. The salt effect permitted investigation of the acylation over a wide range of conditions. Variation of pH from 5.4 to 8.6 produced at most a two-fold change in the acylation rate. One proton was taken up per site acylated at pH8.0. By using a chromophoric H+ indicator the rate of proton uptake could be monitored during the acylation and was also almost invariant in the pH range 5.5–8.5. Transient kinetic studies of the overall enzyme-catalysed reaction indicated that acylation was the process involving proton uptake at pH8.0. The enzyme mechanism is discussed in the light of these results. PMID:4360248

  11. A chimeric LysK-lysostaphin fusion enzyme lysing Staphylococcus aureus cells: a study of both kinetics of inactivation and specifics of interaction with anionic polymers (United States)

    A staphylolytic fusion protein (K-L) was created, harboring three unique lytic activities comprised of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of K-L...

  12. Kinetic characterization of glucose aerodehydrogenase from Aspergillus niger EMS-150-F after optimizing the dose of mutagen for enhanced production of enzyme


    Umbreen, Huma; Zia, Muhammad Anjum; Rasul, Samreen


    In the present study enhanced production of glucose aerodehydrogenase from Aspergillus niger has been achieved after optimizing the dose of chemical mutagen ethyl methane sulfonate (EMS) that has not been reported earlier. Different doses of mutagen were applied and a strain was developed basing upon the best production. The selected strain Aspergillus niger EMS-150-F was optimized for nutrient requirements in order to produce enzyme through fermentation and the results showed the best yield ...

  13. The role of Val-265 for flavin adenine dinucleotide (FAD) binding in pyruvate oxidase: FTIR, kinetic, and crystallographic studies on the enzyme variant V265A. (United States)

    Wille, Georg; Ritter, Michaela; Weiss, Manfred S; König, Stephan; Mäntele, Werner; Hübner, Gerhard


    In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.

  14. Peroxisomal multifunctional enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics. (United States)

    Haataja, Tatu J K; Koski, M Kristian; Hiltunen, J Kalervo; Glumoff, Tuomo


    All of the peroxisomal β-oxidation pathways characterized thus far house at least one MFE (multifunctional enzyme) catalysing two out of four reactions of the spiral. MFE type 2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for Drosophila melanogaster MFE-2 (DmMFE-2), complements the Saccharomyces cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure-function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both the structural features, as well as by the fact that hydration and dehydrogenation activities of MFE-2, if produced as separate enzymes, are equally efficient in catalysis as the full-length MFE-2.

  15. Pancreatic Enzymes (United States)

    ... Contact Us DONATE NOW GENERAL DONATION PURPLESTRIDE Pancreatic enzymes Home Facing Pancreatic Cancer Living with Pancreatic Cancer ... and see a registered dietitian. What are pancreatic enzymes? Pancreatic enzymes help break down fats, proteins and ...

  16. Determination of Glutamic Acid Decarboxylase (GAD65 in Pancreatic Islets and Its In Vitro and In Vivo Degradation Kinetics in Serum Using a Highly Sensitive Enzyme Immunoassay

    Directory of Open Access Journals (Sweden)

    Michael Schlosser


    Full Text Available Glutamic acid decarboxylase GAD65 autoantibodies (GADA are an established marker for autoimmune diabetes. Recently, the autoantigen GAD65 itself was proposed as biomarker of beta-cell loss for prediction of autoimmune diabetes and graft rejection after islet transplantation. Therefore, the GAD65 content in pancreatic islets of different species and its serum degradation kinetics were examined in this study using a sensitive immunoassay. GAD65 was found in quantities of 78 (human, 43.7 (LEW.1A rat and 37.4 (BB/OK rat ng per 1,000 islets, respectively, but not in mouse islets. The in vitro half-life of porcine GAD65 and human recombinant GAD65 ranged from 1.27 to 2.35 hours at 37°C in human serum, plasma and blood, and was unaffected by presence of GAD65 autoantibodies. After injecting 2,000 ng recombinant human GAD65 into LEW.1A rats, the in vivo half-life was 2.77 hours. GAD65 was undetectable after 24 hours in these animals, and for up to 48 hours following diabetes induction by streptozotocin in LEW.1A rats. Estimated from these data, at least 13 islets in rat and 1,875 in human must be simultaneously destroyed to detect GAD65 in circulation. These results should be taken into consideration in further studies aimed at examining the diagnostic relevance of GAD65.

  17. Combined pressure and cosolvent effects on enzyme activity - a high-pressure stopped-flow kinetic study on α-chymotrypsin. (United States)

    Luong, Trung Quan; Winter, Roland


    We investigated the combined effects of cosolvents and pressure on the hydrolysis of a model peptide catalysed by α-chymotrypsin. The enzymatic activity was measured in the pressure range from 0.1 to 200 MPa using a high-pressure stopped-flow systems with 10 ms time resolution. A kosmotropic (trimethalymine-N-oxide, TMAO) and chaotropic (urea) cosolvent and mixtures thereof were used as cosolvents. High pressure enhances the hydrolysis rate as a consequence of a negative activation volume, ΔV(#), which, depending on the cosolvent system, amounts to -2 to -4 mL mol(-1). A more negative activation volume can be explained by a smaller compression of the ES complex relative to the transition state. Kinetic constants, such as kcat and the Michaelis constant KM, were determined for all solution conditions as a function of pressure. With increasing pressure, kcat increases by about 35% and its pressure dependence by a factor of 1.9 upon addition of 2 M urea, whereas 1 M TMAO has no significant effect on kcat and its pressure dependence. Similarly, KM increases upon addition of urea 6-fold. Addition of TMAO compensates the urea-effect on kcat and KM to some extent. The maximum rate of the enzymatic reaction increases with increasing pressure in all solutions except in the TMAO : urea 1 : 2 mixture, where, remarkably, pressure is found to have no effect on the rate of the enzymatic reaction anymore. Our data clearly show that compatible solutes can easily override deleterious effects of harsh environmental conditions, such as high hydrostatic pressures in the 100 MPa range, which is the maximum pressure encountered in the deep biosphere on Earth.

  18. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes. (United States)

    Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika


    Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

  19. Palmito de pupunha (Bactris gasipaes Kunth. composição mineral e cinética de enzimas oxidativas Heart of palm (Bactris gasipaes Kunth.: mineral composition and kinetics of oxidative enzymes

    Directory of Open Access Journals (Sweden)

    Nathália Ottoboni Galdino


    Full Text Available A análise da presença de enzimas oxidativas como a peroxidase (POD e a polifenoloxidase (PPO e o controle da atividade destas enzimas são importantes na preservação e no processamento de alimentos. Este trabalho teve por objetivo determinar a atividade enzimática da polifenoloxidase (PPO e da peroxidase (POD do palmito de pupunha, bem como avaliar o comportamento destas enzimas frente ao tratamento térmico e assim calcular a cinética de inativação térmica das mesmas para suas porções termorresistente e termolábil. Para a extração de peroxidase (POD e polifenoloxidase (PPO de palmito, utilizou-se solução tampão fosfato de sódio 100 mM com diferentes pHs (5,5; 6,0; 6,5 e 7,0. O melhor pH de extração da POD foi 5,5 e da PPO, 6,5. Estes extratos foram tratados em diferentes temperaturas (65, 70, 75 e 80 °C por períodos de 1 a 10 minutos. A POD e a PPO sofreram um decréscimo de 70 e 80%, respectivamente, em relação às suas atividades iniciais. As energias de ativação, nas temperaturas estudadas, para a porção termolábil e termorresistente da peroxidase foram 154,0 e 153,0 kJ.mol-1, respectivamente, enquanto que para a polifenoloxidase foram 26,3 e 27,0 kJ.mol-1, respectivamente. Resultados apresentaram valores que estão dentro da faixa de energia de ativação reportada para o processo de inativação térmica de enzimas.Analysis of oxidative enzymes such as peroxidase (POD and polyphenoloxidase (PPO and the control of the activity of these enzymes are important in food preservation and also in food processing. The aim of this work was to determine polyphenoloxidase (PPO and peroxidase (POD enzymatic activity in heart of palm, as well as to evaluate enzyme behavior during thermal treatment, determining the kinetics of thermal inactivation of the heat resistant and heat labile portions. For the extraction of peroxidase (POD and polyphenoloxidase (PPO from the heart of palm solution, 100 mM sodium phosphate buffer with

  20. Measurement of enzyme activity. (United States)

    Harris, T K; Keshwani, M M


    To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

  1. Enzyme Molecules in Solitary Confinement

    Directory of Open Access Journals (Sweden)

    Raphaela B. Liebherr


    Full Text Available Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

  2. Enzymes: principles and biotechnological applications (United States)

    Robinson, Peter K.


    Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed. PMID:26504249

  3. 21 CFR 862.2500 - Enzyme analyzer for clinical use. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enzyme analyzer for clinical use. 862.2500 Section... Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a device intended to measure enzymes in plasma or serum by nonkinetic or kinetic measurement of...

  4. Kinetic Typography

    DEFF Research Database (Denmark)

    van Leeuwen, Theo; Djonov, Emilia


    After discussing broad cultural drivers behind the development of kinetic typography, the chapter outlines an approach to analysing kinetic typography which is based on Halliday's theory of transitivity, as applied by Kress and Van Leeuwen to visual images.......After discussing broad cultural drivers behind the development of kinetic typography, the chapter outlines an approach to analysing kinetic typography which is based on Halliday's theory of transitivity, as applied by Kress and Van Leeuwen to visual images....

  5. Enzyme technology: Key to selective biorefining

    DEFF Research Database (Denmark)

    Meyer, Anne S.


    to the reaction is a unique trait of enzyme catalysis. Since enzyme selectivity means that a specific reaction is catalysed between particular species to produce definite products, enzymes are particularly fit for converting specific compounds in mixed biomass streams. Since enzymes are protein molecules...... their rational use in biorefinery processes requires an understanding of the basic features of enzymes and reaction traits with respect to specificity, kinetics, reaction optima, stability and structure-function relations – we are now at a stage where it is possible to use nature’s enzyme structures as starting...... point and then improve the functional traits by targeted mutation of the protein. The talk will display some of our recent hypotheses related to enzyme action, recently obtained results within knowledge-based enzyme improvements as well as cast light on research methods used in optimizing enzyme...

  6. In vivo doses of butadiene epoxides as estimated from in vitro enzyme kinetics by using cob(I)alamin and measured hemoglobin adducts: An inter-species extrapolation approach

    International Nuclear Information System (INIS)

    Motwani, Hitesh V.; Törnqvist, Margareta


    1,3-Butadiene (BD) is a rodent and human carcinogen. In the cancer tests, mice have been much more susceptible than rats with regard to BD-induced carcinogenicity. The species-differences are dependent on metabolic formation/disappearance of the genotoxic BD epoxy-metabolites that lead to variations in the respective in vivo doses, i.e. “area under the concentration-time curve” (AUC). Differences in AUC of the most gentoxic BD epoxy-metabolite, diepoxybutane (DEB), are considered important with regard to cancer susceptibility. The present work describes: the application of cob(I)alamin for accurate measurements of in vitro enzyme kinetic parameters associated with BD epoxy-metabolites in human, mouse and rat; the use of published data on hemoglobin (Hb) adduct levels of BD epoxides from BD exposure studies on the three species to calculate the corresponding AUCs in blood; and a parallelogram approach for extrapolation of AUC of DEB based on the in vitro metabolism studies and adduct data from in vivo measurements. The predicted value of AUC of DEB for humans from the parallelogram approach was 0.078 nM · h for 1 ppm · h of BD exposure compared to 0.023 nM · h/ppm · h as calculated from Hb adduct levels observed in occupational exposure. The corresponding values in nM · h/ppm · h were for mice 41 vs. 38 and for rats 1.26 vs. 1.37 from the parallelogram approach vs. experimental exposures, respectively, showing a good agreement. This quantitative inter-species extrapolation approach will be further explored for the clarification of metabolic rates/pharmacokinetics and the AUC of other genotoxic electrophilic compounds/metabolites, and has a potential to reduce and refine animal experiments. - Highlights: • In vitro metabolism to in vivo dose extrapolation of butadiene metabolites was proposed. • A parallelogram approach was introduced to estimate dose (AUC) in humans and rodents. • AUC of diepoxybutane predicted in humans was 0.078 nM h/ppm h

  7. In vivo doses of butadiene epoxides as estimated from in vitro enzyme kinetics by using cob(I)alamin and measured hemoglobin adducts: An inter-species extrapolation approach

    Energy Technology Data Exchange (ETDEWEB)

    Motwani, Hitesh V., E-mail:; Törnqvist, Margareta


    1,3-Butadiene (BD) is a rodent and human carcinogen. In the cancer tests, mice have been much more susceptible than rats with regard to BD-induced carcinogenicity. The species-differences are dependent on metabolic formation/disappearance of the genotoxic BD epoxy-metabolites that lead to variations in the respective in vivo doses, i.e. “area under the concentration-time curve” (AUC). Differences in AUC of the most gentoxic BD epoxy-metabolite, diepoxybutane (DEB), are considered important with regard to cancer susceptibility. The present work describes: the application of cob(I)alamin for accurate measurements of in vitro enzyme kinetic parameters associated with BD epoxy-metabolites in human, mouse and rat; the use of published data on hemoglobin (Hb) adduct levels of BD epoxides from BD exposure studies on the three species to calculate the corresponding AUCs in blood; and a parallelogram approach for extrapolation of AUC of DEB based on the in vitro metabolism studies and adduct data from in vivo measurements. The predicted value of AUC of DEB for humans from the parallelogram approach was 0.078 nM · h for 1 ppm · h of BD exposure compared to 0.023 nM · h/ppm · h as calculated from Hb adduct levels observed in occupational exposure. The corresponding values in nM · h/ppm · h were for mice 41 vs. 38 and for rats 1.26 vs. 1.37 from the parallelogram approach vs. experimental exposures, respectively, showing a good agreement. This quantitative inter-species extrapolation approach will be further explored for the clarification of metabolic rates/pharmacokinetics and the AUC of other genotoxic electrophilic compounds/metabolites, and has a potential to reduce and refine animal experiments. - Highlights: • In vitro metabolism to in vivo dose extrapolation of butadiene metabolites was proposed. • A parallelogram approach was introduced to estimate dose (AUC) in humans and rodents. • AUC of diepoxybutane predicted in humans was 0.078 nM h/ppm h

  8. Enzymes in CO2 Capture

    DEFF Research Database (Denmark)

    Fosbøl, Philip Loldrup; Gladis, Arne; Thomsen, Kaj

    The enzyme Carbonic Anhydrase (CA) can accelerate the absorption rate of CO2 into aqueous solutions by several-fold. It exist in almost all living organisms and catalyses different important processes like CO2 transport, respiration and the acid-base balances. A new technology in the field...... of carbon capture is the application of enzymes for acceleration of typically slow ternary amines or inorganic carbonates. There is a hidden potential to revive currently infeasible amines which have an interesting low energy consumption for regeneration but too slow kinetics for viable CO2 capture. The aim...... of this work is to discuss the measurements of kinetic properties for CA promoted CO2 capture solvent systems. The development of a rate-based model for enzymes will be discussed showing the principles of implementation and the results on using a well-known ternary amine for CO2 capture. Conclusions...

  9. Illustrating Enzyme Inhibition Using Gibbs Energy Profiles (United States)

    Bearne, Stephen L.


    Gibbs energy profiles have great utility as teaching and learning tools because they present students with a visual representation of the energy changes that occur during enzyme catalysis. Unfortunately, most textbooks divorce discussions of traditional kinetic topics, such as enzyme inhibition, from discussions of these same topics in terms of…

  10. Enzyme Catalysis and the Gibbs Energy (United States)

    Ault, Addison


    Gibbs-energy profiles are often introduced during the first semester of organic chemistry, but are less often presented in connection with enzyme-catalyzed reactions. In this article I show how the Gibbs-energy profile corresponds to the characteristic kinetics of a simple enzyme-catalyzed reaction. (Contains 1 figure and 1 note.)

  11. Advances in enzyme bioelectrochemistry

    Directory of Open Access Journals (Sweden)


    Full Text Available ABSTRACT Bioelectrochemistry can be defined as a branch of Chemical Science concerned with electron-proton transfer and transport involving biomolecules, as well as electrode reactions of redox enzymes. The bioelectrochemical reactions and system have direct impact in biotechnological development, in medical devices designing, in the behavior of DNA-protein complexes, in green-energy and bioenergy concepts, and make it possible an understanding of metabolism of all living organisms (e.g. humans where biomolecules are integral to health and proper functioning. In the last years, many researchers have dedicated itself to study different redox enzymes by using electrochemistry, aiming to understand their mechanisms and to develop promising bioanodes and biocathodes for biofuel cells as well as to develop biosensors and implantable bioelectronics devices. Inside this scope, this review try to introduce and contemplate some relevant topics for enzyme bioelectrochemistry, such as the immobilization of the enzymes at electrode surfaces, the electron transfer, the bioelectrocatalysis, and new techniques conjugated with electrochemistry vising understand the kinetics and thermodynamics of redox proteins. Furthermore, examples of recent approaches in designing biosensors and biofuel developed are presented.

  12. Physical kinetics

    International Nuclear Information System (INIS)

    Lifschitz, E.M.; Pitajewski, L.P.


    The textbook covers the subject under the following headings: kinetic gas theory, diffusion approximation, collisionless plasma, collisions within the plasma, plasma in the magnetic field, theory of instabilities, dielectrics, quantum fluids, metals, diagram technique for nonequilibrium systems, superconductors, and kinetics of phase transformations

  13. Heparin kinetics

    International Nuclear Information System (INIS)

    Swart, C.A.M. de.


    The author has studied the kinetics of heparin and heparin fractions after intravenous administration in humans and in this thesis the results of this study are reported. Basic knowledge about the physico-chemical properties of heparin and its interactions with proteins resulting in anticoagulant and lipolytic effects are discussed in a review (chapter II), which also comprises some clinical aspects of heparin therapy. In chapter III the kinetics of the anticoagulant effect are described after intravenous administration of five commercial heparin preparations. A mathematical model is presented that fits best to these kinetics. The kinetics of the anticoagulant and lipolytic effects after intravenous injection of various 35 S-radiolabelled heparin fractions and their relationship with the disappearance of the radiolabel are described in chapter IV. Chapter V gives a description of the kinetics of two radiolabels after injection of in vitro formed complexes consisting of purified, 125 I-radiolabelled antithrombin III and various 35 S-radiolabelled heparin fractions. (Auth.)

  14. Application of HPLC to study the kinetics of a branched bi-enzyme system consisting of hypoxanthine-guanine phosphoribosyltransferase and xanthine oxidase--an important biochemical system to evaluate the efficiency of the anticancer drug 6-mercaptopurine in ALL cell line. (United States)

    Kalra, Sukirti; Paul, Manash K; Balaram, Hemalatha; Mukhopadhyay, Anup Kumar


    The thiopurine antimetabolite 6-mercaptopurine (6MP) is an important chemotherapeutic drug in the conventional treatment of childhood acute lymphoblastic leukemia (ALL). 6MP is mainly catabolized by both hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine oxidase (XOD) to form thioinosinic monophosphate (TIMP) (therapeutically active metabolite) and 6-thiouric acid (6TUA) (inactive metabolite), respectively. The activity of both the enzymes varies among ALL patients governing the active and the inactive metabolite profile within the immature lymphocytes. Therefore, an attempt was made to study the kinetic nature of the branched bi-enzyme system acting on 6MP and to quantitate TIMP and 6TUA formed when the two enzymes are present in equal and variable ratios. The quantification of the branched kinetics using spectrophotometric method presents problem due to the closely apposed lambda(max) of the substrates and products. Hence, employing an HPLC method, the quantification of the products was done with the progress of time. The limit of quantification (LOQ) of substrate was found to be 10nM and for products as 50 nM. The limit of detection (LOD) was found to be 1 nM for the substrate and the products. The method exhibited linearity in the range of 0.01-100 microM for 6MP and 0.05-100 microM for both 6TUA and TIMP. The amount of TIMP formed was higher than that of 6TUA in the bi-enzyme system when both the enzymes were present in equivalent enzymatic ratio. It was further found that enzymatic ratios play an important role in determining the amounts of TIMP and 6TUA. This method was further validated using actively growing T-ALL cell line (Jurkat) to study the branched kinetics, wherein it was observed that treatment of 50 microM 6MP led to the generation of 12 microM TIMP and 0.8 microM 6TUA in 6 h at 37 degrees C.

  15. Rethinking fundamentals of enzyme action. (United States)

    Northrop, D B


    Despite certain limitations, investigators continue to gainfully employ concepts rooted in steady-state kinetics in efforts to draw mechanistically relevant inferences about enzyme catalysis. By reconsidering steady-state enzyme kinetic behavior, this review develops ideas that allow one to arrive at the following new definitions: (a) V/K, the ratio of the maximal initial velocity divided by the Michaelis-Menten constant, is the apparent rate constant for the capture of substrate into enzyme complexes that are destined to yield product(s) at some later point in time; (b) the maximal velocity V is the apparent rate constant for the release of substrate from captured complexes in the form of free product(s); and (c) the Michaelis-Menten constant K is the ratio of the apparent rate constants for release and capture. The physiologic significance of V/K is also explored to illuminate aspects of antibiotic resistance, the concept of "perfection" in enzyme catalysis, and catalytic proficiency. The conceptual basis of congruent thermodynamic cycles is also considered in an attempt to achieve an unambiguous way for comparing an enzyme-catalyzed reaction with its uncatalyzed reference reaction. Such efforts promise a deeper understanding of the origins of catalytic power, as it relates to stabilization of the reactant ground state, stabilization of the transition state, and reciprocal stabilizations of ground and transition states.

  16. Application of dhurrin for kinetics and thermodynamic ...

    African Journals Online (AJOL)

    The entropy change (ΔS) increased with enzyme purity from 0.588 J/mol.deg. to 1.4625Jmol degree. The enthalpy change KJ/mol followed the same pattern whereby increases influenced by enzyme purity ranged from 1892 KJ/mol to 13104KJ/mol. Keywords: kinetics, thermodynamic, characterization, dhurrin, genetically ...

  17. Kinetic Interface

    DEFF Research Database (Denmark)


    A kinetic interface for orientation detection in a video training system is disclosed. The interface includes a balance platform instrumented with inertial motion sensors. The interface engages a participant's sense of balance in training exercises.......A kinetic interface for orientation detection in a video training system is disclosed. The interface includes a balance platform instrumented with inertial motion sensors. The interface engages a participant's sense of balance in training exercises....

  18. Kinetics and

    Directory of Open Access Journals (Sweden)

    Mojtaba Ahmadi


    Full Text Available The aqueous degradation of Reactive Yellow 84 (RY84 by potassium peroxydisulfate (K2S2O8 has been studied in laboratory scale experiments. The effect of the initial concentrations of potassium peroxydisulfate and RY84, pH and temperature on RY84 degradation were also examined. Experimental data were analyzed using first and second-order kinetics. The degradation kinetics of RY84 of the potassium peroxydisulfate process followed the second-order reaction kinetics. These rate constants have an extreme values similar to of 9.493 mM−1min−1 at a peroxydisulfate dose of 4 mmol/L. Thermodynamic parameters such as activation (Ea and Gibbs free energy (ΔG° were also evaluated. The negative value of ΔGo and Ea shows the spontaneous reaction natural conditions and exothermic nature.

  19. Microsecond reaction kinetics and catalytic mechanism of bacterial cytochrome oxidases

    NARCIS (Netherlands)

    Paulus, A.


    Fundamental biochemical research is of crucial importance for a complete and detailed
    understanding of what drives enzyme activity and how enzyme kinetic properties are
    optimized towards survival of the host organism. When cells fail to produce a fully functional
    enzyme, the organism’s

  20. Kinetic studies of alkaline phosphatase extracted from rabbit (Lepus ...

    African Journals Online (AJOL)


    activity, and the kinetic constants-maximum enzyme velocity (Vmax) and Michealis-Menten constant (Km) were evaluated. ... the enzyme a readily available parameter for diagnostic and research .... procedure while treatment means were separated by the least .... mammalian enzymes are responsible for this increase in ...

  1. Granulocyte kinetics

    International Nuclear Information System (INIS)

    Peters, A.M.; Lavender, J.P.; Saverymuttu, S.H.


    By using density gradient materials enriched with autologous plasma, the authors have been able to isolate granulocutes from other cellular elements and label them with In-111 without separation from a plasma environment. The kinetic behavior of these cells suggests that phenomena attributed to granulocyte activation are greatly reduced by this labeling. Here, they review their study of granulocyte kinetics in health and disease in hope of quantifying sites of margination and identifying principal sites of destruction. The three principle headings of the paper are distribution, life-span, and destruction

  2. Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic (United States)

    Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.


    The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

  3. Physisorption kinetics

    CERN Document Server

    Kreuzer, Hans Jürgen


    This monograph deals with the kinetics of adsorption and desorption of molecules physisorbed on solid surfaces. Although frequent and detailed reference is made to experiment, it is mainly concerned with the theory of the subject. In this, we have attempted to present a unified picture based on the master equation approach. Physisorption kinetics is by no means a closed and mature subject; rather, in writing this monograph we intended to survey a field very much in flux, to assess its achievements so far, and to give a reasonable basis from which further developments can take off. For this reason we have included many papers in the bibliography that are not referred to in the text but are of relevance to physisorption. To keep this monograph to a reasonable size, and also to allow for some unity in the presentation of the material, we had to omit a number of topics related to physisorption kinetics. We have not covered to any extent the equilibrium properties of physisorbed layers such as structures, phase tr...

  4. Purification, characterization of phytase enzyme from Lactobacillus ...

    African Journals Online (AJOL)

    Purification, characterization of phytase enzyme from Lactobacillus plantarum bacteria and determination of its kinetic properties. ... Many of the cereal grains, legumes and oilseeds store phosphorus in phytate form. Phytases can be produced by plants, animals and microorganisms. However, the ones with microbial origin ...

  5. Protein engineering of enzymes for process applications

    DEFF Research Database (Denmark)

    Woodley, John M


    opportunities will be targeted on modification to enable process application. This article discusses the challenges involved in enzyme modification focused on process requirements, such as the need to fulfill reaction thermodynamics, specific activity under the required conditions, kinetics at required...... concentrations, and stability. Finally, future research directions are discussed, including the integration of biocatalysis with neighboring chemical steps....

  6. Modeling metabolic response to changes of enzyme amount in ...

    African Journals Online (AJOL)



    Oct 11, 2010 ... In this work, we first introduced the enzyme parameter (ɑ) into the kinetic equations and consequently established an in silico glycolysis model of Saccharomyces cerevisiae in XML format (Figure 1), based on the work of Hynn et al. (2001). Equation 1 shows how the ɑis introduced into the kinetic equation.

  7. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)


    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  8. Stochastic kinetics

    International Nuclear Information System (INIS)

    Colombino, A.; Mosiello, R.; Norelli, F.; Jorio, V.M.; Pacilio, N.


    A nuclear system kinetics is formulated according to a stochastic approach. The detailed probability balance equations are written for the probability of finding the mixed population of neutrons and detected neutrons, i.e. detectrons, at a given level for a given instant of time. Equations are integrated in search of a probability profile: a series of cases is analyzed through a progressive criterium. It tends to take into account an increasing number of physical processes within the chosen model. The most important contribution is that solutions interpret analytically experimental conditions of equilibrium (moise analysis) and non equilibrium (pulsed neutron measurements, source drop technique, start up procedures)

  9. Modeling metabolic response to changes of enzyme amount in ...

    African Journals Online (AJOL)

    Based on the work of Hynne et al. (2001), in an in silico model of glycolysis, Saccharomyces cerevisiae is established by introducing an enzyme amount multiple factor (.) into the kinetic equations. The model is aimed to predict the metabolic response to the change of enzyme amount. With the help of .α, the amounts of ...

  10. Representing Rate Equations for Enzyme-Catalyzed Reactions (United States)

    Ault, Addison


    Rate equations for enzyme-catalyzed reactions are derived and presented in a way that makes it easier for the nonspecialist to see how the rate of an enzyme-catalyzed reaction depends upon kinetic constants and concentrations. This is done with distribution equations that show how the rate of the reaction depends upon the relative quantities of…

  11. Elevated Liver Enzymes (United States)

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  12. Calcite Dissolution Kinetics (United States)

    Berelson, W.; Subhas, A.; Dong, S.; Naviaux, J.; Adkins, J. F.


    A geological buffer for high atmospheric CO2 concentrations is neutralization via reaction with CaCO3. We have been studying the dissolution kinetics of carbonate minerals using labeled 13C calcite and Picarro-based measurements of 13C enrichments in solution DIC. This methodology has greatly facilitated our investigation of dissolution kinetics as a function of water carbonate chemistry, temperature and pressure. One can adjust the saturation state Omega by changing the ion activity product (e.g. adjusting carbonate ion concentration), or by changing the solubility product (e.g. adjusting temperature or pressure). The canonical formulation of dissolution rate vs. omega has been refined (Subhas et al. 2015) and shows distinct non-linear behavior near equilibrium and rates in sea water of 1-3 e-6 g/cm2day at omega = 0.8. Carbonic anhydrase (CA), an enzyme that catalyzes the hydration of dissolved CO2 to carbonic acid, was shown (in concentrations 500x. This result points to the importance of carbonic acid in enhancing dissolution at low degrees of undersaturation. CA activity and abundance in nature must be considered regarding the role it plays in catalyzing dissolution. We also have been investigating the role of temperature on dissolution kinetics. An increase of 16C yields an order of magnitude increase in dissolution rate. Temperature (and P) also change Omega critical, the saturation state where dissolution rates change substantially. Increasing pressure (achieved in a pressure reaction chamber we built) also shifts Omega critical closer to equilibrium and small pressure increases have large impact on dissolution kinetics. Dissolution rates are enhanced by an order of magnitude for a change in pressure of 1500 psi relative to the dissolution rate achieved by water chemistry effects alone for an omega of 0.8. We've shown that the thermodynamic determination of saturation state does not adequately describe the kinetics of dissolution. The interplay of mineral

  13. Enzymes as Biocatalysts for Lipid-based Bioproducts Processing

    DEFF Research Database (Denmark)

    Cheong, Ling-Zhi; Guo, Zheng; Fedosov, Sergey


    Bioproducts are materials, chemicals and energy derived from renewable biological resources such as agriculture, forestry, and biologically-derived wastes. To date, the use of enzymes as biocatalysts for lipid-based bioproducts processing has shown marked increase. This is mainly due to the fact...... that cost benefit derived from enzymatic processing such as enzyme specificity, higher product purity and lesser or none toxic waste disposal has surpassed the cost of biocatalysts itself. This chapter provided insights into distinct enzymes characteristics essential in industrial processing especially...... enzymes kinetics. Understanding of enzyme kinetics is important especially in designing efficient reaction set-ups including type of bioreactors, reaction conditions and reusability of biocatalysts to ensure efficient running cost. A brief review of state-of-the-art in industrial applications of enzymes...

  14. Tolrestat kinetics

    International Nuclear Information System (INIS)

    Hicks, D.R.; Kraml, M.; Cayen, M.N.; Dubuc, J.; Ryder, S.; Dvornik, D.


    The kinetics of tolrestat, a potent inhibitor of aldose reductase, were examined. Serum concentrations of tolrestat and of total 14 C were measured after dosing normal subjects and subjects with diabetes with 14 C-labeled tolrestat. In normal subjects, tolrestat was rapidly absorbed and disappearance from serum was biphasic. Distribution and elimination t 1/2s were approximately 2 and 10 to 12 hr, respectively, after single and multiple doses. Unchanged tolrestat accounted for the major portion of 14 C in serum. Radioactivity was rapidly and completely excreted in urine and feces in an approximate ratio of 2:1. Findings were much the same in subjects with diabetes. In normal subjects, the kinetics of oral tolrestat were independent of dose in the 10 to 800 mg range. Repetitive dosing did not result in unexpected cumulation. Tolrestat was more than 99% bound to serum protein; it did not compete with warfarin for binding sites but was displaced to some extent by high concentrations of tolbutamide or salicylate

  15. Substrate-Dependent Kinetics in Tyrosinase-based Biosensing: Amperometry vs. Spectrophotometry

    NARCIS (Netherlands)

    Rassaei, Liza; Cui, Jin; Goluch, E.D.; Lemay, Serge Joseph Guy


    Despite the broad use of enzymes in electroanalytical biosensors, the influence of enzyme kinetics on the function of prototype sensors is often overlooked or neglected. In the present study, we employ amperometry as an alternative or complementary method to study the kinetics of tyrosinase, whose

  16. Ceramic membrane microfilter as an immobilized enzyme reactor. (United States)

    Harrington, T J; Gainer, J L; Kirwan, D J


    This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.

  17. Descriptive and predictive assessment of enzyme activity and enzyme related processes in biorefinery using IR spectroscopy and chemometrics

    DEFF Research Database (Denmark)

    Baum, Andreas

    the understanding of the structural properties of the extracted pectin. Secondly, enzyme kinetics of biomass converting enzymes was examined in terms of measuring enzyme activity by spectral evolution profiling utilizing FTIR. Chemometric multiway methods were used to analyze the tensor datasets enabling the second......-order calibration advantage (reference Theory of Analytical chemistry). As PAPER 3 illustrates the method is universally applicable without the need of any external standards and was exemplified by performing quantitative enzyme activity determinations for glucose oxidase, pectin lyase and a cellolytic enzyme blend...... (Celluclast 1.5L). In PAPER 4, the concept is extended to quantify enzyme activity of two simultaneously acting enzymes, namely pectin lyase and pectin methyl esterase. By doing so the multiway methods PARAFAC, TUCKER3 and NPLS were compared and evaluated towards accuracy and precision....

  18. Thermal inactivation kinetics of β-galactosidase during bread baking. (United States)

    Zhang, Lu; Chen, Xiao Dong; Boom, Remko M; Schutyser, Maarten A I


    In this study, β-galactosidase was utilized as a model enzyme to investigate the mechanism of enzyme inactivation during bread baking. Thermal inactivation of β-galactosidase was investigated in a wheat flour/water system at varying temperature-moisture content combinations, and in bread during baking at 175 or 205°C. In the wheat flour/water system, the thermostability of β-galactosidase increased with decreased moisture content, and a kinetic model was accurately fitted to the corresponding inactivation data (R 2 =0.99). Interestingly, the residual enzyme activity in the bread crust (about 30%) was hundredfold higher than that in the crumb (about 0.3%) after baking, despite the higher temperature in the crust throughout baking. This result suggested that the reduced moisture content in the crust increased the thermostability of the enzyme. Subsequently, the kinetic model reasonably predicted the enzyme inactivation in the crumb using the same parameters derived from the wheat flour/water system. However, the model predicted a lower residual enzyme activity in the crust compared with the experimental result, which indicated that the structure of the crust may influence the enzyme inactivation mechanism during baking. The results reported can provide a quantitative understanding of the thermal inactivation kinetics of enzyme during baking, which is essential to better retain enzymatic activity in bakery products supplemented with heat-sensitive enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Enzyme inhibition by iminosugars

    DEFF Research Database (Denmark)

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus


    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

  20. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. (United States)

    Wei, Hui; Wang, Erkang


    Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references).

  1. Computational Biochemistry-Enzyme Mechanisms Explored. (United States)

    Culka, Martin; Gisdon, Florian J; Ullmann, G Matthias


    Understanding enzyme mechanisms is a major task to achieve in order to comprehend how living cells work. Recent advances in biomolecular research provide huge amount of data on enzyme kinetics and structure. The analysis of diverse experimental results and their combination into an overall picture is, however, often challenging. Microscopic details of the enzymatic processes are often anticipated based on several hints from macroscopic experimental data. Computational biochemistry aims at creation of a computational model of an enzyme in order to explain microscopic details of the catalytic process and reproduce or predict macroscopic experimental findings. Results of such computations are in part complementary to experimental data and provide an explanation of a biochemical process at the microscopic level. In order to evaluate the mechanism of an enzyme, a structural model is constructed which can be analyzed by several theoretical approaches. Several simulation methods can and should be combined to get a reliable picture of the process of interest. Furthermore, abstract models of biological systems can be constructed combining computational and experimental data. In this review, we discuss structural computational models of enzymatic systems. We first discuss various models to simulate enzyme catalysis. Furthermore, we review various approaches how to characterize the enzyme mechanism both qualitatively and quantitatively using different modeling approaches. © 2017 Elsevier Inc. All rights reserved.

  2. Activity assessment of microbial fibrinolytic enzymes. (United States)

    Kotb, Essam


    Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

  3. Online Measurement of Oxygen-Dependent Enzyme Reaction Kinetics

    DEFF Research Database (Denmark)

    Meissner, Murray Peter; Nordblad, Mathias; Woodley, John M


    accurate measurement of the oxygen mass balance in the gas-phase of a reactor. The method was successfully validated and demonstrated using two model reactions: firstly the oxidation of glucose by glucose oxidase and secondly the Baeyer-Villiger oxidation of macrocyclic ketones to lactones. Initial...

  4. Effective use of enzyme microreactors : thermal, kinetic and ethical guidelines

    NARCIS (Netherlands)

    Swarts, J.W.


    Microreactor technology is reported to have many benefits over regular chemical methods. Due to the small dimensions over which temperature and concentration gradients can exist, mass and heat transfer can be very quick. This could minimize the time needed for heating and mixing, due to a reduction

  5. Bisubstrate Kinetics of Glutathione S-Transferase: A Colorimetric Experiment for the Introductory Biochemistry Laboratory (United States)

    Stefanidis, Lazaros; Scinto, Krystal V.; Strada, Monica I.; Alper, Benjamin J.


    Most biochemical transformations involve more than one substrate. Bisubstrate enzymes catalyze multiple chemical reactions in living systems and include members of the transferase, oxidoreductase, and ligase enzyme classes. Working knowledge of bisubstrate enzyme kinetic models is thus of clear importance to the practicing biochemist. However,…

  6. Cesium removal and kinetics equilibrium: Precipitation kinetics

    International Nuclear Information System (INIS)

    Barnes, M.J.


    This task consisted of both non-radioactive and radioactive (tracer) tests examining the influence of potentially significant variables on cesium tetraphenylborate precipitation kinetics. The work investigated the time required to reach cesium decontamination and the conditions that affect the cesium precipitation kinetics

  7. Enzymes for improved biomass conversion (United States)

    Brunecky, Roman; Himmel, Michael E.


    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  8. Co-immobilized Coupled Enzyme Systems in Biotechnology (United States)


    coimmobilized by ~n­ capsulation in silica spheres that were formed by a polymer -templated silicificatiOn reaction (Betancor et al., 2006). Nitrobenzene...F. , FERNANDEZ-LAFUENTE, R. , GUISAN J. M. (2005). Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol... polymer monoliths in microftuidic devices for steady- state kinetic analysis and spatially separated multi-enzyme reactions. Analytical Chemistry, 79

  9. Immobilized enzymes and cells

    Energy Technology Data Exchange (ETDEWEB)

    Bucke, C; Wiseman, A


    This article reviews the current state of the art of enzyme and cell immobilization and suggests advances which might be made during the 1980's. Current uses of immobilized enzymes include the use of glucoamylase in the production of glucose syrups from starch and glucose isomerase in the production of high fructose corn syrup. Possibilities for future uses of immobilized enzymes and cells include the utilization of whey and the production of ethanol.

  10. Profiling the orphan enzymes (United States)


    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new

  11. Plasma kinetic theory

    International Nuclear Information System (INIS)

    Elliott, J.A.


    Plasma kinetic theory is discussed and a comparison made with the kinetic theory of gases. The plasma is described by a modified set of fluid equations and it is shown how these fluid equations can be derived. (UK)

  12. Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enxymes? Testing biochemistry.

    NARCIS (Netherlands)

    Teusink, B.; Passarge, J.R.; Reijenga, C.A.; Esgalhado, M.E.L.M.; van der Weijden, C.C.; Schepper, M.; Walsh, M.C.; Bakker, B.M.; van Dam, K.; Westerhoff, H.V.; Snoep, J.L.


    This paper examines whether the in vivo behavior of yeast glycolysis can be understood in terms of the in vitro kinetic properties of the constituent enzymes. In nongrowing, anaerobic, compressed Saccharomyces cerevisiae the values of the kinetic parameters of most glycolytic enzymes were

  13. Concentration profiles near an activated enzyme. (United States)

    Park, Soohyung; Agmon, Noam


    When a resting enzyme is activated, substrate concentration profile evolves in its vicinity, ultimately tending to steady state. We use modern theories for many-body effects on diffusion-influenced reactions to derive approximate analytical expressions for the steady-state profile and the Laplace transform of the transient concentration profiles. These show excellent agreement with accurate many-particle Brownian-dynamics simulations for the Michaelis-Menten kinetics. The steady-state profile has a hyperbolic dependence on the distance of the substrate from the enzyme, albeit with a prefactor containing the complexity of the many-body effects. These are most conspicuous for the substrate concentration at the surface of the enzyme. It shows an interesting transition as a function of the enzyme turnover rate. When it is high, the contact concentration decays monotonically to steady state. However, for slow turnover it is nonmonotonic, showing a minimum due to reversible substrate binding, then a maximum due to diffusion of new substrate toward the enzyme, and finally decay to steady state. Under certain conditions one can obtain a good estimate for the critical value of the turnover rate constant at the transition.

  14. Artificial Enzymes, "Chemzymes"

    DEFF Research Database (Denmark)

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M


    Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successf......Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...... that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well...

  15. Magnetically responsive enzyme powders

    Energy Technology Data Exchange (ETDEWEB)

    Pospiskova, Kristyna, E-mail: [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)


    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

  16. Targeted enzyme prodrug therapies. (United States)

    Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C


    The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

  17. Enzymes in Fermented Fish. (United States)

    Giyatmi; Irianto, H E

    Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

  18. Asymmetric effect of mechanical stress on the forward and reverse reaction catalyzed by an enzyme.

    Directory of Open Access Journals (Sweden)

    Collin Joseph

    Full Text Available The concept of modulating enzymatic activity by exerting a mechanical stress on the enzyme has been established in previous work. Mechanical perturbation is also a tool for probing conformational motion accompanying the enzymatic cycle. Here we report measurements of the forward and reverse kinetics of the enzyme Guanylate Kinase from yeast (Saccharomyces cerevisiae. The enzyme is held in a state of stress using the DNA spring method. The observation that mechanical stress has different effects on the forward and reverse reaction kinetics suggests that forward and reverse reactions follow different paths, on average, in the enzyme's conformational space. Comparing the kinetics of the stressed and unstressed enzyme we also show that the maximum speed of the enzyme is comparable to the predictions of the relaxation model of enzyme action, where we use the independently determined dissipation coefficient [Formula: see text] for the enzyme's conformational motion. The present experiments provide a mean to explore enzyme kinetics beyond the static energy landscape picture of transition state theory.

  19. Standards for Reporting Enzyme Data: The STRENDA Consortium: What it aims to do and why it should be helpful

    Directory of Open Access Journals (Sweden)

    Keith F. Tipton


    Full Text Available Data on enzyme activities and kinetics have often been reported with insufficient experimental detail to allow their repetition. This paper discusses the objectives and recommendations of the Standards for Reporting Enzyme Data (STRENDA project to define minimal experimental standards for the reporting enzyme functional data.

  20. Drying of liquid food droplets : enzyme inactivation and multicomponent diffusion

    NARCIS (Netherlands)

    Meerdink, G.


    In this thesis the drying of liquid food droplets is studied from three different points of view: drying kinetics, enzyme inactivation and multicomponent diffusion. Mathematical models are developed and validated experimentally.

    Drying experiments are performed with suspended

  1. The Kinetics of Carrier Transport Inhibition

    DEFF Research Database (Denmark)

    Rosenberg, T.; Wilbrandt, Robert Walter


    The kinetical treatment of enzymatic carrier transports as given in previous communications has been extended to conditions of inhibition. Various possible types of inhibitors have been considered differing in the site of attack (enzyme or carrier), in the mode of action (competing with the subst......The kinetical treatment of enzymatic carrier transports as given in previous communications has been extended to conditions of inhibition. Various possible types of inhibitors have been considered differing in the site of attack (enzyme or carrier), in the mode of action (competing...... with the substrate for the enzyme or the carrier or for both, competing with the carrier for the enzyme, or non-competitive) and in the ability of penetrating the membrane. Experiments are reported on the inhibition of glucose and fructose transport across the human red cell membrane by phlorizine, phloretine...... and polyphloretinephosphate. The results of the analysis for these inhibitors indicate a substrate competitive mode of action. The effect of reversing the transport direction by interchanging the substrate concentration has been treated for the case of a non-penetrating substrate competitive inhibitor in the external medium...

  2. Molecular Imaging of Hydrolytic Enzymes Using PET and SPECT. (United States)

    Rempel, Brian P; Price, Eric W; Phenix, Christopher P


    Hydrolytic enzymes are a large class of biological catalysts that play a vital role in a plethora of critical biochemical processes required to maintain human health. However, the expression and/or activity of these important enzymes can change in many different diseases and therefore represent exciting targets for the development of positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radiotracers. This review focuses on recently reported radiolabeled substrates, reversible inhibitors, and irreversible inhibitors investigated as PET and SPECT tracers for imaging hydrolytic enzymes. By learning from the most successful examples of tracer development for hydrolytic enzymes, it appears that an early focus on careful enzyme kinetics and cell-based studies are key factors for identifying potentially useful new molecular imaging agents.

  3. Structural analysis of enzymes used for bioindustry and bioremediation. (United States)

    Tanokura, Masaru; Miyakawa, Takuya; Guan, Lijun; Hou, Feng


    Microbial enzymes have been widely applied in the large-scale, bioindustrial manufacture of food products and pharmaceuticals due to their high substrate specificity and stereoselectivity, and their effectiveness under mild conditions with low environmental burden. At the same time, bioremedial techniques using microbial enzymes have been developed to solve the problem of industrial waste, particularly with respect to persistent chemicals and toxic substances. And finally, structural studies of these enzymes have revealed the mechanistic basis of enzymatic reactions, including the stereoselectivity and binding specificity of substrates and cofactors. The obtained structural insights are useful not only to deepen our understanding of enzymes with potential bioindustrial and/or bioremedial application, but also for the functional improvement of enzymes through rational protein engineering. This review shows the structural bases for various types of enzymatic reactions, including the substrate specificity accompanying cofactor-controlled and kinetic mechanisms.

  4. Enzyme-Embedded, Microstructural Reactors for Industrial Biocatalysis

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Sarah E. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Knipe, J. M. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Oakdale, J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Stolaroff, J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)


    In this project we explored enzyme-catalyzed methane conversion to methanol. Industrial biological approaches to methane conversion using whole organisms are predicted to be more energy efficient than chemical approaches, but are limited by mass transfer of the gas phase reactants, methane and oxygen, to the organisms. We demonstrated that 3D printing the enzyme particulate Methane Mono Oxygenase (pMMO) embedded in a polymer can improve the kinetics of methane to methanol conversion. This improvement was likely due to the ability to increase the surface area of the catalytic material using 3D printing. We also demonstrated the first continuous use of pMMO in a flow-through reactor. In order to understand the fundamental kinetic properties of pMMO, we conducted an in-depth study of pMMO kinetics using analytical tools developed in our lab. Finally, we developed a new copolymer system that allowed tuning of the gas permeability of the biocatalytic material.

  5. Enzymic lactose hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Miller, J J; Brand, J C


    Acid or enzymic hydrolysis can be used to hydrolyze lactose. Advantages of both are compared and details of enzymic hydrolysis using yeast or fungal enzymes given. The new scheme outlined involves recycling lactase. Because lactose and lactase react to ultrafiltration (UF) membranes differently separation is possible. Milk or milk products are ultrafiltered to separate a concentrate from a lactose-rich permeate which is treated with lactase in a reactor until hydrolysis reaches a required level. The lactase can be removed by UF as it does not permeate the membrane, and it is recycled back to the reactor. Permeate from the second UF stage may or may not be recombined with the concentrate from the first stage to produce a low lactose product (analysis of a typical low-lactose dried whole milk is given). Batch or continuous processes are explained and a batch process without enzyme recovery is discussed. (Refs. 4).

  6. Indicators: Sediment Enzymes (United States)

    Sediment enzymes are proteins that are produced by microorganisms living in the sediment or soil. They are indicators of key ecosystem processes and can help determine which nutrients are affecting the biological community of a waterbody.

  7. Enzyme Vs. Extremozyme -32 ...

    Indian Academy of Sciences (India)

    Enzymes are biocatalytic protein molecules that enhance the rates of ... to physical forces (hydrogen bonds, hydrophobic 1, electrostatic and Van der ... conformation. In 1995 ... surface against 14.7% in Klenow poll (some of the hydrophobic.

  8. Evaluation method for the drying performance of enzyme containing formulations

    DEFF Research Database (Denmark)

    Sloth, Jakob; Bach, P.; Jensen, Anker Degn


    A method is presented for fast and cheap evaluation of the performance of enzyme containing formulations in terms of preserving the highest enzyme activity during spray drying. The method is based on modeling the kinetics of the thermal inactivation reaction which occurs during the drying process....... Relevant kinetic parameters are determined from differential scanning calorimeter (DSC) experiments and the model is used to simulate the severity of the inactivation reaction for temperatures and moisture levels relevant for spray drying. After conducting experiments and subsequent simulations...... for a number of different formulations it may be deduced which formulation performs best. This is illustrated by a formulation design study where 4 different enzyme containing formulations are evaluated. The method is validated by comparison to pilot scale spray dryer experiments....

  9. Overproduction of ligninolytic enzymes (United States)

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas


    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  10. Measuring Intracellular Enzyme Concentrations: Assessing the Effect of Oxidative Stress on the Amount of Glyoxalase I (United States)

    Miranda, Hugo Vicente; Ferreira, Antonio E. N.; Quintas, Alexandre; Cordeiro, Carlos; Freire, Ana Ponces


    Enzymology is one of the fundamental areas of biochemistry and involves the study of the structure, kinetics, and regulation of enzyme activity. Research in this area is often conducted with purified enzymes and extrapolated to "in vivo" conditions. The specificity constant, k[subscript S], is the ratio between k[subscript cat] (the catalytic…

  11. Single-enzyme analysis in a droplet-based micro- and nanofluidic system

    NARCIS (Netherlands)

    Arayanarakool, Rerngchai; Shui, Lingling; Kengen, Servé W.M.; van den Berg, Albert; Eijkel, Jan C.T.


    The kinetic activity of individual enzyme molecules was determined in aqueous droplets generated in a nano- and microfluidic device. To avoid high background noise, the enzyme and substrate solution was confined into femtoliter carriers, achieving high product concentrations from single-molecule

  12. Kinetic studies on the inhibition of Proteus vulgaris beta-lactamase by imipenem.


    Hashizume, T; Yamaguchi, A; Hirata, T; Sawai, T


    Imipenem was found to inhibit Proteus vulgaris beta-lactamase in a progressive manner. Kinetic experiments confirmed that the inactivated enzyme was not completely recovered after intact imipenem had been exhausted.

  13. Principles of chemical kinetics

    CERN Document Server

    House, James E


    James House's revised Principles of Chemical Kinetics provides a clear and logical description of chemical kinetics in a manner unlike any other book of its kind. Clearly written with detailed derivations, the text allows students to move rapidly from theoretical concepts of rates of reaction to concrete applications. Unlike other texts, House presents a balanced treatment of kinetic reactions in gas, solution, and solid states. The entire text has been revised and includes many new sections and an additional chapter on applications of kinetics. The topics covered include quantitative rela

  14. Introduction to chemical kinetics

    CERN Document Server

    Soustelle, Michel


    This book is a progressive presentation of kinetics of the chemical reactions. It provides complete coverage of the domain of chemical kinetics, which is necessary for the various future users in the fields of Chemistry, Physical Chemistry, Materials Science, Chemical Engineering, Macromolecular Chemistry and Combustion. It will help them to understand the most sophisticated knowledge of their future job area. Over 15 chapters, this book present the fundamentals of chemical kinetics, its relations with reaction mechanisms and kinetic properties. Two chapters are then devoted to experimental re

  15. Kinetic equation solution by inverse kinetic method

    International Nuclear Information System (INIS)

    Salas, G.


    We propose a computer program (CAMU) which permits to solve the inverse kinetic equation. The CAMU code is written in HPL language for a HP 982 A microcomputer with a peripheral interface HP 9876 A ''thermal graphic printer''. The CAMU code solves the inverse kinetic equation by taking as data entry the output of the ionization chambers and integrating the equation with the help of the Simpson method. With this program we calculate the evolution of the reactivity in time for a given disturbance

  16. Random-walk enzymes (United States)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.


    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  17. Kinetics in radiation chemistry

    International Nuclear Information System (INIS)

    Hummel, A.


    In this chapter the authors first briefly review the kinetics of first- and second-order processes for continuous and pulsed irradiation, without taking the effects of nonhomogeneous formation of the species into consideration. They also discuss diffusion controlled reactions under conditions where interactions of more than two particles can be neglected, first the kinetics of the diffusion-controlled reaction of randomly generated species (homogeneous reaction) and then that of isolated pairs of reactants. The latter is often called geminate kinetics when dealing with pairs of oppositely charged species; they shall use this term for the kinetics of isolated pairs in general. In the last section they discuss briefly the kinetics of groups of more than two reactants

  18. Non-kinetic capabilities: complementing the kinetic prevalence to targeting


    Ducheine, P.


    Targeting is used in military doctrine to describe a military operational way, using (military) means to influence a target (or addressee) in order to achieve designated political and/or military goals. The four factors italicized are used to analyse non-kinetic targeting, complementing our knowledge and understanding of the kinetic prevalence. Paradoxically, non-kinetic targeting is not recognized as a separate concept: kinetic and non-kinetic are intertwined facets of targeting. Kinetic tar...

  19. Comparison of the Kinetic Promoters Piperazine and Carbonic Anhydrase for CO2 Absorption

    DEFF Research Database (Denmark)

    Gladis, Arne; Gundersen, Maria T.; Thomsen, Kaj


    Kinetic promoter that enhance the reaction kinetics with CO2 are enabling the use of the low heat of reaction of slow absorbing solvents like MDEA. Mass transfer experiments with 30 wt% MDEA promoted by either by 1.7 and 8.5g/L enzyme carbonic anhydrase (CA) or 5 wt% piperazine (PZ) where conduct...

  20. Multiple Reaction Monitoring for quantitative laccase kinetics by LC-MS

    DEFF Research Database (Denmark)

    Perna, Valentina; Agger, Jane W.; Holck, Jesper


    as substrates to assess enzyme kinetics by HPLC-MS on two fungal laccases Trametes versicolor laccase, Tv and Ganoderma lucidum laccase, Gl. The method allowed accurate kinetic measurements and detailed insight into the product profiles of both laccases. Both Tv and Gl laccase are active...

  1. Enzyme Teaching by a Virtual Laboratory

    Directory of Open Access Journals (Sweden)

    J.K. Sugai


    Full Text Available Biochemistry learning demands skills to obtaining and interpreting the experimental data. In a classical model of teaching involve student’s hands-on participation. However this model is expensive, not safe and should be carried out in a short and limited time course. With utilization of educational software these disadvantages are overcome, since the virtual activity could be realized at free full access, and is a tool for individual study. The aim of the present work is to present educational software focused on a virtual for undergraduate student of biochemistry courses. The software development was performed with the help of concept maps, ISIS Draw, ADOBE Photoshop and FLASH MX Program applied on the subject salivary amylase. It was possible to present the basic methodologies for study of the kinetic of enzyme. The substrate (starch consumption was determinate by iodine reaction, while the products (reducing sugars formation was evaluated by cupper-alkaline reaction. The protocols of the virtual experiments are present verbally as well as a subtitle. A set of exercises are disposable, which allowed an auto evaluation and a review of the subject. The experimental treatment involved the presentation of this hypermedia for Nutrition and Dentistry/UFSC undergraduate students as a tool for better comprehension of the theme and promoted the understanding of the kinetic of enzyme.

  2. Matrix Metalloproteinase Enzyme Family

    Directory of Open Access Journals (Sweden)

    Ozlem Goruroglu Ozturk


    Full Text Available Matrix metalloproteinases play an important role in many biological processes such as embriogenesis, tissue remodeling, wound healing, and angiogenesis, and in some pathological conditions such as atherosclerosis, arthritis and cancer. Currently, 24 genes have been identified in humans that encode different groups of matrix metalloproteinase enzymes. This review discuss the members of the matrix metalloproteinase family and their substrate specificity, structure, function and the regulation of their enzyme activity by tissue inhibitors. [Archives Medical Review Journal 2013; 22(2.000: 209-220

  3. Friction analysis of kinetic schemes : the friction coefficient

    NARCIS (Netherlands)

    Lolkema, Juke S.


    Friction analysis is proposed as the application of general control analysis to single enzymes to describe the control of elementary kinetic steps on the overall catalytic rate. For each transition, a friction coefficient is defined that measures the sensitivity of the turnover rate to the free

  4. Purification, product characterization and kinetic properties of soluble tomato lipoxygenase

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Suurmeijer, C.N.S.P.; Pérez-Gilabert, M.; Hijden, H.T.W.M. van der; Veldink, G.A.


    Soluble lipoxygenase (EC from tomato fruits (Lycopersicon esculentum, var. Trust) was purified to apparent homogeneity as judged by SDS-PAGE, and the products and kinetics of the enzyme were studied in order to clarify the contradictory results that were obtained with a less purified

  5. The surface science of enzymes

    DEFF Research Database (Denmark)

    Rod, Thomas Holm; Nørskov, Jens Kehlet


    One of the largest challenges to science in the coming years is to find the relation between enzyme structure and function. Can we predict which reactions an enzyme catalyzes from knowledge of its structure-or from its amino acid sequence? Can we use that knowledge to modify enzyme function......? To solve these problems we must understand in some detail how enzymes interact with reactants from its surroundings. These interactions take place at the surface of the enzyme and the question of enzyme function can be viewed as the surface science of enzymes. In this article we discuss how to describe...... catalysis by enzymes, and in particular the analogies between enzyme catalyzed reactions and surface catalyzed reactions. We do this by discussing two concrete examples of reactions catalyzed both in nature (by enzymes) and in industrial reactors (by inorganic materials), and show that although analogies...

  6. Biosensors for the determination of environmental inhibitors of enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Evtugyn, Gennadii A; Budnikov, Herman C [Kazan State University, Kazan (Russian Federation); Nikolskaya, Elena B [I.M. Sechenov Institute of Evolution Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg (Russian Federation)


    Characteristic features of functioning and practical application of enzyme-based biosensors for the determination of environmental pollutants as enzyme inhibitors are considered with special emphasis on the influence of the methods used for the measurement of the rates of enzymic reactions, of enzyme immobilisation procedure and of the composition of the reaction medium on the analytical characteristics of inhibitor assays. The published data on the development of biosensors for detecting pesticides and heavy metals are surveyed. Special attention is given to the use of cholinesterase-based biosensors in environmental and analytical monitoring. The approaches to the estimation of kinetic parameters of inhibition are reviewed and the factors determining the selectivity and sensitivity of inhibitor assays in environmental objects are analysed. The bibliography includes 195 references.

  7. Biosensors for the determination of environmental inhibitors of enzymes

    International Nuclear Information System (INIS)

    Evtugyn, Gennadii A; Budnikov, Herman C; Nikolskaya, Elena B


    Characteristic features of functioning and practical application of enzyme-based biosensors for the determination of environmental pollutants as enzyme inhibitors are considered with special emphasis on the influence of the methods used for the measurement of the rates of enzymic reactions, of enzyme immobilisation procedure and of the composition of the reaction medium on the analytical characteristics of inhibitor assays. The published data on the development of biosensors for detecting pesticides and heavy metals are surveyed. Special attention is given to the use of cholinesterase-based biosensors in environmental and analytical monitoring. The approaches to the estimation of kinetic parameters of inhibition are reviewed and the factors determining the selectivity and sensitivity of inhibitor assays in environmental objects are analysed. The bibliography includes 195 references.

  8. Molecular Modeling of Enzyme Dynamics Towards Understanding Solvent Effects

    DEFF Research Database (Denmark)

    Wedberg, Nils Hejle Rasmus Ingemar

    This thesis describes the development of a molecular simulation methodology to study properties of enzymes in non-aqueous media at fixed thermodynamic water activities. The methodology is applied in a molecular dynamics study of the industrially important enzyme Candida antarctica lipase B (CALB...... of enzyme kinetics in non-aqueous media, it has been a fruitful approach to fix the enzyme hydration level by controlling the water activity of the medium. In this work, a protocol is therefore developed for determining the water activity in non-aqueous protein simulations. The method relies on determining...... integration, while for small systems, it seems to be even better. The method is applied to compute the excess Gibbs energy of the mixtures of water and organic solvents used in the simulations of CALB. This allows to determine the water activity of the simulated systems and thus to compare protein properties...

  9. Kinetics of phase transformations

    International Nuclear Information System (INIS)

    Thompson, M.O.; Aziz, M.J.; Stephenson, G.B.


    This volume contains papers presented at the Materials Research Society symposium on Kinetics of Phase Transformations held in Boston, Massachusetts from November 26-29, 1990. The symposium provided a forum for research results in an exceptionally broad and interdisciplinary field. Presentations covered nearly every major class of transformations including solid-solid, liquid-solid, transport phenomena and kinetics modeling. Papers involving amorphous Si, a dominant topic at the symposium, are collected in the first section followed by sections on four major areas of transformation kinetics. The symposium opened with joint sessions on ion and electron beam induced transformations in conjunction with the Surface Chemistry and Beam-Solid Interactions: symposium. Subsequent sessions focused on the areas of ordering and nonlinear diffusion kinetics, solid state reactions and amorphization, kinetics and defects of amorphous silicon, and kinetics of melting and solidification. Seven internationally recognized invited speakers reviewed many of the important problems and recent results in these areas, including defects in amorphous Si, crystal to glass transformations, ordering kinetics, solid-state amorphization, computer modeling, and liquid/solid transformations

  10. Improvement of Student Understanding of How Kinetic Data Facilitates the Determination of Amino Acid Catalytic Function through an Alkaline Phosphatase Structure/Mechanism Bioinformatics Exercise (United States)

    Grunwald, Sandra K.; Krueger, Katherine J.


    Laboratory exercises, which utilize alkaline phosphatase as a model enzyme, have been developed and used extensively in undergraduate biochemistry courses to illustrate enzyme steady-state kinetics. A bioinformatics laboratory exercise for the biochemistry laboratory, which complements the traditional alkaline phosphatase kinetics exercise, was…

  11. Magnetically responsive enzyme powders

    Czech Academy of Sciences Publication Activity Database

    Pospišková, K.; Šafařík, Ivo


    Roč. 380, APR 2015 (2015), s. 197-200 ISSN 0304-8853 R&D Projects: GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : enzyme powders * cross-linking * magnetic modification * magnetic separation * magnetic iron oxides particles * microwave-assisted synthesis Subject RIV: CE - Biochemistry Impact factor: 2.357, year: 2015

  12. Enzyme with rhamnogalacturonase activity.

    NARCIS (Netherlands)

    Kofod, L.V.; Andersen, L.N.; Dalboge, H.; Kauppinen, M.S.; Christgau, S.; Heldt-Hansen, H.P.; Christophersen, C.; Nielsen, P.M.; Voragen, A.G.J.; Schols, H.A.


    An enzyme exhibiting rhamnogalacturonase activity, capable of cleaving a rhamnogalacturonan backbone in such a manner that galacturonic acids are left as the non-reducing ends, and which exhibits activity on hairy regions from a soy bean material and/or on saponified hairy regions from a sugar beet

  13. Implantable enzyme amperometric biosensors. (United States)

    Kotanen, Christian N; Moussy, Francis Gabriel; Carrara, Sandro; Guiseppi-Elie, Anthony


    The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Cold-Adapted Enzymes (United States)

    Georlette, D.; Bentahir, M.; Claverie, P.; Collins, T.; D'amico, S.; Delille, D.; Feller, G.; Gratia, E.; Hoyoux, A.; Lonhienne, T.; Meuwis, M.-a.; Zecchinon, L.; Gerday, Ch.

    In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship.

  15. Embedded enzymes catalyse capture (United States)

    Kentish, Sandra


    Membrane technologies for carbon capture can offer economic and environmental advantages over conventional amine-based absorption, but can suffer from limited gas flux and selectivity to CO2. Now, a membrane based on enzymes embedded in hydrophilic pores is shown to exhibit combined flux and selectivity that challenges the state of the art.

  16. Photoperiodism and Enzyme Activity (United States)

    Queiroz, Orlando; Morel, Claudine


    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  17. ISFET based enzyme sensors

    NARCIS (Netherlands)

    van der Schoot, Bart H.; Bergveld, Piet


    This paper reviews the results that have been reported on ISFET based enzyme sensors. The most important improvement that results from the application of ISFETs instead of glass membrane electrodes is in the method of fabrication. Problems with regard to the pH dependence of the response and the

  18. Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

    DEFF Research Database (Denmark)

    Hurtado-Guerrrero, Ramón; Pena Diaz, Javier; Montalvetti, Andrea


    A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower propo...

  19. Irreversible processes kinetic theory

    CERN Document Server

    Brush, Stephen G


    Kinetic Theory, Volume 2: Irreversible Processes deals with the kinetic theory of gases and the irreversible processes they undergo. It includes the two papers by James Clerk Maxwell and Ludwig Boltzmann in which the basic equations for transport processes in gases are formulated, together with the first derivation of Boltzmann's ""H-theorem"" and a discussion of this theorem, along with the problem of irreversibility.Comprised of 10 chapters, this volume begins with an introduction to the fundamental nature of heat and of gases, along with Boltzmann's work on the kinetic theory of gases and s

  20. The Enzyme Function Initiative† (United States)

    Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.


    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID

  1. The Enzyme Function Initiative. (United States)

    Gerlt, John A; Allen, Karen N; Almo, Steven C; Armstrong, Richard N; Babbitt, Patricia C; Cronan, John E; Dunaway-Mariano, Debra; Imker, Heidi J; Jacobson, Matthew P; Minor, Wladek; Poulter, C Dale; Raushel, Frank M; Sali, Andrej; Shoichet, Brian K; Sweedler, Jonathan V


    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts.


    African Journals Online (AJOL)

    IV) catalyzes the discoloring reaction of DBS-arsenazo oxidized by potassium bromate, a new catalytic kinetic spectrophotometric method for the determination of trace titanium (IV) was developed. The linear range of the determination of ...

  3. Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Sim Edith


    Full Text Available Abstract Background Arylamine N-acetyltransferases (NATs are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme. Results We have determined that NAT from Pseudomonas aeruginosa, which has been described as a model for NAT from M. tuberculosis, follows a Ping Pong Bi Bi kinetic mechanism. We also describe substrate inhibition by 5-aminosalicylic acid, in which the substrate binds both to the free form of the enzyme and the acetyl coenzyme A-enzyme complex in non-productive reaction pathways. The true kinetic parameters for the NAT-catalysed acetylation of 5-aminosalicylic acid with acetyl coenzyme A as the co-factor have been established, validating earlier approximations. Conclusion This is the first reported study investigating the kinetic mechanism of a bacterial NAT enzyme. Additionally, the methods used herein can be applied to investigations of the interactions of NAT enzymes with new chemical entities which are NAT ligands. This is likely to be useful in the design of novel potential anti-tubercular agents.

  4. Inhibition of ligand exchange kinetics via active-site trapping with an antibody fragment. (United States)

    Oyen, David; Steyaert, Jan; Barlow, John N


    We describe the first example of an inhibitory antibody fragment (nanobody ca1697) that binds simultaneously to an enzyme (the enzyme dihydrofolate reductase from Escherichia coli) and its bound substrate (folate). Binding of the antibody to the substrate causes a 20-fold reduction in the rate of folate exchange kinetics. This work opens up the prospect of designing new types of antibody-based inhibitors of enzymes and receptors through suitable design of immunogens.


    Directory of Open Access Journals (Sweden)

    Israel Hernández


    Full Text Available In this paper the degrading activity of enzymes derived from orange peels (Citrus x sinensis, grapefruit (Citrus paradise and pineapple (Ananas comosus on the organic matter in wastewater is evaluated. This activity is measured indirectly by quantifying the biochemical oxygen demand (COD before and after degradation process based on a period of time using the HACH DR / 2010, and then the kinetic study was performed by the differential method and integral with the experimental data, obtaining a reaction order of 1 to pectinase (orange, and order 2 for bromelain (pineapple.

  6. Ultrasound assisted intensification of enzyme activity and its properties: a mini-review. (United States)

    Nadar, Shamraja S; Rathod, Virendra K


    Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (k m and V max ) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

  7. Threonine deaminase from extremely halophilic bacteria - Cooperative substrate kinetics and salt dependence. (United States)

    Lieberman, M. M.; Lanyi, J. K.


    The effect of salt on the activity, stability, and allosteric properties of catabolic threonine deaminase from Halobacterium cutirubrum was studied. The enzyme exhibits sigmoidal kinetics with the substrate, threonine. The Hill slope is 1.55 at pH 10. The enzyme is activated by ADP at low substrate concentrations. In the presence of this effector, sigmoidal kinetics are no longer observed. At pH 10, in the absence of ADP, enzyme activity increases with increasing NaCl concentration from 0 to 4 M.

  8. Evaluation of rate law approximations in bottom-up kinetic models of metabolism

    DEFF Research Database (Denmark)

    Du, Bin; Zielinski, Daniel C.; Kavvas, Erol S.


    mass action rate law that removes the role of the enzyme from the reaction kinetics. We utilized in vivo data for the human red blood cell to compare the effect of rate law choices against the backdrop of physiological flux and concentration differences. We found that the Michaelis-Menten rate law......Background: The mechanistic description of enzyme kinetics in a dynamic model of metabolism requires specifying the numerical values of a large number of kinetic parameters. The parameterization challenge is often addressed through the use of simplifying approximations to form reaction rate laws....... These approximate rate laws were: 1) a Michaelis-Menten rate law with measured enzyme parameters, 2) a Michaelis-Menten rate law with approximated parameters, using the convenience kinetics convention, 3) a thermodynamic rate law resulting from a metabolite saturation assumption, and 4) a pure chemical reaction...

  9. Kinetics of phosphomevalonate kinase from Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    David E Garcia

    Full Text Available The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3 and purified on a Ni²⁺ column. The KM of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K(M of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V(max was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²⁺ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP.

  10. Control of DNA strand displacement kinetics using toehold exchange. (United States)

    Zhang, David Yu; Winfree, Erik


    DNA is increasingly being used as the engineering material of choice for the construction of nanoscale circuits, structures, and motors. Many of these enzyme-free constructions function by DNA strand displacement reactions. The kinetics of strand displacement can be modulated by toeholds, short single-stranded segments of DNA that colocalize reactant DNA molecules. Recently, the toehold exchange process was introduced as a method for designing fast and reversible strand displacement reactions. Here, we characterize the kinetics of DNA toehold exchange and model it as a three-step process. This model is simple and quantitatively predicts the kinetics of 85 different strand displacement reactions from the DNA sequences. Furthermore, we use toehold exchange to construct a simple catalytic reaction. This work improves the understanding of the kinetics of nucleic acid reactions and will be useful in the rational design of dynamic DNA and RNA circuits and nanodevices.

  11. Kinetics of hydrogen peroxide decomposition by catalase: hydroxylic solvent effects. (United States)

    Raducan, Adina; Cantemir, Anca Ruxandra; Puiu, Mihaela; Oancea, Dumitru


    The effect of water-alcohol (methanol, ethanol, propan-1-ol, propan-2-ol, ethane-1,2-diol and propane-1,2,3-triol) binary mixtures on the kinetics of hydrogen peroxide decomposition in the presence of bovine liver catalase is investigated. In all solvents, the activity of catalase is smaller than in water. The results are discussed on the basis of a simple kinetic model. The kinetic constants for product formation through enzyme-substrate complex decomposition and for inactivation of catalase are estimated. The organic solvents are characterized by several physical properties: dielectric constant (D), hydrophobicity (log P), concentration of hydroxyl groups ([OH]), polarizability (α), Kamlet-Taft parameter (β) and Kosower parameter (Z). The relationships between the initial rate, kinetic constants and medium properties are analyzed by linear and multiple linear regression.

  12. NRSA enzyme decomposition model data (United States)

    U.S. Environmental Protection Agency — Microbial enzyme activities measured at more than 2000 US streams and rivers. These enzyme data were then used to predict organic matter decomposition and microbial...

  13. Cellulase enzyme and biomass utilization

    African Journals Online (AJOL)



    Jun 3, 2009 ... human population grows and economic development. However, the current .... conditions and the production cost of the related enzyme system. Therefore ... Given the importance of this enzyme to these so many industries,.

  14. Relativistic Chiral Kinetic Theory

    International Nuclear Information System (INIS)

    Stephanov, Mikhail


    This very brief review of the recent progress in chiral kinetic theory is based on the results of Refs. [J.-Y. Chen, D. T. Son, M. A. Stephanov, H.-U. Yee, Y. Yin, Lorentz Invariance in Chiral Kinetic Theory, Phys. Rev. Lett. 113 (18) (2014) 182302. doi: (10.1103/PhysRevLett.113.182302); J.-Y. Chen, D. T. Son, M. A. Stephanov, Collisions in Chiral Kinetic Theory, Phys. Rev. Lett. 115 (2) (2015) 021601. doi: (10.1103/PhysRevLett.115.021601); M. A. Stephanov, H.-U. Yee, The no-drag frame for anomalous chiral fluid, Phys. Rev. Lett. 116 (12) (2016) 122302. doi: (10.1103/PhysRevLett.116.122302)].

  15. Relativistic Chiral Kinetic Theory

    Energy Technology Data Exchange (ETDEWEB)

    Stephanov, Mikhail


    This very brief review of the recent progress in chiral kinetic theory is based on the results of Refs. [J.-Y. Chen, D. T. Son, M. A. Stephanov, H.-U. Yee, Y. Yin, Lorentz Invariance in Chiral Kinetic Theory, Phys. Rev. Lett. 113 (18) (2014) 182302. doi: (10.1103/PhysRevLett.113.182302); J.-Y. Chen, D. T. Son, M. A. Stephanov, Collisions in Chiral Kinetic Theory, Phys. Rev. Lett. 115 (2) (2015) 021601. doi: (10.1103/PhysRevLett.115.021601); M. A. Stephanov, H.-U. Yee, The no-drag frame for anomalous chiral fluid, Phys. Rev. Lett. 116 (12) (2016) 122302. doi: (10.1103/PhysRevLett.116.122302)].

  16. Erbium hydride decomposition kinetics.

    Energy Technology Data Exchange (ETDEWEB)

    Ferrizz, Robert Matthew


    Thermal desorption spectroscopy (TDS) is used to study the decomposition kinetics of erbium hydride thin films. The TDS results presented in this report are analyzed quantitatively using Redhead's method to yield kinetic parameters (E{sub A} {approx} 54.2 kcal/mol), which are then utilized to predict hydrogen outgassing in vacuum for a variety of thermal treatments. Interestingly, it was found that the activation energy for desorption can vary by more than 7 kcal/mol (0.30 eV) for seemingly similar samples. In addition, small amounts of less-stable hydrogen were observed for all erbium dihydride films. A detailed explanation of several approaches for analyzing thermal desorption spectra to obtain kinetic information is included as an appendix.

  17. Kinetic mechanism and nucleotide specificity of NADH peroxidase

    International Nuclear Information System (INIS)

    Stoll, V.S.; Blanchard, J.S.


    NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [ 14 C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols

  18. Relativistic Kinetic Theory (United States)

    Vereshchagin, Gregory V.; Aksenov, Alexey G.


    Preface; Acknowledgements; Acronyms and definitions; Introduction; Part I. Theoretical Foundations: 1. Basic concepts; 2. Kinetic equation; 3. Averaging; 4. Conservation laws and equilibrium; 5. Relativistic BBGKY hierarchy; 6. Basic parameters in gases and plasmas; Part II. Numerical Methods: 7. The basics of computational physics; 8. Direct integration of Boltzmann equations; 9. Multidimensional hydrodynamics; Part III. Applications: 10. Wave dispersion in relativistic plasma; 11. Thermalization in relativistic plasma; 12. Kinetics of particles in strong fields; 13. Compton scattering in astrophysics and cosmology; 14. Self-gravitating systems; 15. Neutrinos, gravitational collapse and supernovae; Appendices; Bibliography; Index.

  19. Quantum kinetic theory

    CERN Document Server

    Bonitz, Michael


    This book presents quantum kinetic theory in a comprehensive way. The focus is on density operator methods and on non-equilibrium Green functions. The theory allows to rigorously treat nonequilibrium dynamics in quantum many-body systems. Of particular interest are ultrafast processes in plasmas, condensed matter and trapped atoms that are stimulated by rapidly developing experiments with short pulse lasers and free electron lasers. To describe these experiments theoretically, the most powerful approach is given by non-Markovian quantum kinetic equations that are discussed in detail, including computational aspects.

  20. Kinetics of cellobiose hydrolysis using cellobiase composites from Trichoderma reesei and Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Grous, W.; Converse, A.; Grethlein, H.; Lynd, L.


    The enzymatic hydrolysis of cellulose to glucose involves the formation of cellobiose as an intermediate. It has been found necessary to add cellobiase from Aspergillus niger (NOVO) to the cellobiase component of Trichoderma reesei mutant Rut C-30 (Natick) cellulase enzymes in order to obtain after 48 h complete conversion of the cellobiose formed in the enzymatic hydrolysis of biomass. This study of the cellobiase activity of these two enzyme sources was undertaken as a first step in the formation of a kinetic model for cellulose hydrolysis that can be used in process design. In order to cover the full range of cellobiose concentrations, it was necessary to develop separate kinetic parameters for high- and low-concentration ranges of cellobiose for the enzymes from each organism. Competitive glucose inhibition was observed with the enzymes from both organisms. Substrate inhibition was observed only with the A. niger enzymes.

  1. Enzyme recycling in lignocellulosic biorefineries

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Pinelo, Manuel


    platform. Cellulases are the most important enzymes required in this process, but the complex nature of lignocellulose requires several other enzymes (hemicellulases and auxiliary enzymes) for efficient hydrolysis. Enzyme recycling increases the catalytic productivity of the enzymes by reusing them...... for several batches of hydrolysis, and thereby reduces the overall cost associated with the hydrolysis. Research on this subject has been ongoing for many years and several promising technologies and methods have been developed and demonstrated. But only in a very few cases have these technologies been...... upscaled and tested in industrial settings, mainly because of many difficulties with recycling of enzymes from the complex lignocellulose hydrolyzate at industrially relevant conditions, i.e., high solids loadings. The challenges are associated with the large number of different enzymes required...

  2. Characterising Complex Enzyme Reaction Data.

    Directory of Open Access Journals (Sweden)

    Handan Melike Dönertaş

    Full Text Available The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG. Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution.

  3. Silica Sol-Gel Entrapment of the Enzyme Chloro peroxidase

    International Nuclear Information System (INIS)

    Le, T.; Chan, S.; Ebaid, B.; Sommerhalter, M.


    The enzyme chloro peroxidase (CPO) was immobilized in silica sol-gel beads prepared from tetramethoxysilane. The average pore diameter of the silica host structure (∼3 nm) was smaller than the globular CPO diameter (∼6 nm) and the enzyme remained entrapped after sol-gel maturation. The catalytic performance of the entrapped enzyme was assessed via the pyrogallol peroxidation reaction. Sol-gel beads loaded with 4 μg CPO per mL sol solution reached 9-12% relative activity compared to free CPO in solution. Enzyme kinetic analysis revealed a decrease in K_cat but no changes in K_M or K_I . Product release or enzyme damage might thus limit catalytic performance. Yet circular dichroism and visible absorption spectra of transparent CPO sol-gel sheets did not indicate enzyme damage. Activity decline due to methanol exposure was shown to be reversible in solution. To improve catalytic performance the sol-gel protocol was modified. The incorporation of 5, 20, or 40% methyltrimethoxysilane resulted in more brittle sol-gel beads but the catalytic performance increased to 14% relative to free CPO in solution. The use of more acidic casting buffers (ph 4.5 or 5.5 instead of 6.5) resulted in a more porous silica host reaching up to 18% relative activity

  4. Oxidative desulfurization: kinetic modelling. (United States)

    Dhir, S; Uppaluri, R; Purkait, M K


    Increasing environmental legislations coupled with enhanced production of petroleum products demand, the deployment of novel technologies to remove organic sulfur efficiently. This work represents the kinetic modeling of ODS using H(2)O(2) over tungsten-containing layered double hydroxide (LDH) using the experimental data provided by Hulea et al. [V. Hulea, A.L. Maciuca, F. Fajula, E. Dumitriu, Catalytic oxidation of thiophenes and thioethers with hydrogen peroxide in the presence of W-containing layered double hydroxides, Appl. Catal. A: Gen. 313 (2) (2006) 200-207]. The kinetic modeling approach in this work initially targets the scope of the generation of a superstructure of micro-kinetic reaction schemes and models assuming Langmuir-Hinshelwood (LH) and Eley-Rideal (ER) mechanisms. Subsequently, the screening and selection of above models is initially based on profile-based elimination of incompetent schemes followed by non-linear regression search performed using the Levenberg-Marquardt algorithm (LMA) for the chosen models. The above analysis inferred that Eley-Rideal mechanism describes the kinetic behavior of ODS process using tungsten-containing LDH, with adsorption of reactant and intermediate product only taking place on the catalyst surface. Finally, an economic index is presented that scopes the economic aspects of the novel catalytic technology with the parameters obtained during regression analysis to conclude that the cost factor for the catalyst is 0.0062-0.04759 US $ per barrel.

  5. Oxidative desulfurization: Kinetic modelling

    International Nuclear Information System (INIS)

    Dhir, S.; Uppaluri, R.; Purkait, M.K.


    Increasing environmental legislations coupled with enhanced production of petroleum products demand, the deployment of novel technologies to remove organic sulfur efficiently. This work represents the kinetic modeling of ODS using H 2 O 2 over tungsten-containing layered double hydroxide (LDH) using the experimental data provided by Hulea et al. [V. Hulea, A.L. Maciuca, F. Fajula, E. Dumitriu, Catalytic oxidation of thiophenes and thioethers with hydrogen peroxide in the presence of W-containing layered double hydroxides, Appl. Catal. A: Gen. 313 (2) (2006) 200-207]. The kinetic modeling approach in this work initially targets the scope of the generation of a superstructure of micro-kinetic reaction schemes and models assuming Langmuir-Hinshelwood (LH) and Eley-Rideal (ER) mechanisms. Subsequently, the screening and selection of above models is initially based on profile-based elimination of incompetent schemes followed by non-linear regression search performed using the Levenberg-Marquardt algorithm (LMA) for the chosen models. The above analysis inferred that Eley-Rideal mechanism describes the kinetic behavior of ODS process using tungsten-containing LDH, with adsorption of reactant and intermediate product only taking place on the catalyst surface. Finally, an economic index is presented that scopes the economic aspects of the novel catalytic technology with the parameters obtained during regression analysis to conclude that the cost factor for the catalyst is 0.0062-0.04759 US $ per barrel

  6. Modeling chemical kinetics graphically

    NARCIS (Netherlands)

    Heck, A.


    In literature on chemistry education it has often been suggested that students, at high school level and beyond, can benefit in their studies of chemical kinetics from computer supported activities. Use of system dynamics modeling software is one of the suggested quantitative approaches that could


    African Journals Online (AJOL)

    Preferred Customer

    acetylchlorophosphonazo(CPApA) by hydrogen peroxide in 0.10 M phosphoric acid. A novel catalytic kinetic-spectrophotometric method is proposed for the determination of copper based on this principle. Copper(II) can be determined spectrophotometrically ...

  8. Kinetic energy budget details

    Indian Academy of Sciences (India)

    Abstract. This paper presents the detailed turbulent kinetic energy budget and higher order statistics of flow behind a surface-mounted rib with and without superimposed acoustic excitation. Pattern recognition technique is used to determine the large-scale structure magnitude. It is observed that most of the turbulence ...

  9. Point kinetics modeling

    International Nuclear Information System (INIS)

    Kimpland, R.H.


    A normalized form of the point kinetics equations, a prompt jump approximation, and the Nordheim-Fuchs model are used to model nuclear systems. Reactivity feedback mechanisms considered include volumetric expansion, thermal neutron temperature effect, Doppler effect and void formation. A sample problem of an excursion occurring in a plutonium solution accidentally formed in a glovebox is presented

  10. Stabilization of oil-in-water emulsions by enzyme catalyzed oxidative gelation of sugar beet pectin

    DEFF Research Database (Denmark)

    Abang Zaidel, Dayang Norulfairuz; Chronakis, Ioannis S.; Meyer, Anne S.


    Enzyme catalyzed oxidative cross-linking of feruloyl groups can promote gelation of sugar beet pectin (SBP). It is uncertain how the enzyme kinetics of this cross-linking reaction are affected in emulsion systems and whether the gelation affects emulsion stability. In this study, SBP (2.5% w...... larger average particle sizes than the emulsions in which the SBP was homogenized into the emulsion system during emulsion preparation (referred as Mix B). Mix B type emulsions were stable. Enzyme catalyzed oxidative gelation of SBP helped stabilize the emulsions in Mix A. The kinetics of the enzyme...... catalyzed oxidative gelation of SBP was evaluated by small angle oscillatory measurements for horseradish peroxidase (HRP) (EC and laccase (EC catalysis, respectively. HRP catalyzed gelation rates, determined from the slopes of the increase of elastic modulus (G0) with time, were higher...

  11. High-Throughput Analysis of Enzyme Activities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Guoxin [Iowa State Univ., Ames, IA (United States)


    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  12. Gene-enzyme relationships in somatic cells and their organismal derivatives in higher plants. Progress report

    International Nuclear Information System (INIS)

    Jensen, R.A.


    Several enzymes involved in the biosynthesis of aromatic amino acids have been isolated from Nicotiana silvestris. Isozymes of chlorismate mutase were isolated, partially purified and subjected to enzyme kinetic analysis. In addition, studies investigating the role of 5-enolpyruvyl-shikimate-3-phosphate synthetase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase, shikimate dehydrogenase, prephenate aminotransferase, arogenate dehydrogenase and phenylalanine ammonia-lyase in regulation of aromatic amino acids levels in tobacco are reported

  13. Competitive enzyme immunoassay for human chorionic somatomammotropin using the avidin-biotin system

    International Nuclear Information System (INIS)

    Rappuoli, R.; Leoncini, P.; Tarli, P.; Neri, P.


    Human chorionic somatomammotropin (HCS) is determined by an enzyme immunoassay where HCS competes with biotin-labeled HCS for insolubilized anti-HCS antibodies. Enzyme-labeled avidin is then used to reveal the amount of bound HCS. The system proves to be sensitive (1 ng/ml of HCS can be detected) and results agree with radioimmunoassay determinations (correlation coefficient = 0.979). Kinetics of the avidin-biotin reaction and coating of polystyrene wells are also investigated

  14. LLNL Chemical Kinetics Modeling Group

    Energy Technology Data Exchange (ETDEWEB)

    Pitz, W J; Westbrook, C K; Mehl, M; Herbinet, O; Curran, H J; Silke, E J


    The LLNL chemical kinetics modeling group has been responsible for much progress in the development of chemical kinetic models for practical fuels. The group began its work in the early 1970s, developing chemical kinetic models for methane, ethane, ethanol and halogenated inhibitors. Most recently, it has been developing chemical kinetic models for large n-alkanes, cycloalkanes, hexenes, and large methyl esters. These component models are needed to represent gasoline, diesel, jet, and oil-sand-derived fuels.

  15. DGAT enzymes and triacylglycerol biosynthesis (United States)

    Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.


    Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases. PMID:18757836

  16. Enzyme stabilization for pesticide degradation

    Energy Technology Data Exchange (ETDEWEB)

    Rivers, D.B.; Frazer, F.R. III; Mason, D.W.; Tice, T.R.


    Enzymes offer inherent advantages and limitations as active components of formulations used to decontaminate soil and equipment contaminated with toxic materials such as pesticides. Because of the catalytic nature of enzymes, each molecule of enzyme has the potential to destroy countless molecules of a contaminating toxic compound. This degradation takes place under mild environmental conditions of pH, temperature, pressure, and solvent. The basic limitation of enzymes is their degree of stability during storage and application conditions. Stabilizing methods such as the use of additives, covalent crosslinking, covalent attachment, gel entrapment, and microencapsulation have been directed developing an enzyme preparation that is stable under extremes of pH, temperature, and exposure to organic solvents. Initial studies were conducted using the model enzymes subtilisin and horseradish peroxidase.

  17. Direct comparison of enzyme histochemical and immunohistochemical methods to localize an enzyme

    NARCIS (Netherlands)

    van Noorden, Cornelis J. F.


    Immunohistochemical localization of enzymes is compared directly with localization of enzyme activity with (catalytic) enzyme histochemical methods. The two approaches demonstrate principally different aspects of an enzyme. The immunohistochemical method localizes the enzyme protein whether it is

  18. Enzyme Mimics: Advances and Applications. (United States)

    Kuah, Evelyn; Toh, Seraphina; Yee, Jessica; Ma, Qian; Gao, Zhiqiang


    Enzyme mimics or artificial enzymes are a class of catalysts that have been actively pursued for decades and have heralded much interest as potentially viable alternatives to natural enzymes. Aside from having catalytic activities similar to their natural counterparts, enzyme mimics have the desired advantages of tunable structures and catalytic efficiencies, excellent tolerance to experimental conditions, lower cost, and purely synthetic routes to their preparation. Although still in the midst of development, impressive advances have already been made. Enzyme mimics have shown immense potential in the catalysis of a wide range of chemical and biological reactions, the development of chemical and biological sensing and anti-biofouling systems, and the production of pharmaceuticals and clean fuels. This Review concerns the development of various types of enzyme mimics, namely polymeric and dendrimeric, supramolecular, nanoparticulate and proteinic enzyme mimics, with an emphasis on their synthesis, catalytic properties and technical applications. It provides an introduction to enzyme mimics and a comprehensive summary of the advances and current standings of their applications, and seeks to inspire researchers to perfect the design and synthesis of enzyme mimics and to tailor their functionality for a much wider range of applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Phage lytic enzymes: a history. (United States)

    Trudil, David


    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  20. [The rise of enzyme engineering in China]. (United States)

    Li, Gaoxiang


    Enzyme engineering is an important part of the modern biotechnology. Industrial biocatalysis is considered the third wave of biotechnology following pharmaceutical and agricultural waves. In 25 years, China has made a mighty advances in enzyme engineering research. This review focuses on enzyme genomics, enzyme proteomics, biosynthesis, microbial conversion and biosensors in the Chinese enzyme engineering symposiums and advances in enzyme preparation industry in China.

  1. Kinetic study of enzymatic hydrolysis of potato starch

    Directory of Open Access Journals (Sweden)

    Óscar Fernando Castellanos Domínguez


    Full Text Available This article describes the kinetic study of potato starch enzymatic hydrolysis using soluble enzymes (Novo Nordisk. Different assays divided into four groups were used: reaction time (with which it was possible to reduce the 48-72 hour duration reported in the literature to 16 hours with comparable productivity levels; selecting the set of enzymes to be used (different types were evaluated - BAN and Termamyl as alfa-amylases during dextrinisation stage, and AMG, Promozyme and Fungamyl for sacarification reaction- identifying those presenting the best performance during hydrolysis.Reaction conditions were optimised for the process's two stages (destrinisation and sacarification. Enzyme dose, calcium cofactor concentration, pH, temperature and agitation speed were studied for the first stage. Enzyme ratio, pH and agitation speed were studied for sacarification; the latter parameter reported values having no antecedents in the literature (60 rpm and 30 rpm for first and second reactions, respectively. Michaelis Menten kinetics were calculated once conditions had been optimised, varying substrate from 10-50% P/V, obtaining km and Vmax kinetic parameters for each reaction. A kinetic model was found according to local working conditions which was able to explain potato starch conversion to glucose syrup, achieving 96 dextrose equivalents by the end of the reaction, being well within the maximum range reported in the literature (94-98.Laboratory equipment was constructed prior to carrying out assays which was able to reproduce and improve the conditions reported in the literature, making it a useful, reliable tool for use in assays returning good results.

  2. Kinetics of adenylate metabolism in human and rat myocardium


    Tavenier, M.; Skladanowski, A.C.; Abreu, R.A. de; Jong, J.W. de


    textabstractPathways producing and converting adenosine have hardly been investigated in human heart, contrasting work in other species. We compared the kinetics of enzymes associated with purine degradation and salvage in human and rat heart cytoplasm assaying for adenosine deaminase, nucleoside phosphorylase, xanthine oxidoreductase, AMP deaminase, AMP- and IMP-specific 5′-nucleotidases, adenosine kinase and hypoxanthine guanine phosphoribosyltransferase (HGPRT). Xanthine oxidoreductase was...

  3. Microsecond time-scale kinetics of transient biochemical reactions

    NARCIS (Netherlands)

    Mitic, S.; Strampraad, M.J.F.; Hagen, W.R.; de Vries, S.


    To afford mechanistic studies in enzyme kinetics and protein folding in the microsecond time domain we have developed a continuous-flow microsecond time-scale mixing instrument with an unprecedented dead-time of 3.8 ± 0.3 μs. The instrument employs a micro-mixer with a mixing time of 2.7 μs

  4. Enzyme structure, enzyme function and allozyme diversity in ...

    African Journals Online (AJOL)

    In estimates of population genetic diversity based on allozyme heterozygosity, some enzymes are regularly more variable than others. Evolutionary theory suggests that functionally less important molecules, or parts of molecules, evolve more rapidly than more important ones; the latter enzymes should then theoretically be ...

  5. Kinetic energy absorbing pad

    International Nuclear Information System (INIS)

    Bricmont, R.J.; Hamilton, P.A.; Ming Long Ting, R.


    Reactors, fuel processing plants etc incorporate pipes and conduits for fluids under high pressure. Fractures, particularly adjacent to conduit elbows, produce a jet of liquid which whips the broken conduit at an extremely high velocity. An enormous impact load would be applied to any stationary object in the conduit's path. The design of cellular, corrugated metal impact pads to absorb the kinetic energy of the high velocity conduits is given. (U.K.)

  6. Computational enzyme design: transitioning from catalytic proteins to enzymes. (United States)

    Mak, Wai Shun; Siegel, Justin B


    The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward. Published by Elsevier Ltd.

  7. Immobilized enzymes: understanding enzyme - surface interactions at the molecular level. (United States)

    Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G


    Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

  8. Stability of Enzymes in Granular Enzyme Products for Laundry Detergents

    DEFF Research Database (Denmark)

    Biran, Suzan; Bach, Poul; Simonsen, Ole

    Enzymes have long been of interest to the detergent industry due to their ability to improve the cleaning efficiency of synthetic detergents, contribute to shortening washing times, and reduce energy and water consumption, provision of environmentally friendlier wash water effluents and fabric care....... However, incorporating enzymes in detergent formulations gives rise to numerous practical problems due to their incompatibility with and stability against various detergent components. In powdered detergent formulations, these issues can be partly overcome by physically isolating the enzymes in separate...... particles. However, enzymes may loose a significant part of their activity over a time period of several weeks. Possible causes of inactivation of enzymes in a granule may be related to the release of hydrogen peroxide from the bleaching chemicals in a moisture-containing atmosphere, humidity, autolysis...

  9. Enzymes in Human Milk. (United States)

    Dallas, David C; German, J Bruce


    Milk proteins are a complex and diverse source of biological activities. Beyond their function, intact milk proteins also act as carriers of encrypted functional sequences that, when released as peptides, exert biological functions, including antimicrobial and immunomodulatory activity, which could contribute to the infant's competitive success. Research has now revealed that the release of these functional peptides begins within the mammary gland itself. A complex array of proteases produced in mother's milk has been shown to be active in the milk, releasing these peptides. Moreover, our recent research demonstrates that these milk proteases continue to digest milk proteins within the infant's stomach, possibly even to a larger extent than the infant's own proteases. As the neonate has relatively low digestive capacity, the activity of milk proteases in the infant may provide important assistance to digesting milk proteins. The coordinated release of these encrypted sequences is accomplished by selective proteolytic action provided by an array of native milk proteases and infant-produced enzymes. The task for scientists is now to discover the selective advantages of this protein-protease-based peptide release system. © 2017 Nestec Ltd., Vevey/S. Karger AG, Basel.

  10. Kinetics of Adsorbable Organic Halides (AOX Reduction in Laccase-Aided Chlorine Dioxide Bleaching of Bagasse Pulp

    Directory of Open Access Journals (Sweden)

    Xueping Song


    Full Text Available This paper presents a kinetic model of the laccase-aided chlorine dioxide bleaching of bagasse pulp. The kinetic model was based on the rate of reduction of adsorbed organic halogen (AOX. The effects of the laccase enzyme dosage, the mediator 1-hydroxybenzotriazole (HBT dosage, and the reaction temperature on the AOX content of the bleaching effluent are discussed. Good fits were obtained for the experimental data obtained from the different laccase enzyme dosages, HBT dosages, and reaction temperatures, indicating the feasibility of the kinetic model as a means of predicting the optimal operation conditions for the laccase-aided chlorine dioxide bleaching of bagasse pulp in the future.

  11. Digestive enzymes of some earthworms. (United States)

    Mishra, P C; Dash, M C


    4 species of tropical earthworms differed with regard to enzyme activity. The maximum activity of protease and of cellulase occurred in the posterior region of the gut of the earthworms. On the average Octochaetona surensis shows maximum activity and Drawida calebi shows minimum activity for all the enzymes studied.

  12. Photoreactivating enzyme from Escherichia coli

    International Nuclear Information System (INIS)

    Snapka, R.M.; Fuselier, C.O.


    Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E.coli strain lysogenic for a defective lambda bacteriophage carrying the phr gene. The resulting enzyme had a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consisted of an apoprotein and cofactor, both of which were necessary for catalytic activity. The apoprotein had a monomer molecular weight of 35,200 and showed stable aggregates under denaturing conditions. The amino acid analysis of the E.coli enzyme was very similar to that of the photoreactivating enzyme from orchid seedlings (Cattelya aurantiaca). Both had arginine at the amino terminus. The cofactor, like the holoenzyme, showed absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme had an action spectrum which peaked at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme showed any detectable absorption between 300 and 400 nm. (author)

  13. Positron emitter labeled enzyme inhibitors

    International Nuclear Information System (INIS)

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.


    This invention involves a new strategy for imagining and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography

  14. Photoreactivating enzyme from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Snapka, R M; Fuselier, C O [California Univ., Irvine (USA)


    Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E.coli strain lysogenic for a defective lambda bacteriophage carrying the phr gene. The resulting enzyme had a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consisted of an apoprotein and cofactor, both of which were necessary for catalytic activity. The apoprotein had a monomer molecular weight of 35,200 and showed stable aggregates under denaturing conditions. The amino acid analysis of the E.coli enzyme was very similar to that of the photoreactivating enzyme from orchid seedlings (Cattelya aurantiaca). Both had arginine at the amino terminus. The cofactor, like the holoenzyme, showed absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme had an action spectrum which peaked at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme showed any detectable absorption between 300 and 400 nm.

  15. Elastinolytic and proteolytic enzymes. (United States)

    Kessler, Efrat; Safrin, Mary


    staphylolytic activity in which the rate of S. aureus cell lysis is determined spectrophotometrically, and methods for determination of peptidase activity of pseudolysin, staphylolysin, lysyl endopeptidase, and the aminopeptidase. The latter methods employ chromogenic or fluorogenic peptide derivatives comprising a short amino acid sequence matching the preferred cleavage site of the protease as substrates. As only one peptide bond is cleaved in each substrate, these assays permit kinetic studies.

  16. Transient competitive complexation in biological kinetic isotope fractionation explains nonsteady isotopic effects: Theory and application to denitrification in soils (United States)

    Maggi, Federico; Riley, William J.


    The theoretical formulation of biological kinetic isotope fractionation often assumes first-order or Michaelis-Menten kinetics, the latter solved under the quasi-steady state assumption. Both formulations lead to a constant isotope fractionation factor, therefore they may return incorrect estimations of isotopic effects and misleading interpretations of isotopic signatures when fractionation is not a steady process. We have analyzed the isotopic signature of denitrification in biogeochemical soil systems by Menyailo and Hungate (2006) in which high and variable 15N-N2O enrichment during N2O production and inverse isotope fractionation during N2O consumption could not be explained with first-order kinetics and the Rayleigh equation, or with Michaelis-Menten kinetics. When Michaelis-Menten kinetics were coupled to Monod kinetics to describe biomass and enzyme dynamics, and the quasi-steady state assumption was relaxed, transient Michaelis-Menten-Monod kinetics accurately reproduced the observed concentrations, and variable and inverse isotope fractionations. These results imply a substantial revision in modeling isotopic effects, suggesting that steady state kinetics such as first-order, Rayleigh, and classic Michaelis-Menten kinetics should be superseded by transient kinetics in conjunction with biomass and enzyme dynamics.

  17. The development, characterization, and application of biomimetic nanoscale enzyme immobilization (United States)

    Haase, Nicholas R.

    The utilization of enzymes is of interest for applications such as biosensors and biofuel cells. Immobilizing enzymes provides a means to develop these applications. Previous immobilization efforts have been accomplished by exposing surfaces on which silica-forming molecules are present to solutions containing an enzyme and a silica precursor. This approach leads to the enzyme being entrapped in a matrix three orders of magnitude larger than the enzyme itself, resulting in low retention of enzyme activity. The research herein introduces a method for the immobilization of enzymes during the layer-by-layer buildup of Si-O and Ti-O coatings which are nanoscale in thickness. This approach is an application of a peptide-induced mineral deposition method developed in the Sandhage and Kroger groups, and it involves the alternating exposure of a surface to solutions containing the peptide protamine and then an aqueous precursor solution of silicon- or titanium-oxide at near-neutral pH. A method has been developed that enables in situ immobilization of enzymes in the protamine/mineral oxide coatings. Depending on the layer and mineral (silica or titania) within which the enzyme is incorporated, the resulting multilayer biocatalytic hybrid materials retain 20 -- 100% of the enzyme activity. Analyses of kinetic properties of the immobilized enzyme, coupled with characterization of physical properties of the mineral-bearing layers (thickness, porosity, pore size distribution), indicates that the catalytic activities of the enzymes immobilized in the different layers are largely determined by substrate diffusion. The enzyme was also found to be substantially stabilized against heat-induced denaturation and largely protected from proteolytic attack. These functional coatings are then developed for use as antimicrobial materials. Glucose oxidase, which catalyzes production of the cytotoxic agent hydrogen peroxide, was immobilized with silver nanoparticles, can release

  18. Kinetics of tetrataenite disordering

    International Nuclear Information System (INIS)

    Dos Santos, E.; Gattacceca, J.; Rochette, P.; Fillion, G.; Scorzelli, R.B.


    Tetrataenite is a chemically ordered L1 0 -type Fe 50 Ni 50 alloy detected for the first time in 1977 by 57 Fe Mössbauer spectroscopy studies in iron meteorites. The thermal history of meteorites, in particular short thermal events like those associated to hypervelocity impacts, can be constrained by tracing the presence of tetrataenite or its disordering into taenite. The knowledge of the disordering kinetics of tetrataenite, that is associated with changes in its magnetic properties, is still very fragmentary so that the time–temperature history of these meteorites cannot be constrained in details. Furthermore, knowledge of disordering kinetics is important due to potential technological application of tetrataenite as a rare-earth free strong magnet. Thus, this work provides the first time–temperature data for disordering reaction of tetrataenite. We have shown that disordering is not an instantaneous process but is a kinetic limited reaction. It was shown that disordering may take place at any temperature above the order–disorder transition for L 10 superstructure phase (∼320 °C) when the appropriate time-scale is considered. This result means that the apparent Curie point for tetrataenite is not an absolute property in the sense that any estimate of this parameter should be referred to a given time-scale. - Highlights: • The first time–temperature data for tetrataenite disordering reaction is provided. • Previous works does not give a complete picture of tetrataenite disordering. • Apparent Curie temperature of tetrataenite should be referred to a time-scale. • Tetrataenite can be used as a probe to detect thermal/shock events recorded in meteorites

  19. Phosphoglycerate Mutase Is a Highly Efficient Enzyme without Flux Control in Lactococcus lactis

    DEFF Research Database (Denmark)

    Solem, Christian; Petranovic, D.; Købmann, Brian


    The glycolytic enzyme phosphoglycerate mutase (PGM), which catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate, was examined in Lactococcus lactis with respect to its function, kinetics and glycolytic flux control. A library of strains with PGM activities ranging between 15-465% ...

  20. In Vitro Optimization of Enzymes Involved in Precorrin-2 Synthesis Using Response Surface Methodology. (United States)

    Fang, Huan; Dong, Huina; Cai, Tao; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin


    In order to maximize the production of biologically-derived chemicals, kinetic analyses are first necessary for predicting the role of enzyme components and coordinating enzymes in the same reaction system. Precorrin-2 is a key precursor of cobalamin and siroheme synthesis. In this study, we sought to optimize the concentrations of several molecules involved in precorrin-2 synthesis in vitro: porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and S-adenosyl-l-methionine-dependent urogen III methyltransferase (SUMT). Response surface methodology was applied to develop a kinetic model designed to maximize precorrin-2 productivity. The optimal molar ratios of PBGS, PBGD, UROS, and SUMT were found to be approximately 1:7:7:34, respectively. Maximum precorrin-2 production was achieved at 0.1966 ± 0.0028 μM/min, agreeing with the kinetic model's predicted value of 0.1950 μM/min. The optimal concentrations of the cofactor S-adenosyl-L-methionine (SAM) and substrate 5-aminolevulinic acid (ALA) were also determined to be 200 μM and 5 mM, respectively, in a tandem-enzyme assay. By optimizing the relative concentrations of these enzymes, we were able to minimize the effects of substrate inhibition and feedback inhibition by S-adenosylhomocysteine on SUMT and thereby increase the production of precorrin-2 by approximately five-fold. These results demonstrate the effectiveness of kinetic modeling via response surface methodology for maximizing the production of biologically-derived chemicals.

  1. Hydrogen peroxide decomposition kinetics in aquaculture water

    DEFF Research Database (Denmark)

    Arvin, Erik; Pedersen, Lars-Flemming


    during the HP decomposition. The model assumes that the enzyme decay is controlled by an inactivation stoichiometry related to the HP decomposition. In order to make the model easily applicable, it is furthermore assumed that the COD is a proxy of the active biomass concentration of the water and thereby......Hydrogen peroxide (HP) is used in aquaculture systems where preventive or curative water treatments occasionally are required. Use of chemical agents can be challenging in recirculating aquaculture systems (RAS) due to extended water retention time and because the agents must not damage the fish...... reared or the nitrifying bacteria in the biofilters at concentrations required to eliminating pathogens. This calls for quantitative insight into the fate of the disinfectant residuals during water treatment. This paper presents a kinetic model that describes the HP decomposition in aquaculture water...

  2. Quantum kinetic Ising models

    International Nuclear Information System (INIS)

    Augusiak, R; Cucchietti, F M; Lewenstein, M; Haake, F


    In this paper, we introduce a quantum generalization of classical kinetic Ising models (KIM), described by a certain class of quantum many-body master equations. Similarly to KIMs with detailed balance that are equivalent to certain Hamiltonian systems, our models reduce to a set of Hamiltonian systems determining the dynamics of the elements of the many-body density matrix. The ground states of these Hamiltonians are well described by the matrix product, or pair entangled projected states. We discuss critical properties of such Hamiltonians, as well as entanglement properties of their low-energy states.


    Directory of Open Access Journals (Sweden)

    Václav Koman


    Full Text Available Normal 0 21 false false false SK X-NONE X-NONE Bread is the most common and traditional food in the world. For years, enzymes such as malt and fungal alpha-amylase have been used in bread making. Due to the changes in the baking industry and the ever-increasing demand for more natural products, enzymes have gained real importance in bread-making. If an enzyme is added, it is often destroyed by the heat during the baking process. For generations, enzymes have been used for the improvement of texture and appearance, enhancement of nutritional values and generation of appealing flavours and aromas. Enzymes used in bakery industry constitute nearly one third of the market. The bakery products have undergone radical improvements in quality over the past years in terms of flavour, texture and shelf-life. The the biggest contributor for these improvementsis the usage of enzymes. Present work seeks to systematically describe bakery enzymes, their classification, benefits, usage and chemical reactions in the bread making process.doi:10.5219/193

  4. An efficient approach to bioconversion kinetic model generation based on automated microscale experimentation integrated with model driven experimental design

    DEFF Research Database (Denmark)

    Chen, B. H.; Micheletti, M.; Baganz, F.


    -erythrulose. Experiments were performed using automated microwell studies at the 150 or 800 mu L scale. The derived kinetic parameters were then verified in a second round of experiments where model predictions showed excellent agreement with experimental data obtained under conditions not included in the original......Reliable models of enzyme kinetics are required for the effective design of bioconversion processes. Kinetic expressions of the enzyme-catalysed reaction rate however, are frequently complex and establishing accurate values of kinetic parameters normally requires a large number of experiments....... These can be both time consuming and expensive when working with the types of non-natural chiral intermediates important in pharmaceutical syntheses. This paper presents ail automated microscale approach to the rapid and cost effective generation of reliable kinetic models useful for bioconversion process...

  5. Modelling of different enzyme productions by solid-state fermentation on several agro-industrial residues. (United States)

    Diaz, Ana Belen; Blandino, Ana; Webb, Colin; Caro, Ildefonso


    A simple kinetic model, with only three fitting parameters, for several enzyme productions in Petri dishes by solid-state fermentation is proposed in this paper, which may be a valuable tool for simulation of this type of processes. Basically, the model is able to predict temporal fungal enzyme production by solid-state fermentation on complex substrates, maximum enzyme activity expected and time at which these maxima are reached. In this work, several fermentations in solid state were performed in Petri dishes, using four filamentous fungi grown on different agro-industrial residues, measuring xylanase, exo-polygalacturonase, cellulose and laccase activities over time. Regression coefficients after fitting experimental data to the proposed model turned out to be quite high in all cases. In fact, these results are very interesting considering, on the one hand, the simplicity of the model and, on the other hand, that enzyme activities correspond to different enzymes, produced by different fungi on different substrates.

  6. Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro kcat measurements (United States)

    Davidi, Dan; Noor, Elad; Liebermeister, Wolfram; Bar-Even, Arren; Flamholz, Avi; Tummler, Katja; Barenholz, Uri; Goldenfeld, Miki; Shlomi, Tomer; Milo, Ron


    Turnover numbers, also known as kcat values, are fundamental properties of enzymes. However, kcat data are scarce and measured in vitro, thus may not faithfully represent the in vivo situation. A basic question that awaits elucidation is: how representative are kcat values for the maximal catalytic rates of enzymes in vivo? Here, we harness omics data to calculate kmaxvivo, the observed maximal catalytic rate of an enzyme inside cells. Comparison with kcat values from Escherichia coli, yields a correlation of r2= 0.62 in log scale (p enzymes and the backward flux dictated by thermodynamics, we further refine the correspondence between kmaxvivo and kcat values. The approach we present here characterizes the quantitative relationship between enzymatic catalysis in vitro and in vivo and offers a high-throughput method for extracting enzyme kinetic constants from omics data. PMID:26951675

  7. [Automated analyzer of enzyme immunoassay]. (United States)

    Osawa, S


    Automated analyzers for enzyme immunoassay can be classified by several points of view: the kind of labeled antibodies or enzymes, detection methods, the number of tests per unit time, analytical time and speed per run. In practice, it is important for us consider the several points such as detection limits, the number of tests per unit time, analytical range, and precision. Most of the automated analyzers on the market can randomly access and measure samples. I will describe the recent advance of automated analyzers reviewing their labeling antibodies and enzymes, the detection methods, the number of test per unit time and analytical time and speed per test.

  8. Accurate label-free reaction kinetics determination using initial rate heat measurements (United States)

    Ebrahimi, Kourosh Honarmand; Hagedoorn, Peter-Leon; Jacobs, Denise; Hagen, Wilfred R.


    Accurate label-free methods or assays to obtain the initial reaction rates have significant importance in fundamental studies of enzymes and in application-oriented high throughput screening of enzyme activity. Here we introduce a label-free approach for obtaining initial rates of enzyme activity from heat measurements, which we name initial rate calorimetry (IrCal). This approach is based on our new finding that the data recorded by isothermal titration calorimetry for the early stages of a reaction, which have been widely ignored, are correlated to the initial rates. Application of the IrCal approach to various enzymes led to accurate enzyme kinetics parameters as compared to spectroscopic methods and enabled enzyme kinetic studies with natural substrate, e.g. proteases with protein substrates. Because heat is a label-free property of almost all reactions, the IrCal approach holds promise in fundamental studies of various enzymes and in use of calorimetry for high throughput screening of enzyme activity. PMID:26574737

  9. Kinetics of coal pyrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Seery, D.J.; Freihaut, J.D.; Proscia, W.M. (United Technologies Research Center, East Hartford, CT (USA)); Howard, J.B.; Peters, W.; Hsu, J.; Hajaligol, M.; Sarofim, A. (Massachusetts Inst. of Tech., Cambridge, MA (USA)); Jenkins, R.; Mallin, J.; Espindola-Merin, B. (Pennsylvania State Univ., University Park, PA (USA)); Essenhigh, R.; Misra, M.K. (Ohio State Univ., Columbus, OH (USA))


    This report contains results of a coordinated, multi-laboratory investigation of coal devolatilization. Data is reported pertaining to the devolatilization for bituminous coals over three orders of magnitude in apparent heating rate (100 to 100,000 + {degree}C/sec), over two orders of magnitude in particle size (20 to 700 microns), final particle temperatures from 400 to 1600{degree}C, heat transfer modes ranging from convection to radiative, ambient pressure ranging from near vacuum to one atmosphere pressure. The heat transfer characteristics of the reactors are reported in detail. It is assumed the experimental results are to form the basis of a devolatilization data base. Empirical rate expressions are developed for each phase of devolatilization which, when coupled to an awareness of the heat transfer rate potential of a particular devolatilization reactor, indicate the kinetics emphasized by a particular system reactor plus coal sample. The analysis indicates the particular phase of devolatilization that will be emphasized by a particular reactor type and, thereby, the kinetic expressions appropriate to that devolatilization system. Engineering rate expressions are developed from the empirical rate expressions in the context of a fundamental understanding of coal devolatilization developed in the course of the investigation. 164 refs., 223 figs., 44 tabs.

  10. Repair kinetics in tissues

    International Nuclear Information System (INIS)

    Thames, H.D.


    Monoexponential repair kinetics is based on the assumption of a single, dose-independent rate of repair of sublethal injury in the target cells for tissue injury after exposure to ionizing radiation. Descriptions of the available data based on this assumption have proved fairly successful for both acutely responding (skin, lip mucosa, gut) and late-responding (lung, spinal cord) normal tissues. There are indications of biphasic exponential repair in both categories, however. Unfortunately, the data usually lack sufficient resolution to permit unambiguous determination of the repair rates. There are also indications that repair kinetics may depend on the size of the dose. The data are conflicting on this account, however, with suggestions of both faster and slower repair after larger doses. Indeed, experiments that have been explicitly designed to test this hypothesis show either no effect (gut, spinal cord), faster repair after higher doses (lung, kidney), or slower repair after higher doses (skin). Monoexponential repair appears to be a fairly accurate description that provides an approximation to a more complicated picture, the elucidation of whose details will, however, require very careful and extensive experimental study. (author). 30 refs.; 1 fig

  11. Diffusion Influenced Adsorption Kinetics. (United States)

    Miura, Toshiaki; Seki, Kazuhiko


    When the kinetics of adsorption is influenced by the diffusive flow of solutes, the solute concentration at the surface is influenced by the surface coverage of solutes, which is given by the Langmuir-Hinshelwood adsorption equation. The diffusion equation with the boundary condition given by the Langmuir-Hinshelwood adsorption equation leads to the nonlinear integro-differential equation for the surface coverage. In this paper, we solved the nonlinear integro-differential equation using the Grünwald-Letnikov formula developed to solve fractional kinetics. Guided by the numerical results, analytical expressions for the upper and lower bounds of the exact numerical results were obtained. The upper and lower bounds were close to the exact numerical results in the diffusion- and reaction-controlled limits, respectively. We examined the validity of the two simple analytical expressions obtained in the diffusion-controlled limit. The results were generalized to include the effect of dispersive diffusion. We also investigated the effect of molecular rearrangement of anisotropic molecules on surface coverage.

  12. Chemical kinetics and reaction mechanism

    International Nuclear Information System (INIS)

    Jung, Ou Sik; Park, Youn Yeol


    This book is about chemical kinetics and reaction mechanism. It consists of eleven chapters, which deal with reaction and reaction speed on reaction mechanism, simple reaction by rate expression, reversible reaction and simultaneous reaction, successive reaction, complicated reaction mechanism, assumption for reaction mechanism, transition state theory, successive reaction and oscillating reaction, reaction by solution, research method high except kinetics on reaction mechanism, high reaction of kinetics like pulsed radiolysis.

  13. Chemical kinetics of gas reactions

    CERN Document Server

    Kondrat'Ev, V N


    Chemical Kinetics of Gas Reactions explores the advances in gas kinetics and thermal, photochemical, electrical discharge, and radiation chemical reactions. This book is composed of 10 chapters, and begins with the presentation of general kinetic rules for simple and complex chemical reactions. The next chapters deal with the experimental methods for evaluating chemical reaction mechanisms and some theories of elementary chemical processes. These topics are followed by discussions on certain class of chemical reactions, including unimolecular, bimolecular, and termolecular reactions. The rema

  14. Adsorption analysis equilibria and kinetics

    CERN Document Server

    Do, Duong D


    This book covers topics of equilibria and kinetics of adsorption in porous media. Fundamental equilibria and kinetics are dealt with for homogeneous as well as heterogeneous particles. Five chapters of the book deal with equilibria and eight chapters deal with kinetics. Single component as well as multicomponent systems are discussed. In kinetics analysis, we deal with the various mass transport processes and their interactions inside a porous particle. Conventional approaches as well as the new approach using Maxwell-Stefan equations are presented. Various methods to measure diffusivity, such

  15. Kinetically Controlled Drug Resistance

    DEFF Research Database (Denmark)

    Sun, Xin E.; Hansen, Bjarne Gram; Hedstrom, Lizbeth


    The filamentous fungus Penicillium brevicompactum produces the immunosuppressive drug mycophenolic acid (MPA), which is a potent inhibitor of eukaryotic IMP dehydrogenases (IMPDHs). IMPDH catalyzes the conversion of IMP to XMP via a covalent enzyme intermediate, E-XMP*; MPA inhibits by trapping E...... of resistance is not apparent. Here, we show that, unlike MPA-sensitive IMPDHs, formation of E-XMP* is rate-limiting for both PbIMPDH-A and PbIMPDH-B. Therefore, MPA resistance derives from the failure to accumulate the drug-sensitive intermediate....

  16. Multi-enzyme Process Modeling

    DEFF Research Database (Denmark)

    Andrade Santacoloma, Paloma de Gracia

    are affected (in a positive or negative way) by the presence of the other enzymes and compounds in the media. In this thesis the concept of multi-enzyme in-pot term is adopted for processes that are carried out by the combination of enzymes in a single reactor and implemented at pilot or industrial scale...... features of the process and provides the information required to structure the process model by using a step-by-step procedure with the required tools and methods. In this way, this framework increases efficiency of the model development process with respect to time and resources needed (fast and effective....... In this way the model parameters that drives the main dynamic behavior can be identified and thus a better understanding of this type of processes. In order to develop, test and verify the methodology, three case studies were selected, specifically the bi-enzyme process for the production of lactobionic acid...

  17. PIXE analysis of Zn enzymes

    International Nuclear Information System (INIS)

    Solis, C.; Oliver, A.; Andrade, E.; Ruvalcaba-Sil, J.L.; Romero, I.; Celis, H.


    Zinc is a necessary component in the action and structural stability of many enzymes. Some of them are well characterized, but in others, Zn stoichiometry and its association is not known. PIXE has been proven to be a suitable technique for analyzing metallic proteins embedded in electrophoresis gels. In this study, PIXE has been used to investigate the Zn content of enzymes that are known to carry Zn atoms. These include the carbonic anhydrase, an enzyme well characterized by other methods and the cytoplasmic pyrophosphatase of Rhodospirillum rubrum that is known to require Zn to be stable but not how many metal ions are involved or how they are bound to the enzyme. Native proteins have been purified by polyacrylamide gel electrophoresis and direct identification and quantification of Zn in the gel bands was performed with an external proton beam of 3.7 MeV energy

  18. GRE Enzymes for Vector Analysis (United States)

    U.S. Environmental Protection Agency — Microbial enzyme data that were collected during the 2004-2006 EMAP-GRE program. These data were then used by Moorhead et al (2016) in their ecoenzyme vector...

  19. Watching Individual Enzymes at Work (United States)

    Blank, Kerstin; Rocha, Susana; De Cremer, Gert; Roeffaers, Maarten B. J.; Uji-i, Hiroshi; Hofkens, Johan

    Single-molecule fluorescence experiments are a powerful tool to analyze reaction mechanisms of enzymes. Because of their unique potential to detect heterogeneities in space and time, they have provided unprecedented insights into the nature and mechanisms of conformational changes related to the catalytic reaction. The most important finding from experiments with single enzymes is the generally observed phenomenon that the catalytic rate constants fluctuate over time (dynamic disorder). These fluctuations originate from conformational changes occurring on time scales, which are similar to or slower than that of the catalytic reaction. Here, we summarize experiments with enzymes that show dynamic disorder and introduce new experimental strategies showing how single-molecule fluorescence experiments can be applied to address other open questions in medical and industrial enzymology, such as enzyme inactivation processes, reactant transfer in cascade reactions, and the mechanisms of interfacial catalysis.

  20. Photosynthetic fuel for heterologous enzymes

    DEFF Research Database (Denmark)

    Mellor, Silas Busck; Vavitsas, Konstantinos; Nielsen, Agnieszka Janina Zygadlo


    of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely...... on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However......, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic...

  1. Perspectives on electrostatics and conformational motions in enzyme catalysis. (United States)

    Hanoian, Philip; Liu, C Tony; Hammes-Schiffer, Sharon; Benkovic, Stephen


    CONSPECTUS: Enzymes are essential for all living organisms, and their effectiveness as chemical catalysts has driven more than a half century of research seeking to understand the enormous rate enhancements they provide. Nevertheless, a complete understanding of the factors that govern the rate enhancements and selectivities of enzymes remains elusive, due to the extraordinary complexity and cooperativity that are the hallmarks of these biomolecules. We have used a combination of site-directed mutagenesis, pre-steady-state kinetics, X-ray crystallography, nuclear magnetic resonance (NMR), vibrational and fluorescence spectroscopies, resonance energy transfer, and computer simulations to study the implications of conformational motions and electrostatic interactions on enzyme catalysis in the enzyme dihydrofolate reductase (DHFR). We have demonstrated that modest equilibrium conformational changes are functionally related to the hydride transfer reaction. Results obtained for mutant DHFRs illustrated that reductions in hydride transfer rates are correlated with altered conformational motions, and analysis of the evolutionary history of DHFR indicated that mutations appear to have occurred to preserve both the hydride transfer rate and the associated conformational changes. More recent results suggested that differences in local electrostatic environments contribute to finely tuning the substrate pKa in the initial protonation step. Using a combination of primary and solvent kinetic isotope effects, we demonstrated that the reaction mechanism is consistent across a broad pH range, and computer simulations suggested that deprotonation of the active site Tyr100 may play a crucial role in substrate protonation at high pH. Site-specific incorporation of vibrational thiocyanate probes into the ecDHFR active site provided an experimental tool for interrogating these microenvironments and for investigating changes in electrostatics along the DHFR catalytic cycle

  2. DGAT enzymes and triacylglycerol biosynthesis


    Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.


    Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, ...

  3. Model for how type I restriction enzymes select cleavage sites in DNA

    International Nuclear Information System (INIS)

    Studier, F.W.; Bandyopadhyay, P.K.


    Under appropriate conditions, digestion of phage T7 DNA by the type I restriction enzyme EcoK produces an orderly progression of discrete DNA fragments. All details of the fragmentation pattern can be explained on the basis of the known properties of type I enzymes, together with two further assumptions: (i) in the ATP-stimulated translocation reaction, the enzyme bound at the recognition sequence translocates DNA toward itself from both directions simultaneously; and (ii) when translocation causes neighboring enzymes to meet, they cut the DNA between them. The kinetics of digestion at 37 degree C indicates that the rate of translocation of DNA from each side of a bound enzyme is about 200 base pairs per second, and the cuts are completed within 15-25 sec of the time neighboring enzymes meet. The resulting DNA fragments each contain a single recognition site with an enzyme (or subunit) remaining bound to it. At high enzyme concentrations, such fragments can bu further degraded, apparently by cooperation between the specifically bound and excess enzymes. This model is consistent with a substantial body of previous work on the nuclease activity of EcoB and EcoK, and it explains in a simple way how cleavage sites are selected

  4. Glutathione reductase: solvent equilibrium and kinetic isotope effects

    International Nuclear Information System (INIS)

    Wong, K.K.; Vanoni, M.A.; Blanchard, J.S.


    Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D 2 O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456

  5. Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes. (United States)

    Wei, Ren; Oeser, Thorsten; Zimmermann, Wolfgang


    Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented. © 2014 Elsevier Inc. All rights reserved.

  6. de novo computational enzyme design. (United States)

    Zanghellini, Alexandre


    Recent advances in systems and synthetic biology as well as metabolic engineering are poised to transform industrial biotechnology by allowing us to design cell factories for the sustainable production of valuable fuels and chemicals. To deliver on their promises, such cell factories, as much as their brick-and-mortar counterparts, will require appropriate catalysts, especially for classes of reactions that are not known to be catalyzed by enzymes in natural organisms. A recently developed methodology, de novo computational enzyme design can be used to create enzymes catalyzing novel reactions. Here we review the different classes of chemical reactions for which active protein catalysts have been designed as well as the results of detailed biochemical and structural characterization studies. We also discuss how combining de novo computational enzyme design with more traditional protein engineering techniques can alleviate the shortcomings of state-of-the-art computational design techniques and create novel enzymes with catalytic proficiencies on par with natural enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Rumen degradation of oil palm fronds is improved through pre-digestion with white rot fungi but not through supplementation with yeast or enzymes

    NARCIS (Netherlands)

    Hassim, H.A.; Lourenco, M.; Goh, Y.M.; Baars, J.J.P.; Fievez, V.


    Rumen fermentation kinetics of oil palm fronds (OPF) supplemented or not with enzymes (Hemicell® or Allzyme SSF®) or yeasts (Levucell®SC or Yea-Sacc®) were studied through an in vitro gas production test (96 h) (exp. 1). In exp. 2, enzymes were supplemented to OPF pre-treated during 3 or 9 wk with

  8. Mathematical modeling of enzyme production using Trichoderma harzianum P49P11 and sugarcane bagasse as carbon source. (United States)

    Gelain, Lucas; da Cruz Pradella, José Geraldo; da Costa, Aline Carvalho


    A mathematical model to describe the kinetics of enzyme production by the filamentous fungus Trichoderma harzianum P49P11 was developed using a low cost substrate as main carbon source (pretreated sugarcane bagasse). The model describes the cell growth, variation of substrate concentration and production of three kinds of enzymes (cellulases, beta-glucosidase and xylanase) in different sugarcane bagasse concentrations (5; 10; 20; 30; 40 gL(-1)). The 10 gL(-1) concentration was used to validate the model and the other to parameter estimation. The model for enzyme production has terms implicitly representing induction and repression. Substrate variation was represented by a simple degradation rate. The models seem to represent well the kinetics with a good fit for the majority of the assays. Validation results indicate that the models are adequate to represent the kinetics for a biotechnological process. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Engineering Cellulase Enzymes for Bioenergy (United States)

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  10. Kinetics of Social Contagion (United States)

    Ruan, Zhongyuan; Iñiguez, Gerardo; Karsai, Márton; Kertész, János


    Diffusion of information, behavioral patterns or innovations follows diverse pathways depending on a number of conditions, including the structure of the underlying social network, the sensitivity to peer pressure and the influence of media. Here we study analytically and by simulations a general model that incorporates threshold mechanism capturing sensitivity to peer pressure, the effect of "immune" nodes who never adopt, and a perpetual flow of external information. While any constant, nonzero rate of dynamically introduced spontaneous adopters leads to global spreading, the kinetics by which the asymptotic state is approached shows rich behavior. In particular, we find that, as a function of the immune node density, there is a transition from fast to slow spreading governed by entirely different mechanisms. This transition happens below the percolation threshold of network fragmentation, and has its origin in the competition between cascading behavior induced by adopters and blocking due to immune nodes. This change is accompanied by a percolation transition of the induced clusters.

  11. The temperature hydration kinetics

    Directory of Open Access Journals (Sweden)

    Mircea Oroian


    Full Text Available The aim of this study is to evaluate the hydration kinetics of lentil seeds (Lens culinaris in water at different temperatures (25, 32.5, 40, 55, 70 and 80 °C for assessing the adequacy of models for describing the absorption phenomena during soaking. The diffusion coefficient values were calculated using Fick’s model for spherical and hemispherical geometries and the values were in the range of 10−6 m2/s. The experimental data were fitted to Peleg, Sigmoidal, Weibull and Exponential models. The models adequacy was determined using regression coefficients (R2, root mean square error (RMSE and reduced chi-square (χ2. The Peleg model is the suitable one for predicting the experimental data. Temperature had a positive and significant effect on the water absorption capacities and absorption was an endothermic process.

  12. Kinetic theory of Jeans instability

    NARCIS (Netherlands)

    Trigger, S.A.; Ershkovic, A.I.; Heijst, van G.J.F.; Schram, P.P.J.M.


    Kinetic treatment of the Jeans gravitational instability, with collisions taken into account, is presented. The initial-value problem for the distribution function which obeys the kinetic equation, with the collision integral conserving the number of particles, is solved. Dispersion relation is

  13. Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses. (United States)

    Azad, Kimi; Banerjee, Manidipa; Johnson, John E


    Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

  14. Enzyme reactions and their time resolved measurements

    International Nuclear Information System (INIS)

    Hajdu, Janos


    This paper discusses experimental strategies in data collection with the Laue method and summarises recent results using synchrotron radiation. Then, an assessment is made of the progress towards time resolved studies with protein crystals and the problems that remain. The paper consists of three parts which respectively describe some aspects of Laue diffraction, recent examples of structural results from Laue diffraction, and kinetic Laue crystallography. In the first part, characteristics of Laue diffraction is discussed first, focusing on the harmonics problems, spatials problem, wavelength normalization, low resolution hole, data completeness, and uneven coverage of reciprocal space. Then, capture of the symmetry unique reflection set is discussed focusing on the effect of wavelength range on the number of reciprocal lattice points occupying diffracting positions, effect of crystal to film distance and the film area and shape on the number of reflections captured, and effect of crystal symmetry on the number of unique reflections within the number of reflections captured. The second part addresses the determination of the structure of turkey egg white lysozyme, and calcium binding in tomato bushy stunt virus. The third part describes the initiation of reactions in enzyme crystals, picosecond Laue diffraction at high energy storage rings, and detectors. (N.K.)

  15. Influence of exogenous fibrolytic enzymes on in vitro and in sacco degradation of forages for ruminants

    Directory of Open Access Journals (Sweden)

    Lorenzo Carreón


    Full Text Available An in vitro assay was carried out to evaluate the effects of exogenous fibrolytic enzymes (1, 2, 3 and 4 g/kg DM powder preparation containing xylanase and cellulase from Aspergillus niger and Trichoderma viride on DM, NDF and ADF degradation of alfalfa hay, corn silage, corn stover, elephant grass, Guinea grass and oat straw. Kinetics data of in vitro degradations were analyzed. The potentially degradable fraction and degradation rate of NDF and ADF of alfalfa increased quadratically (P<0.05 as the inclusion level of enzyme increased up to 3 g. The others forages were not affected by the enzyme. An in sacco trail was performed using four Holstein steers fitted with ruminal cannulas to evaluate the effects of the exogenous fibrolytic enzymes (3 g/kg DM on DM, NDF and ADF degradation of alfalfa hay and corn stover. Kinetics data were also analyzed. The potentially degradable fraction degradation of NDF (62.0 vs 65.7% and ADF (52.8 vs 56.9%, of alfalfa hay were increased (P<0.05 by the exogenous fibrolytic enzymes, but no differences were found for corn stover. These results suggest that the enzymes increased in vitro and in sacco fibre degradation only for alfalfa hay.

  16. Analytical systems as a basis for immobilized enzymes. 3. Use of a glucose enzyme electrode to determine carbohydrates in biological solutions

    Energy Technology Data Exchange (ETDEWEB)

    Kulys, J; Pesliakiene, M


    A method is described for determination of glucose, sucrose, and lactose in biological solutions using a glucose enzyme electrode characterized by high sensitivity and selectivity. The enzyme membrane (15 nm thick) is prepared from glucose oxidase isolated from Penicillium vitale. Glucose is determined in one minute (using static currents) or in 12 s (using registered current in a kinetic regime). Phosphate buffer (5-10 mM) is the only reagent required for analysis. Determination of sucrose and lactose require prior hydrolysis with 17.8% HCl at 70 degrees Celcius for O.5 and lO.7 minutes, respectively.

  17. Thermal and high pressure inactivation kinetics of blueberry peroxidase. (United States)

    Terefe, Netsanet Shiferaw; Delon, Antoine; Versteeg, Cornelis


    This study for the first time investigated the stability and inactivation kinetics of blueberry peroxidase in model systems (McIlvaine buffer, pH=3.6, the typical pH of blueberry juice) during thermal (40-80°C) and combined high pressure-thermal processing (0.1-690MPa, 30-90°C). At 70-80°C, the thermal inactivation kinetics was best described by a biphasic model with ∼61% labile and ∼39% stable fractions at temperature between 70 and 75°C. High pressure inhibited the inactivation of the enzyme with no inactivation at pressures as high as 690MPa and temperatures less than 50°C. The inactivation kinetics of the enzyme at 60-70°C, and pressures higher than 500MPa was best described by a first order biphasic model with ∼25% labile fraction and 75% stable fraction. The activation energy values at atmospheric pressure were 548.6kJ/mol and 324.5kJ/mol respectively for the stable and the labile fractions. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  18. Distribution of enzyme activity hotspots induced by earthworms in top- and subsoil (United States)

    Hoang, D. T. T.


    Earthworms (Lumbricus terrestris L.) not only affect soil physics, but they also boost microbial activities and consequently create important hotspots of microbial mediated carbon and nutrient turnover through their burrowing activity. However, it is still unknown to which extend earthworms change the enzyme distribution and activity inside their burrows in top- and subsoil horizons. We hypothesized that earthworm burrows, which are enriched in available substrates, have higher percentage of enzyme activity hotspots than soil without earthworms, and that enzyme activities decreased with increasing depth because of the increasing recalcitrance of organic matter in subsoil. We visualized enzyme distribution inside and outside of worm burrows (biopores) by in situ soil zymography and measured enzyme kinetics of 6 enzymes - β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) - in pore and bulk soil material up to 105 cm. Zymography showed a heterogeneous distribution of hotspots in worm burrows. The hotspot areas was 2.4 to 14 times larger in the burrows than in soil without earthworms. However, the dispersion index of hotspot distribution showed more aggregated hotspots in soil without earthworms than in soil with earthworms and burrow wall. Enzyme activities decreased with depth, by a factor of 2 to 8 due to fresh C input from the soil surface. Compared to bulk soil, enzyme activities in topsoil biopores were up to 11 times higher for all enzymes, but in the subsoil activities of XYL, NAG and APT were lower in earthworm biopores than bulk soil. In conclusion, hotspots were twice as concentrated close to earthworm burrows as in surrounding soil. Earthworms exerted stronger effects on enzyme activities in biopores in the topsoil than in subsoil. Keywords: Earthworms, hotspots, enzyme activities, enzyme distribution, subsoil

  19. A kinetic study of soluble glucose oxidase using a rotating-disc electrode

    NARCIS (Netherlands)

    Stroe-Biezen, van S.A.M.; Janssen, A.P.M.; Janssen, L.J.J.


    In order to determine the kinetic parameters of glucose oxidation catalysed by the enzyme glucose oxidase (GO) the initial velocity of hydrogen peroxide formation was measured using a rotating disc electrode (RDE). The major advantage of this method is the possibility of continuous measurement of

  20. Modelling and simulation of a transketolase mediated reaction: Sensitivity analysis of kinetic parameters

    DEFF Research Database (Denmark)

    Sayar, N.A.; Chen, B.H.; Lye, G.J.


    In this paper we have used a proposed mathematical model, describing the carbon-carbon bond format ion reaction between beta-hydroxypyruvate and glycolaldehyde to synthesise L-erythrulose, catalysed by the enzyme transketolase, for the analysis of the sensitivity of the process to its kinetic...

  1. Kinetic modeling and dynamic analysis of simultaneous saccharification and fermentation of cellulose to bioethanol

    International Nuclear Information System (INIS)

    Shadbahr, Jalil; Khan, Faisal; Zhang, Yan


    Highlights: • Deeper understanding of saccharification and fermentation process. • A new kinetic model for dynamic analysis of the simultaneous saccharification and fermentation. • Testing and validation of kinetic model. - Abstract: Kinetic modeling and dynamic analysis of the simultaneous saccharification and fermentation (SSF) of cellulose to ethanol was carried out in this study to determine the key reaction kinetics parameters and product inhibition features of the process. To obtain the more reliable kinetic parameters which can be applied for a wide range of operating conditions, batch SSF experiments were carried out at three enzyme loadings (10, 15 and 20 FPU/g cellulose) and two levels of initial concentrations of fermentable sugars (glucose and mannose). Results indicated that the maximum ethanol yield and concentration were achieved at high level of sugar concentrations with intermediate enzyme loading (15 FPU/g cellulose). Dynamic analysis of the acquired experimental results revealed that cellulase inhibition by cellobiose plays the most important role at high level of enzyme loading and low level of initial sugar concentrations. The inhibition of glucose becomes significant when high concentrations of sugars were present in the feedstock. Experimental results of SSF process also reveal that an efficient mixing between the phases helps to improve the ethanol yield significantly.

  2. A simple and fast kinetic assay for the determination of fructan exohydrolase activity in perennial ryegrass (Lolium perenne L.

    Directory of Open Access Journals (Sweden)

    Anna eGasperl


    Full Text Available Despite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLC equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP by fructan exohydrolases (FEHs to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding.

  3. Subcellular localization of pituitary enzymes (United States)

    Smith, R. E.


    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  4. Kinetic Study of Acetone-Butanol-Ethanol Fermentation in Continuous Culture (United States)

    Buehler, Edward A.; Mesbah, Ali


    Acetone-butanol-ethanol (ABE) fermentation by clostridia has shown promise for industrial-scale production of biobutanol. However, the continuous ABE fermentation suffers from low product yield, titer, and productivity. Systems analysis of the continuous ABE fermentation will offer insights into its metabolic pathway as well as into optimal fermentation design and operation. For the ABE fermentation in continuous Clostridium acetobutylicum culture, this paper presents a kinetic model that includes the effects of key metabolic intermediates and enzymes as well as culture pH, product inhibition, and glucose inhibition. The kinetic model is used for elucidating the behavior of the ABE fermentation under the conditions that are most relevant to continuous cultures. To this end, dynamic sensitivity analysis is performed to systematically investigate the effects of culture conditions, reaction kinetics, and enzymes on the dynamics of the ABE production pathway. The analysis provides guidance for future metabolic engineering and fermentation optimization studies. PMID:27486663

  5. Substrate mediated enzyme prodrug therapy

    DEFF Research Database (Denmark)

    Fejerskov, Betina; Jarlstad Olesen, Morten T; Zelikin, Alexander N


    Substrate mediated enzyme prodrug therapy (SMEPT) is a biomedical platform developed to perform a localized synthesis of drugs mediated by implantable biomaterials. This approach combines the benefits and at the same time offers to overcome the drawbacks for traditional pill-based drug administra......Substrate mediated enzyme prodrug therapy (SMEPT) is a biomedical platform developed to perform a localized synthesis of drugs mediated by implantable biomaterials. This approach combines the benefits and at the same time offers to overcome the drawbacks for traditional pill-based drug...

  6. Kinetics of thyroid hormones

    International Nuclear Information System (INIS)

    Inada, Mitsuo; Nishikawa, Mitsushige; Naito, Kimikazu; Ishii, Hitoshi; Tanaka, Kiyoshi


    Kinetics of thyroid hormones were outlined, and recent progress in metabolism of these hormones was also described. Recently, not only T 4 and T 3 but also rT 3 , 3,3'-T 2 , 3',5'-T 2 , and 3,5-T 2 can be measured by RIA. To clarify metabolic pathways of these hormones, metabolic clearance rate and production rate of these hormones were calculated. As single-compartment analysis was insufficient to clarify disappearance curves of thyroid hormones in blood such as T 3 and T 2 of which metabolic speed was so fast, multi-compartment analysis or non-compartment analysis were also performed. Thyroid hormones seemed to be measured more precisely by constant infusion method. At the first step of T 4 metabolism, T 3 was formed by 5'-monodeiodination of T 4 , and rT 3 was formed by 5-monodeiodination of T 4 . As metabolic pathways of T 3 and rT 3 , conversion of them to 3,3'-T 2 or to 3',5'-T 2 and 3,5-T 2 was supposed. This subject will be an interesting research theme in future. (Tsunoda, M.)

  7. Characterization of nicotinamidases: steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism. (United States)

    French, Jarrod B; Cen, Yana; Vrablik, Tracy L; Xu, Ping; Allen, Eleanor; Hanna-Rose, Wendy; Sauve, Anthony A


    Nicotinamidases are metabolic enzymes that hydrolyze nicotinamide to nicotinic acid. These enzymes are widely distributed across biology, with examples found encoded in the genomes of Mycobacteria, Archaea, Eubacteria, Protozoa, yeast, and invertebrates, but there are none found in mammals. Although recent structural work has improved our understanding of these enzymes, their catalytic mechanism is still not well understood. Recent data show that nicotinamidases are required for the growth and virulence of several pathogenic microbes. The enzymes of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans regulate life span in their respective organisms, consistent with proposed roles in the regulation of NAD(+) metabolism and organismal aging. In this work, the steady state kinetic parameters of nicotinamidase enzymes from C. elegans, Sa. cerevisiae, Streptococcus pneumoniae (a pathogen responsible for human pneumonia), Borrelia burgdorferi (the pathogen that causes Lyme disease), and Plasmodium falciparum (responsible for most human malaria) are reported. Nicotinamidases are generally efficient catalysts with steady state k(cat) values typically exceeding 1 s(-1). The K(m) values for nicotinamide are low and in the range of 2 -110 μM. Nicotinaldehyde was determined to be a potent competitive inhibitor of these enzymes, binding in the low micromolar to low nanomolar range for all nicotinamidases tested. A variety of nicotinaldehyde derivatives were synthesized and evaluated as inhibitors in kinetic assays. Inhibitions are consistent with reaction of the universally conserved catalytic Cys on each enzyme with the aldehyde carbonyl carbon to form a thiohemiacetal complex that is stabilized by a conserved oxyanion hole. The S. pneumoniae nicotinamidase can catalyze exchange of (18)O into the carboxy oxygens of nicotinic acid with H(2)(18)O. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic

  8. Kinetic effects on magnetohydrodynamic phenomena

    International Nuclear Information System (INIS)

    Naito, Hiroshi; Matsumoto, Taro


    Resistive and ideal magnetohydrodynamic (MHD) theories are insufficient to adequately explain MHD phenomena in the high-temperature plasma. Recent progress in numerical simulations concerning kinetic effects on magnetohydrodynamic phenomena is summarized. The following three topics are studied using various models treating extended-MHD phenomena. (1) Kinetic modifications of internal kink modes in tokamaks with normal and reversed magnetic shear configurations. (2) Temporal evolution of the toroidal Alfven eigenmode and fishbone mode in tokamaks with energetic ions. (3) Kinetic stabilization of a title mode in field-reversed configurations by means of anchoring ions and beam ions. (author)

  9. Thermodynamics of accuracy in kinetic proofreading: dissipation and efficiency trade-offs

    International Nuclear Information System (INIS)

    Rao, Riccardo; Peliti, Luca


    The high accuracy exhibited by biological information transcription processes is due to kinetic proofreading, i.e. by a mechanism which reduces the error rate of the information-handling process by driving it out of equilibrium. We provide a consistent thermodynamic description of enzyme-assisted assembly processes involving competing substrates, in a master equation framework. We introduce and evaluate a measure of the efficiency based on rigorous non-equilibrium inequalities. The performance of several proofreading models are thus analyzed and the related time, dissipation and efficiency versus error trade-offs exhibited for different discrimination regimes. We finally introduce and analyze in the same framework a simple model which takes into account correlations between consecutive enzyme-assisted assembly steps. This work highlights the relevance of the distinction between energetic and kinetic discrimination regimes in enzyme-substrate interactions. (paper)

  10. Kinetic properties of ATP sulfurylase and APS kinase from Thiobacillus denitrificans. (United States)

    Gay, Sean C; Fribourgh, Jennifer L; Donohoue, Paul D; Segel, Irwin H; Fisher, Andrew J


    The Thiobacillus denitrificans genome contains two sequences corresponding to ATP sulfurylase (Tbd_0210 and Tbd_0874). Both genes were cloned and expressed protein characterized. The larger protein (Tbd_0210; 544 residues) possesses an N-terminal ATP sulfurylase domain and a C-terminal APS kinase domain and was therefore annotated as a bifunctional enzyme. But, the protein was not bifunctional because it lacked ATP sulfurylase activity. However, the enzyme did possess APS kinase activity and displayed substrate inhibition by APS. Truncated protein missing the N-terminal domain had APS kinase activity suggesting the function of the inactive sulfurylase domain is to promote the oligomerization of the APS kinase domains. The smaller gene product (Tbd_0874; 402 residues) possessed strong ATP sulfurylase activity with kinetic properties that appear to be kinetically optimized for the direction of APS utilization and ATP+sulfate production, which is consistent with an enzyme that functions physiologically to produce inorganic sulfate.

  11. Characterization of the human cytochrome P450 enzymes involved in the metabolism of dihydrocodeine (United States)

    Kirkwood, L. C.; Nation, R. L.; Somogyi, A. A.


    Aims Using human liver microsomes from donors of the CYP2D6 poor and extensive metabolizer genotypes, the role of individual cytochromes P-450 in the oxidative metabolism of dihydrocodeine was investigated. Methods The kinetics of formation of N- and O-demethylated metabolites, nordihydrocodeine and dihydromorphine, were determined using microsomes from six extensive and one poor metabolizer and the effects of chemical inhibitors selective for individual P-450 enzymes of the 1A, 2A, 2C, 2D, 2E and 3A families and of LKM1 (anti-CYP2D6) antibodies were studied. Results Nordihydrocodeine was the major metabolite in both poor and extensive metabolizers. Kinetic constants for N-demethylation derived from the single enzyme Michaelis-Menten model did not differ between the two groups. Troleandomycin and erythromycin selectively inhibited N-demethylation in both extensive and poor metabolizers. The CYP3A inducer, α-naphthoflavone, increased N-demethylation rates. The kinetics of formation of dihydromorphine in both groups were best described by a single enzyme Michaelis-Menten model although inhibition studies in extensive metabolizers suggested involvement of two enzymes with similar Km values. The kinetic constants for O-demethylation were significantly different in extensive and poor metabolizers. The extensive metabolizers had a mean intrinsic clearance to dihydromorphine more than ten times greater than the poor metabolizer. The CYP2D6 chemical inhibitors, quinidine and quinine, and LKM1 antibodies inhibited O-demethylation in extensive metabolizers; no effect was observed in microsomes from a poor metabolizer. Conclusions CYP2D6 is the major enzyme mediating O-demethylation of dihydrocodeine to dihydromorphine. In contrast, nordihydrocodeine formation is predominantly catalysed by CYP3A. PMID:9431830

  12. Infrared Analysis Of Enzymes Adsorbed Onto Model Surfaces (United States)

    Story, Gloria M.; Rauch, Deborah S.; Brode, Philip F.; Marcott, Curtis A.


    The adsorption of the enzymes, subtilisin BPN' and lysozyme, onto model surfaces was examined using attenuated total reflectance (ATR) infrared (IR) spectroscopy. Using a cylindrical internal reflection (CIRcle) cell with a Germanium (Ge) internal reflection element (IRE), model hydrophilic surfaces were made by plasma cleaning the IRE and model hydrophobic surfaces were made by precoating the IRE with a thin film of polystyrene. Gas chromatography (GC)-IR data collection software was used to monitor adsorption kinetics during the first five minutes after injection of the enzyme into the CIRcle cell. It was found that for both lysozyme and BPN', most of the enzyme that was going to adsorb onto the model surface did so within ten seconds after injection. Nearly an order-of-magnitude more BPN' adsorbed on the hydrophobic Ge surface than the hydrophilic one, while lysozyme adsorbed somewhat more strongly to the hydrophilic Ge surface. Overnight, the lysozyme layer continued to increase in thickness, while BPN' maintained its initial coverage. The appearance of carboxylate bands in some of the adsorbed BPN' spectra suggests the occurrence of peptide bond hydrolysis. A Au/Pd coating on the CIRcle cell o-rings had a significant effect on the adsorption of BPN'. (This coating was applied in an attempt to eliminate interfering Teflon absorption bands.) An apparent electrochemical reaction occurred, involving BPN', Ge, Au/Pd, and the salt solution used to stabilize BPN'. The result of this reaction was enhanced adsorption of the enzyme around the coated o-rings, etching of the Ge IRE at the o-ring site, and some autolysis of the enzyme. No such reaction was observed with lysozyme.

  13. Kinetics and mechanism of jack bean urease inhibition by Hg2+

    Directory of Open Access Journals (Sweden)

    Du Nana


    Full Text Available Abstract Background Jack bean urease (EC is a metalloenzyme, which catalyzes the hydrolysis of urea to produce ammonia and carbon dioxide. The heavy metal ions are common inhibitors to control the rate of the enzymatic urea hydrolysis, which take the Hg2+ as the representative. Hg2+ affects the enzyme activity causing loss of the biological function of the enzyme, which threatens the survival of many microorganism and plants. However, inhibitory kinetics of urease by the low concentration Hg2+ has not been explored fully. In this study, the inhibitory effect of the low concentration Hg2+ on jack bean urease was investigated in order to elucidate the mechanism of Hg2+ inhibition. Results According to the kinetic parameters for the enzyme obtained from Lineweaver–Burk plot, it is shown that the Km is equal to 4.6±0.3 mM and Vm is equal to 29.8±1.7 μmol NH3/min mg. The results show that the inhibition of jack bean urease by Hg2+ at low concentration is a reversible reaction. Equilibrium constants have been determined for Hg2+ binding with the enzyme or the enzyme-substrate complexes (Ki =0.012 μM. The results show that the Hg2+ is a noncompetitive inhibitor. In addition, the kinetics of enzyme inhibition by the low concentration Hg2+ has been studied using the kinetic method of the substrate reaction. The results suggest that the enzyme first reversibly and quickly binds Hg2+ and then undergoes a slow reversible course to inactivation. Furthermore, the rate constant of the forward reactions (k+0 is much larger than the rate constant of the reverse reactions (k-0. By combining with the fact that the enzyme activity is almost completely lost at high concentration, the enzyme is completely inactivated when the Hg2+ concentration is high enough. Conclusions These results suggest that Hg2+ has great impacts on the urease activity and the established inhibition kinetics model is suitable.

  14. Thermodynamics of Enzyme-Catalyzed Reactions Database (United States)

    SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

  15. Curious Cases of the Enzymes. (United States)

    Ulusu, Nuriye Nuray


    Life as we know it heavily relies on biological catalysis, in fact, in a very nonromantic version of it, life could be considered as a series of chemical reactions, regulated by the guarding principles of thermodynamics. In ancient times, a beating heart was a good sign of vitality, however, to me, it is actually the presence of active enzymes that counts… Though we do not usually pay attention, the history of enzymology is as old as humanity itself, and dates back to the ancient times. This paper is dedicated to these early moments of this remarkable science that touched our lives in the past and will make life a lot more efficient for humanity in the future. There was almost always a delicate, fundamentally essential relationship between mankind and the enzymes. Challenged by a very alien and hostile Nature full of predators, prehistoric men soon discovered the medicinal properties of the plants, through trial and error. In fact, they accidently discovered the enzyme inhibitors and thus, in crude terms, kindled a sparkling area of research. These plant-derivatives that acted as enzyme inhibitors helped prehistoric men in their pursuit of survival and protection from predators; in hunting and fishing… Later in history, while the underlying purposes of survival and increasing the quality of life stayed intact, the ways and means of enzymology experienced a massive transformation, as the 'trial and error' methodology of the ancients is now replaced with rational scientific theories.

  16. Enzymes with activity toward Xyloglucan

    NARCIS (Netherlands)

    Vincken, J.P.


    Xyloglucans are plant cell wall polysaccharides, which belong to the hemicellulose class. Here the structural variations of xyloglucans will be reviewed. Subsequently, the anchoring of xyloglucan in the plant cell wall will be discussed. Enzymes involved in degradation or modification of xyloglucan

  17. Bicarbonate kinetics in Indian males

    Indian Academy of Sciences (India)


    ized kinetics of bicarbonate using a three-compartment model, to assess which compartmental fluxes changed dur- .... total VCO2 was < 3 % and the average respiratory quotient ..... a part of the nonrespiratory losses of 13CO2 occur to this.

  18. Kinetic equations in dirty superconductors

    International Nuclear Information System (INIS)

    Kraehenbuehl, Y.


    Kinetic equations for superconductors in the dirty limit are derived using a method developed for superfluid systems, which allows a systematic expansion in small parameters; exact charge conservation is obeyed. (orig.)

  19. Cell kinetics and therapeutic efficiency

    International Nuclear Information System (INIS)

    Andreeff, M.; Abenhardt, W.; Gruner, B.; Stoffner, D.; Mainz Univ.


    The study shows that cell kinetics effects correlate with the effects of cytostatic drugs in the tumour model investigated here. It should, however, be noted that even genetically related tumour cell types may react differently to the same cytostatic drug, and that the cell kinetics effects, due to the changes in the cell cycle, cannot be predicted but should be followed with a very fast method, e.g. sequential flan fluorescence cytophotometry, for optimal therapeutic results. (orig./GSE) [de

  20. Kinetic Studies on Microbial Production of Tannase Using Redgram Husk


    S. K. Mohan; T. Viruthagiri; C. Arunkumar


    Tannase (tannin acyl hydrolase, E.C. is an important hydrolysable enzyme with innumerable applications and industrial potential. In the present study, a kinetic model has been developed for the batch fermentation used for the production of tannase by A.flavus MTCC 3783. Maximum tannase activity of 143.30 U/ml was obtained at 96 hours under optimum operating conditions at 35oC, an initial pH of 5.5 and with an inducer tannic acid concentration of 3% (w/v) for a fe...

  1. Kinetic parameters from thermogravimetric analysis (United States)

    Kiefer, Richard L.


    High performance polymeric materials are finding increased use in aerospace applications. Proposed high speed aircraft will require materials to withstand high temperatures in an oxidative atmosphere for long periods of time. It is essential that accurate estimates be made of the performance of these materials at the given conditions of temperature and time. Temperatures of 350 F (177 C) and times of 60,000 to 100,000 hours are anticipated. In order to survey a large number of high performance polymeric materials on a reasonable time scale, some form of accelerated testing must be performed. A knowledge of the rate of a process can be used to predict the lifetime of that process. Thermogravimetric analysis (TGA) has frequently been used to determine kinetic information for degradation reactions in polymeric materials. Flynn and Wall studied a number of methods for using TGA experiments to determine kinetic information in polymer reactions. Kinetic parameters, such as the apparent activation energy and the frequency factor, can be determined in such experiments. Recently, researchers at the McDonnell Douglas Research Laboratory suggested that a graph of the logarithm of the frequency factor against the apparent activation energy can be used to predict long-term thermo-oxidative stability for polymeric materials. Such a graph has been called a kinetic map. In this study, thermogravimetric analyses were performed in air to study the thermo-oxidative degradation of several high performance polymers and to plot their kinetic parameters on a kinetic map.

  2. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes. (United States)


    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

  3. Heavy enzymes--experimental and computational insights in enzyme dynamics. (United States)

    Swiderek, Katarzyna; Ruiz-Pernía, J Javier; Moliner, Vicent; Tuñón, Iñaki


    The role of protein motions in the chemical step of enzyme-catalyzed reactions is the subject of an open debate in the scientific literature. The systematic use of isotopically substituted enzymes has been revealed as a useful tool to quantify the role of these motions. According to the Born-Oppenheimer approximation, changing the mass of the protein does not change the forces acting on the system but alters the frequencies of the protein motions, which in turn can affect the rate constant. Experimental and theoretical studies carried out in this field are presented in this article and discussed in the framework of Transition State Theory. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Canceling effect leads temperature insensitivity of hydrolytic enzymes in soil (United States)

    Razavi, Bahar S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov


    Extracellular enzymes are important for decomposition of many macromolecules abundant in soil such as cellulose, hemicelluloses and proteins (Allison et al., 2010; Chen et al., 2012). The temperature sensitivity of enzymes responsible for organic matter decomposition is the most crucial parameter for prediction of the effects of global warming on carbon cycle. Temperature responses of biological systems are often expressed as a Q10 functions; The Q10 describes how the rate of a chemical reaction changes with a temperature increase for 10 °C The aim of this study was to test how the canceling effect will change with variation in temperature interval, during short-term incubation. We additionally investigated, whether canceling effect occurs in a broad range of concentrations (low to high) and whether it is similar for the set of hydrolytic enzymes within broad range of temperatures. To this end, we performed soil incubation over a temperature range of 0-40°C (with 5°C steps). We determined the activities of three enzymes involved in plant residue decomposition: β-glucosidase and cellobiohydrolase, which are commonly measured as enzymes responsible for degrading cellulose (Chen et al., 2012), and xylanase, which degrades xylooligosaccharides (short xylene chain) in to xylose, thus being responsible for breaking down hemicelluloses (German et al., 2011). Michaelis-Menten kinetics measured at each temperature allowed to calculate Q10 values not only for the whole reaction rates, but specifically for maximal reaction rate (Vmax) and substrate affinity (Km). Subsequently, the canceling effect - simultaneous increase of Vmax and Km with temperature was analyzed within 10 and 5 degree of temperature increase. Three temperature ranges (below 10, between 15 and 25, and above 30 °C) clearly showed non-linear but stepwise increase of temperature sensitivity of all three enzymes and allowed to conclude for predominance of psychrophilic, mesophilic and thermophilic

  5. [Flavonoid oxidation kinetics in aqueous and aqueous organic media in the presence of peroxidase, tyrosynase, and hemoglobin]. (United States)

    Barsukova, M E; Tokareva, A I; Buslova, T S; Malinina, L I; Veselova, I A; Shekhovtsova, T N


    The kinetics of oxidation reactions of flavonoids, quercetin, dihydroquercetin, and epicatechin has been studied in the presence of biocatalysts of different natures: horseradish peroxidase, mushroom tyrosinase, and hemoglobin from bull blood. Comparison of the kinetic parameters of the oxidation reaction showed that peroxidase appeared to be the most effective biocatalyst in these processes. The specificity of the enzyme for quercetin increased with increasing the polarity of the solvent in a series of ethanol–acetonitrile–dimethyl sulfoxide.

  6. Elimination Kinetics of Ethanol in a 5-Week-Old Infant and a Literature Review of Infant Ethanol Pharmacokinetics

    Directory of Open Access Journals (Sweden)

    Jonathan B. Ford


    Full Text Available Primary ethanol metabolism occurs through alcohol dehydrogenase, but minor metabolic pathways such as the P450 enzymes CYP2E1 and CYP1A2 and the enzyme catalase exist. These enzymes have distinct developmental stages. Elimination kinetics of ethanol in the infant is limited. We report the elimination kinetics of ethanol in a 5-week-old African-American male who had a serum ethanol level of 270 mg/dL on admission. A previously healthy 5-week-old African-American male was brought to the ED with a decreased level of consciousness. His initial blood ethanol level was 270 mg/dL. Serial blood ethanol levels were obtained. The elimination rate of ethanol was calculated to be in a range from 17.1 to 21.2 mg/dL/hr and appeared to follow zero-order elimination kinetics with a R2=0.9787. Elimination kinetics for ethanol in the young infant has been reported in only four previously published reports. After reviewing these reports, there appears to be variability in the elimination rates of ethanol in infants. Very young infants may not eliminate ethanol as quickly as previously described. Given that there are different stages of enzyme development in children, caution should be used when generalizing the elimination kinetics in young infants and children.

  7. Equilibrium isotope exchange kinetics of native and site-specific mutant forms of E. coli aspartate transcarbamoylase

    International Nuclear Information System (INIS)

    Wedler, F.C.; Hsuanyu, Y.; Kantrowitz, E.R.


    Isotope exchange kinetics at equilibrium (EIEK) have been used to probe the kinetic and regulatory mechanisms of native aspartate transcarbamoylase (ATCase) from E. coli at pH 7.0, 30 0 . Substrate saturation patterns were most consistent with a preferred order random kinetic mechanism: C-P prior to L-Asp, C-Asp released before Pi, with the Asp ↔ C-Asp exchange rate 5X faster than C-P ↔ Pi. Computer simulations allow one to fit the EIEK experimental data and to arrive at the best set of kinetic constants for a given enzyme state. These approaches have been applied to modified ATCase. Bound CTP and ATP were observed, respectively, to inhibit and activate differentially Asp ↔ C-Asp, but not C-P ↔ Pi, indicating that these modifiers alter the association-dissociation rates of L-Asp and C-Asp but not of C-P or Pi. Low levels of PALA activated both exchange rates (due to shifting the T-R equilibrium), but higher [PALA] completely blocked both exchanges. The effects of a site-specific mutation of Tyr240 Phe have been similarly probed by EIEK methods. The Phe240 mutant enzyme exhibited kinetic properties markedly different from native ATCase: the data indicate that Phe240 ATCase is much closer to an R-state enzyme than is native enzyme

  8. Substrate Specificity and Enzyme Recycling Using Chitosan Immobilized Laccase

    Directory of Open Access Journals (Sweden)

    Everton Skoronski


    Full Text Available The immobilization of laccase (Aspergillus sp. on chitosan by cross-linking and its application in bioconversion of phenolic compounds in batch reactors were studied. Investigation was performed using laccase immobilized via chemical cross-linking due to the higher enzymatic operational stability of this method as compared to immobilization via physical adsorption. To assess the influence of different substrate functional groups on the enzyme’s catalytic efficiency, substrate specificity was investigated using chitosan-immobilized laccase and eighteen different phenol derivatives. It was observed that 4-nitrophenol was not oxidized, while 2,5-xylenol, 2,6-xylenol, 2,3,5-trimethylphenol, syringaldazine, 2,6-dimetoxyphenol and ethylphenol showed reaction yields up 90% at 40 °C. The kinetic of process, enzyme recyclability and operational stability were studied. In batch reactors, it was not possible to reuse the enzyme when it was applied to syringaldazne bioconversion. However, when the enzyme was applied to bioconversion of 2,6-DMP, the activity was stable for eight reaction batches.

  9. Gyrocenter-gauge kinetic theory

    International Nuclear Information System (INIS)

    Qin, H.; Tang, W.M.; Lee, W.W.


    Gyrocenter-gauge kinetic theory is developed as an extension of the existing gyrokinetic theories. In essence, the formalism introduced here is a kinetic description of magnetized plasmas in the gyrocenter coordinates which is fully equivalent to the Vlasov-Maxwell system in the particle coordinates. In particular, provided the gyroradius is smaller than the scale-length of the magnetic field, it can treat high frequency range as well as the usual low frequency range normally associated with gyrokinetic approaches. A significant advantage of this formalism is that it enables the direct particle-in-cell simulations of compressional Alfven waves for MHD applications and of RF waves relevant to plasma heating in space and laboratory plasmas. The gyrocenter-gauge kinetic susceptibility for arbitrary wavelength and arbitrary frequency electromagnetic perturbations in a homogeneous magnetized plasma is shown to recover exactly the classical result obtained by integrating the Vlasov-Maxwell system in the particle coordinates. This demonstrates that all the waves supported by the Vlasov-Maxwell system can be studied using the gyrocenter-gauge kinetic model in the gyrocenter coordinates. This theoretical approach is so named to distinguish it from the existing gyrokinetic theory, which has been successfully developed and applied to many important low-frequency and long parallel wavelength problems, where the conventional meaning of gyrokinetic has been standardized. Besides the usual gyrokinetic distribution function, the gyrocenter-gauge kinetic theory emphasizes as well the gyrocenter-gauge distribution function, which sometimes contains all the physics of the problems being studied, and whose importance has not been realized previously. The gyrocenter-gauge distribution function enters Maxwell's equations through the pull-back transformation of the gyrocenter transformation, which depends on the perturbed fields. The efficacy of the gyrocenter-gauge kinetic approach is

  10. Kinetics of enzymatic trans-esterification of glycerides for biodiesel production. (United States)

    Calabrò, Vincenza; Ricca, Emanuele; De Paola, Maria Gabriela; Curcio, Stefano; Iorio, Gabriele


    In this paper, the reaction of enzymatic trans-esterification of glycerides with ethanol in a reaction medium containing hexane at a temperature of 37 degrees C has been studied. The enzyme was Lipase from Mucor miehei, immobilized on ionic exchange resin, aimed at achieving high catalytic specific surface and recovering, regenerating and reusing the biocatalyst. A kinetic analysis has been carried out to identify the reaction path; the rate equation and kinetic parameters have been also calculated. The kinetic model has been validated by comparison between predicted and experimental results. Mass transport resistances estimation was undertaken in order to verify that the kinetics found was intrinsic. Model potentialities in terms of reactors design and optimization are also shown.

  11. Crystallization Kinetics within a Generic Modelling Framework

    DEFF Research Database (Denmark)

    Meisler, Kresten Troelstrup; von Solms, Nicolas; Gernaey, Krist


    An existing generic modelling framework has been expanded with tools for kinetic model analysis. The analysis of kinetics is carried out within the framework where kinetic constitutive models are collected, analysed and utilized for the simulation of crystallization operations. A modelling...... procedure is proposed to gain the information of crystallization operation kinetic model analysis and utilize this for faster evaluation of crystallization operations....

  12. Inhibition study of alanine aminotransferase enzyme using sequential online capillary electrophoresis analysis. (United States)

    Liu, Lina; Chen, Yuanfang; Yang, Li


    We report the study of several inhibitors on alanine aminotransferase (ALT) enzyme using sequential online capillary electrophoresis (CE) assay. Using metal ions (Na(+) and Mg(2+)) as example inhibitors, we show that evolution of the ALT inhibition reaction can be achieved by automatically and simultaneously monitoring the substrate consumption and product formation as a function of reaction time. The inhibition mechanism and kinetic constants of ALT inhibition with succinic acid and two traditional Chinese medicines were derived from the sequential online CE assay. Our study could provide valuable information about the inhibition reactions of ALT enzyme. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes (United States)

    Guerra, Nelson P.; Pastrana Castro, Lorenzo


    The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, v max decreased significantly (P enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G]. PMID:22666116

  14. Infrared-thermographic screening of the activity and enantioselectivity of enzymes. (United States)

    Reetz, M T; Hermes, M; Becker, M H


    The infrared radiation caused by the heat of reaction of an enantioselective enzyme-catalyzed transformation can be detected by modern photovoltaic infrared (IR)-thermographic cameras equipped with focal-plane array detectors. Specifically, in the lipase-catalyzed enantioselective acylation of racemic 1-phenylethanol, the (R)- and (S)-substrates are allowed to react separately in the wells of microtiter plates, the (R)-alcohol showing hot spots in the IR-thermographic images. Thus, highly enantioselective enzymes can be identified at kinetic resolution.

  15. Computer-assisted enzyme immunoassays and simplified immunofluorescence assays: applications for the diagnostic laboratory and the veterinarian's office. (United States)

    Jacobson, R H; Downing, D R; Lynch, T J


    A computer-assisted enzyme-linked immunosorbent assay (ELISA) system, based on kinetics of the reaction between substrate and enzyme molecules, was developed for testing large numbers of sera in laboratory applications. Systematic and random errors associated with conventional ELISA technique were identified leading to results formulated on a statistically validated, objective, and standardized basis. In a parallel development, an inexpensive system for field and veterinary office applications contained many of the qualities of the computer-assisted ELISA. This system uses a fluorogenic indicator (rather than the enzyme-substrate interaction) in a rapid test (15 to 20 minutes' duration) which promises broad application in serodiagnosis.

  16. Kinetics and Thermal Properties of Crude and Purified β-Galactosidase with Potential for the Production of Galactooligosaccharides

    Directory of Open Access Journals (Sweden)

    Anna Rafaela Cavalcante Braga


    Full Text Available β-Galactosidase is an enzyme that catalyzes the hydrolysis of lactose. It has potential importance due to various applications in the food and dairy industries, involving lactose-reduced ingredients. The properties of two β-galactosidase enzymes, crude and purified, from different sources, Kluyveromyces marxianus CCT 7082 and Kluyveromyces marxianus ATCC 16045, were analyzed. The pH and temperature optima, deactivation energy, thermal stability and kinetic and thermodynamic parameters were determined, as well as the ability to hydrolyze lactose and produce galactooligosaccharides. Purification process improved the properties of the enzymes, and the results showed that purified enzymes from both strains had a higher optimum temperature, and lower values of Km, thus showing greater affinity for o-nitrophenyl-β-D-galactopiranoside than the crude enzymes. The production of galactooligosaccharides was also greater when using purified enzymes, increasing the synthesis by more than 30 % by both strains.

  17. Study of DNA reconstruction enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Sekiguchi, M [Kyushu Univ., Fukuoka (Japan). Faculty of Science


    Description was made of the characteristics and mechanism of 3 reconstructive enzymes which received from M. luteus or E. coli or T4, and of which natures were clarified as reconstructive enzymes of DNA irradiated with ultraviolet rays. As characteristics, the site of breaking, reaction, molecular weight, electric charge in the neutrality and a specific adhesion to DNA irradiated with ultraviolet rays were mentioned. As to mutant of ultraviolet ray sensitivity, hereditary control mechanism of removal and reconstruction by endo-nuclease activation was described, and suggestion was referred to removal and reconstruction of cells of xedoderma pigmentosum which is a hereditary disease of human. Description was also made as to the mechanism of exonuclease activation which separates dimer selectively from irradiated DNA.

  18. Metrological aspects of enzyme production

    International Nuclear Information System (INIS)

    Kerber, T M; Pereira-Meirelles, F V; Dellamora-Ortiz, G M


    Enzymes are frequently used in biotechnology to carry out specific biological reactions, either in industrial processes or for the production of bioproducts and drugs. Microbial lipases are an important group of biotechnologically valuable enzymes that present widely diversified applications. Lipase production by microorganisms is described in several published papers; however, none of them refer to metrological evaluation and the estimation of the uncertainty in measurement. Moreover, few of them refer to process optimization through experimental design. The objectives of this work were to enhance lipase production in shaken-flasks with Yarrowia lipolytica cells employing experimental design and to evaluate the uncertainty in measurement of lipase activity. The highest lipolytic activity obtained was about three- and fivefold higher than the reported activities of CRMs BCR-693 and BCR-694, respectively. Lipase production by Y. lipolytica cells aiming the classification as certified reference material is recommended after further purification and stability studies

  19. Quantitative enzyme activity determination with zeptomole sensitivity by microfluidic gradient-gel zymography. (United States)

    Hughes, Alex J; Herr, Amy E


    We describe a sensitive zymography technique that utilizes an automated microfluidic platform to report enzyme molecular weight, amount, and activity (including k(cat) and K(m)) from dilute protein mixtures. Calf intestinal alkaline phosphatase (CIP) is examined in detail as a model enzyme system, and the method is also demonstrated for horseradish peroxidase (HRP). The 40 min assay has a detection limit of 5 zmol ( approximately 3 000 molecules) of CIP. Two-step pore-limit electrophoresis with enzyme assay (PLENZ) is conducted in a single, straight microchannel housing a polyacrylamide (PA) pore-size gradient gel. In the first step, pore limit electrophoresis (PLE) sizes and pseudoimmobilizes resolved proteins. In the second step, electrophoresis transports both charged and neutral substrates into the PLE channel to the entrapped proteins. Arrival of substrate at the resolved enzyme band generates fluorescent product that reveals enzyme molecular weight against a fluorescent protein ladder. Additionally, the PLENZ zymography assay reports the kinetic properties of CIP in a fully quantitative manner. In contrast to covalent enzyme immobilization, physical pseudoimmobilization of CIP in the PA gel does not significantly reduce its maximum substrate turnover rate. However, an 11-fold increase in the Michaelis constant (over the free solution value) is observed, consistent with diffusional limitations on substrate access to the enzyme active site. PLENZ offers a robust platform for rapid and multiplexed functional analysis of heterogeneous protein samples in drug discovery, clinical diagnostics, and biocatalyst engineering.

  20. Consumer attitudes to enzymes in food production

    DEFF Research Database (Denmark)

    Søndergaard, Helle Alsted; Grunert, Klaus G.; Scholderer, Joachim


    The use of enzymes in food production has potential benefits for both food manufacturers and consumers. A central question is how consumers react to new ways of producing foods with enzymes. This study investigates the formation of consumer attitudes to different enzyme production methods in three...... European countries. Results show that consumers are most positive towards non-GM enzyme production methods. The enzyme production method is by far the most important factor for the formation of buying intentions compared to price and benefits. Results also show that environmental concern and attitudes...... to technological progress are the socio-political attitudes that have the highest predictive value regarding attitudes to enzyme production methods....

  1. Research progress of nanoparticles as enzyme mimetics (United States)

    Hu, XiaoNa; Liu, JianBo; Hou, Shuai; Wen, Tao; Liu, WenQi; Zhang, Ke; He, WeiWei; Ji, YingLu; Ren, HongXuan; Wang, Qi; Wu, XiaoChun


    Natural enzymes as biological catalysts possess remarkable advantages, especially their highly efficient and selective catalysis under mild conditions. However, most natural enzymes are proteins, thus exhibiting an inherent low durability to harsh reaction conditions. Artificial enzyme mimetics have been pursued extensively to avoid this drawback. Quite recently, some inorganic nanoparticles (NPs) have been found to exhibit unique enzyme mimetics. In addition, their much higher stability overcomes the inherent disadvantage of natural enzymes. Furthermore, easy mass-production and low cost endow them more benefits. As a new member of artificial enzyme mimetics, they have received intense attention. In this review article, major progress in this field is summarized and future perspectives are highlighted.

  2. Allosteric regulation of epigenetic modifying enzymes. (United States)

    Zucconi, Beth E; Cole, Philip A


    Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Silica-Immobilized Enzyme Reactors (United States)


    Silica-IMERs 14 implicated in neurological disorders such as Schizophrenia and Parkinson’s disease.[86] Drug discovery for targets that can alter the...primarily the activation of prodrugs and proantibiotics for cancer treatments or antibiotic therapy , respectively.[87] Nitrobenzene nitroreductase was...BuChE) Monolith disks* Packed Silica Biosilica Epoxide- Silica Silica-gel Enzyme Human AChE Human AChE Human AChE Equine BuChE Human

  4. Immobilised enzymes in biorenewable production


    Franssen, M.C.R.; Steunenberg, P.; Scott, E.L.; Zuilhof, H.; Sanders, J.P.M.


    Oils, fats, carbohydrates, lignin, and amino acids are all important raw materials for the production of biorenewables. These compounds already play an important role in everyday life in the form of wood, fabrics, starch, paper and rubber. Enzymatic reactions do, in principle, allow the transformation of these raw materials into biorenewables under mild and sustainable conditions. There are a few examples of processes using immobilised enzymes that are already applied on an industrial scale, ...

  5. Immobilization of enzymes by radiation

    International Nuclear Information System (INIS)

    Kaetsu, I.; Kumakura, M.; Yoshida, M.; Asano, M.; Himei, M.; Tamura, M.; Hayashi, K.


    Immobilization of various enzymes was performed by radiation-induced polymerization of glass-forming monomers at low temperatures. Alpha-amylase and glucoamylase were effectively immobilized in hydrophilic polymer carrier such as poly(2-hydroxyethyl methacrylate) and also in rather hydrophobic carrier such as poly(tetraethylene-glycol diacrylate). Immobilized human hemoglobin underwent the reversible oxygenation concomitantly with change of oxygen concentration outside of the matrices. (author)

  6. Lignin-degrading enzyme activities. (United States)

    Chen, Yi-ru; Sarkanen, Simo; Wang, Yun-Yan


    Over the past three decades, the activities of four kinds of enzyme have been purported to furnish the mechanistic foundations for macromolecular lignin depolymerization in decaying plant cell walls. The pertinent fungal enzymes comprise lignin peroxidase (with a relatively high redox potential), manganese peroxidase, an alkyl aryl etherase, and laccase. The peroxidases and laccase, but not the etherase, are expressed extracellularly by white-rot fungi. A number of these microorganisms exhibit a marked preference toward lignin in their degradation of lignocellulose. Interestingly, some white-rot fungi secrete both kinds of peroxidase but no laccase, while others that are equally effective express extracellular laccase activity but no peroxidases. Actually, none of these enzymes has been reported to possess significant depolymerase activity toward macromolecular lignin substrates that are derived with little chemical modification from the native biopolymer. Here, the assays commonly employed for monitoring the traditional fungal peroxidases, alkyl aryl etherase, and laccase are described in their respective contexts. A soluble native polymeric substrate that can be isolated directly from a conventional milled-wood lignin preparation is characterized in relation to its utility in next-generation lignin-depolymerase assays.

  7. Immobilised enzymes in biorenewables production. (United States)

    Franssen, Maurice C R; Steunenberg, Peter; Scott, Elinor L; Zuilhof, Han; Sanders, Johan P M


    Oils, fats, carbohydrates, lignin, and amino acids are all important raw materials for the production of biorenewables. These compounds already play an important role in everyday life in the form of wood, fabrics, starch, paper and rubber. Enzymatic reactions do, in principle, allow the transformation of these raw materials into biorenewables under mild and sustainable conditions. There are a few examples of processes using immobilised enzymes that are already applied on an industrial scale, such as the production of High-Fructose Corn Syrup, but these are still rather rare. Fortunately, there is a rapid expansion in the research efforts that try to improve this, driven by a combination of economic and ecological reasons. This review focusses on those efforts, by looking at attempts to use fatty acids, carbohydrates, proteins and lignin (and their building blocks), as substrates in the synthesis of biorenewables using immobilised enzymes. Therefore, many examples (390 references) from the recent literature are discussed, in which we look both at the specific reactions as well as to the methods of immobilisation of the enzymes, as the latter are shown to be a crucial factor with respect to stability and reuse. The applications of the renewables produced in this way range from building blocks for the pharmaceutical and polymer industry, transport fuels, to additives for the food industry. A critical evaluation of the relevant factors that need to be improved for large-scale use of these examples is presented in the outlook of this review.

  8. Self-powered enzyme micropumps (United States)

    Sengupta, Samudra; Patra, Debabrata; Ortiz-Rivera, Isamar; Agrawal, Arjun; Shklyaev, Sergey; Dey, Krishna K.; Córdova-Figueroa, Ubaldo; Mallouk, Thomas E.; Sen, Ayusman


    Non-mechanical nano- and microscale pumps that function without the aid of an external power source and provide precise control over the flow rate in response to specific signals are needed for the development of new autonomous nano- and microscale systems. Here we show that surface-immobilized enzymes that are independent of adenosine triphosphate function as self-powered micropumps in the presence of their respective substrates. In the four cases studied (catalase, lipase, urease and glucose oxidase), the flow is driven by a gradient in fluid density generated by the enzymatic reaction. The pumping velocity increases with increasing substrate concentration and reaction rate. These rechargeable pumps can be triggered by the presence of specific analytes, which enables the design of enzyme-based devices that act both as sensor and pump. Finally, we show proof-of-concept enzyme-powered devices that autonomously deliver small molecules and proteins in response to specific chemical stimuli, including the release of insulin in response to glucose.

  9. Substrate mediated enzyme prodrug therapy.

    Directory of Open Access Journals (Sweden)

    Betina Fejerskov

    Full Text Available In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol, β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose - dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering.

  10. Processing of poultry feathers by alkaline keratin hydrolyzing enzyme from Serratia sp. HPC 1383. (United States)

    Khardenavis, Anshuman A; Kapley, Atya; Purohit, Hemant J


    The present study describes the production and characterization of a feather hydrolyzing enzyme by Serratia sp. HPC 1383 isolated from tannery sludge, which was identified by the ability to form clear zones around colonies on milk agar plates. The proteolytic activity was expressed in terms of the micromoles of tyrosine released from substrate casein per ml per min (U/mL min). Induction of the inoculum with protein was essential to stimulate higher activity of the enzyme, with 0.03% feathermeal in the inoculum resulting in increased enzyme activity (45U/mL) that further increased to 90U/mL when 3d old inoculum was used. The highest enzyme activity, 130U/mL, was observed in the presence of 0.2% yeast extract. The optimum assay temperature and pH for the enzyme were found to be 60 degrees C and 10.0, respectively. The enzyme had a half-life of 10min at 60 degrees C, which improved slightly to 18min in presence of 1mM Ca(2+). Inhibition of the enzyme by phenylmethyl sulfonyl fluoride (PMSF) indicated that the enzyme was a serine protease. The enzyme was also partially inhibited (39%) by the reducing agent beta-mercaptoethanol and by divalent metal ions such as Zn(2+) (41% inhibition). However, Ca(2+) and Fe(2+) resulted in increases in enzyme activity of 15% and 26%, respectively. The kinetic constants of the keratinase were found to be 3.84 microM (K(m)) and 108.7 microM/mLmin (V(max)). These results suggest that this extracellular keratinase may be a useful alternative and eco-friendly route for handling the abundant amount of waste feathers or for applications in other industrial processes.

  11. Enzyme catalyzed oxidative cross-linking of feruloylated pectic polysaccharides from sugar beet

    DEFF Research Database (Denmark)

    Abang Zaidel, Dayang Norulfairuz

    beet pulp as a potential starting material for production of pectin derived products which could help maintain the competitiveness of the sugar beet based industry. The overall objective of this study has been focusing on understanding the kinetics of enzyme catalyzed oxidative crosslinking......-linked by HRP catalysis in the presence of hydrogen peroxide (H2O2) to form ferulic acid dehydrodimers (diFAs). The composition of the substrate was analyzed by HPAEC, HPLC and MALDI-TOF, confirming the structural make up of the arabinan-oligosaccharide (Arabinose: 2.9- 3.4 mmol?g-1 DM; FA: 2.5-7.0 mg?g-1 DM......, identically composed, oil-in-water emulsion systems to study the effect of different methods of emulsion preparation on the emulsion stability in the presence of SBP and the kinetics of enzyme catalyzed oxidative gelation of SBP. The result shows that the different methods of emulsion preparation affect...

  12. Binding of Cimetidine to Balb/C Mouse Liver Catalase; Kinetics and Conformational Studies. (United States)

    Jahangirvand, Mahboubeh; Minai-Tehrani, Dariush; Yazdi, Fatemeh; Minai-Tehrani, Arash; Razmi, Nematollah


    Catalase is responsible for converting hydrogen peroxide (H2O2) into water and oxygen in cells. This enzyme has high affinity for hydrogen peroxide and can protect the cells from oxidative stress damage. Catalase is a tetramer protein and each monomer contains a heme group. Cimetidine is a histamine H2 receptor blocker which inhibits acid release from stomach and is used for gasterointestinal diseases. In this research, effect of cimetidine on the activity of liver catalase was studied and the kinetic parameters of this enzyme and its conformational changes were investigated. Cell free extract of mouse liver was used for the catalase assay. The activity of the catalase was detected in the absence and presence of cimetidine by monitoring hydrogen peroxide reduction absorbance at 240 nm. The purified enzyme was used for conformational studies by Fluorescence spectrophotometry. The data showed that cimetidine could inhibit the enzyme in a non-competitive manner. Ki and IC50 values of the drug were determined to be about 0.75 and 0.85 uM, respectively. The Arrhenius plot showed that activation energy was 6.68 and 4.77 kJ/mol in the presence and absence of the drug, respectively. Fluorescence spectrophotometry revealed that the binding of cimetidine to the purified enzyme induced hyperchromicity and red shift which determined the conformational change on the enzyme. Cimetidine could non-competitively inhibit the liver catalase with high affinity. Binding of cimetidine to the enzyme induced conformational alteration in the enzyme.

  13. High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

    Directory of Open Access Journals (Sweden)

    Philippe Urban


    Full Text Available The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

  14. Saccharification of ozonated sugarcane bagasse using enzymes from Myceliophthora thermophila JCP 1-4 for sugars release and ethanol production. (United States)

    de Cassia Pereira, Josiani; Travaini, Rodolfo; Paganini Marques, Natalia; Bolado-Rodríguez, Silvia; Bocchini Martins, Daniela Alonso


    The saccharification of ozonated sugarcane bagasse (SCB) by enzymes from Myceliophthora thermophila JCP 1-4 was studied. Fungal enzymes provided slightly higher sugar release than commercial enzymes, working at 50°C. Sugar release increased with temperature increase. Kinetic studies showed remarkable glucose release (4.99 g/L, 3%w/w dry matter) at 60°C, 8 h of hydrolysis, using an enzyme load of 10 FPU (filter paper unit). FPase and β-glucosidase activities increased during saccharification (284% and 270%, respectively). No further significant improvement on glucose release was observed increasing the enzyme load above 7.5 FPU per g of cellulose. Higher dry matter contents increased sugars release, but not yields. The fermentation of hydrolysates by Saccharomyces cerevisiae provided glucose-to-ethanol conversions around to 63%. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes. (United States)

    Urban, Philippe; Truan, Gilles; Pompon, Denis


    The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

  16. Fractal-like kinetics, a possible link between preconditioning and sepsis immunodepression. On the chemical basis of innate immunity. (United States)

    Vasilescu, C; Olteanu, M; Flondor, P


    In a recent paper the authors hypothesized that the so called fractal-like enzyme kinetics of intracellular reactions may explain the preconditioning effect in biology (Vasilescu C, Olteanu M, Flondor P, Revue Roumaine de Chimie. 2011; 56(7): 751-7). Inside cells the reaction kinetics is very well described by fractal-like kinetics. In the present work some clinical implications of this model are analyzed. Endotoxin tolerance is a particular case of preconditioning and shows similarities with the immunodepression seen in some sepsis patients. This idea offers a theoretical support for modulation of the enzymatic activity of the cell by changing the fractal dimension of the cytoskeleton.

  17. Lipase-catalyzed synthesis of palmitanilide: Kinetic model and antimicrobial activity study. (United States)

    Liu, Kuan-Miao; Liu, Kuan-Ju


    Enzymatic syntheses of fatty acid anilides are important owing to their wide range of industrial applications in detergents, shampoo, cosmetics, and surfactant formulations. The amidation reaction of Mucor miehei lipase Lipozyme IM20 was investigated for direct amidation of triacylglycerol in organic solvents. The process parameters (reaction temperature, substrate molar ratio, enzyme amount) were optimized to achieve the highest yield of anilide. The maximum yield of palmitanilide (88.9%) was achieved after 24 h of reaction at 40 °C at an enzyme concentration of 1.4% (70 mg). Kinetics of lipase-catalyzed amidation of aniline with tripalmitin has been investigated. The reaction rate could be described in terms of the Michaelis-Menten equation with a Ping-Pong Bi-Bi mechanism and competitive inhibition by both the substrates. The kinetic constants were estimated by using non-linear regression method using enzyme kinetic modules. The enzyme operational stability study showed that Lipozyme IM20 retained 38.1% of the initial activity for the synthesis of palmitanilide (even after repeated use for 48 h). Palmitanilide, a fatty acid amide, exhibited potent antimicrobial activity toward Bacillus cereus. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Kinetics of enzymatic high-solid hydrolysis of lignocellulosic biomass studied by calorimetry. (United States)

    Olsen, Søren N; Lumby, Erik; McFarland, Kc; Borch, Kim; Westh, Peter


    Enzymatic hydrolysis of high-solid biomass (>10% w/w dry mass) has become increasingly important as a key step in the production of second-generation bioethanol. To this end, development of quantitative real-time assays is desirable both for empirical optimization and for detailed kinetic analysis. In the current work, we have investigated the application of isothermal calorimetry to study the kinetics of enzymatic hydrolysis of two substrates (pretreated corn stover and Avicel) at high-solid contents (up to 29% w/w). It was found that the calorimetric heat flow provided a true measure of the hydrolysis rate with a detection limit of about 500 pmol glucose s(-1). Hence, calorimetry is shown to be a highly sensitive real-time method, applicable for high solids, and independent on the complexity of the substrate. Dose-response experiments with a typical cellulase cocktail enabled a multidimensional analysis of the interrelationships of enzyme load and the rate, time, and extent of the reaction. The results suggest that the hydrolysis rate of pretreated corn stover is limited initially by available attack points on the substrate surface (conversion) but becomes proportional to enzyme dosage (excess of attack points) at later stages (>10% conversion). This kinetic profile is interpreted as an increase in polymer end concentration (substrate for CBH) as the hydrolysis progresses, probably due to EG activity in the enzyme cocktail. Finally, irreversible enzyme inactivation did not appear to be the source of reduced hydrolysis rate over time.

  19. Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase. (United States)

    Bomati, Erin K; Noel, Joseph P


    We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.


    Directory of Open Access Journals (Sweden)



    Full Text Available Robust immobilization techniques that preserve the activity of biomolecules have manypotential applications. In recent years, a number of new bioimobilisation methods in solgel-derived materials were reported. The interactions between the biomolecule and theinorganic material determine the degree to which the biomolecule retains its nativeproperties. The newer technological developments in the field of immobilizedbiocatalysts can offer the possibility of a wider and more economical exploitation ofbiocatalysts in biological applications, food and feed industry, medicine, and in thedevelopment of bioprocess monitoring devices, like the biosensors.The aim of this study was to obtain immobilized enzymatic preparations by methodswhich affect enzyme conformations and kinetic parameters as less as possible. Weimmobilized the enzymatic preparation with protease activity produced by a Bacilluslicheniformis B 40 local strain by physical bonding on ceramics and entrapment into solgel-derived glasses obtained from tetraethyl orthosilicate (TEOS, deposited in thin layeron a ceramic support (entrapment/deposition. Both physically adsorbed andentrapped/deposited enzymes follow Michaelis-Menten kinetics, similar with the solubleenzyme. In the case of immobilized enzymes, the apparent Michaelis constant, Km, wasgreater than that of the native one, as it was expected. The kinetic parameters indicatethat the enzymatic preparations adsorbed on ceramic support and entrapped/depositedshow less affinity for the substrate, Km being 1.3 and 2.1 times higher than that of thenative enzyme, respectively. The maximum velocity increased also by 3.5 and 7.9 timesrespectively, compared with the free counterpart (according to Lineweaver-Burklinearization.

  1. Electro-ultrafiltration of industrial enzyme solutions

    DEFF Research Database (Denmark)

    Enevoldsen, Ann Dorrit; Hansen, Erik Børresen; Jonsson, Gunnar Eigil


    To reduce the problems with fouling and concentration polarization during crossflow ultrafiltration of industrial enzyme solutions an electric field is applied across the membrane. The filtration performance during electro-ultrafiltration (EUF) has been tested with several enzymes. Results show...

  2. Biochemical characterization of thermostable cellulase enzyme from ...

    African Journals Online (AJOL)



    May 29, 2012 ... tested for their ability to produce cellulase complex enzyme by growing on a defined substrates as well ... In the current industrial processes, cellulolytic enzymes ... energy sources such as glucose, ethanol, hydrogen and.

  3. Epigenetics of dominance for enzyme activity

    Indian Academy of Sciences (India)


    dimer over a wide range of H+ concentrations accounts for the epigenetics of dominance for enzyme activity. [Trehan K S ... The present study has been carried on acid phosphatase .... enzyme activity over mid parent value (table 3, col. 13),.

  4. Castor Oil Transesterification Catalysed by Liquid Enzymes

    DEFF Research Database (Denmark)

    Andrade, Thalles; Errico, Massimiliano; Christensen, Knud Villy


    In the present work, biodiesel production by reaction of non-edible castor oil with methanol under enzymatic catalysis is investigated. Two liquid enzymes were tested: Eversa Transform and Resinase HT. Reactions were performed at 35 °C and with a molar ratio of methanol to oil of 6:1. The reaction...... time was 8 hours. Stepwise addition of methanol was necessary to avoid enzyme inhibition by methanol. In order to minimize the enzyme costs, the influence of enzyme activity loss during reuse of both enzymes was evaluated under two distinct conditions. In the former, the enzymes were recovered...... and fully reused; in the latter, a mixture of 50 % reused and 50 % fresh enzymes was tested. In the case of total reuse after three cycles, both enzymes achieved only low conversions. The biodiesel content in the oil-phase using Eversa Transform was 94.21 % for the first cycle, 68.39 % in the second, and 33...

  5. Measurement of the enzymes lactate dehydrogenase and creatine kinase using reflectance spectroscopy and reagent strips.


    Stevens, J F; Tsang, W; Newall, R G


    Two new methods for the assay of total activities of lactate dehydrogenase and creatine kinase are described, in which the enzyme activities are measured from a solid-state reagent strip during a kinetic reaction, the reaction being monitored in the ultra-violet region of the spectrum by reflectance spectroscopy. The performances of these methods are evaluated, and compared to conventional "wet" chemistry methods. The solid-phase reagent methods demonstrated precision and accuracy acceptable ...

  6. Transformation kinetics for nucleus clusters

    International Nuclear Information System (INIS)

    Villa, Elena; Rios, Paulo R.


    A rigorous mathematical approach based on stochastic geometry concepts is presented to extend previous Johnson-Mehl, Avrami, Kolmogorov treatment of transformation kinetics to situations in which nuclei are not homogeneously located in space but are located in clusters. An exact analytical solution is presented here for the first time assuming that nucleation sites follow a Matern cluster process. The influence of Matern cluster process parameters on subsequent growth kinetics and the microstructural path are illustrated by means of numerical examples. Moreover, using the superposition principle, exact analytical solutions are also obtained when nucleation takes place by a combination of a Matern cluster process and an inhomogeneous Poisson point process. The new solutions presented here significantly increase the number of exactly solvable cases available to formal kinetics.

  7. Chapter 22. Cell population kinetics

    International Nuclear Information System (INIS)

    Tubiana, M.


    The main contribution of radioisotopes to the development of a new discipline, cell population kinetics, was shown. The aim of this science is to establish, for each tissue of the organism, the life span of its component cells and the mechanisms governing its growth, its differentiation and its homeostasis with respect to outside attacks. Labelling techniques have been used to follow the cells during these various processes. The case of non-dividing cells was considered first, taking as example, the red blood cells of which the lifetime was studied, after which the case of proliferating cells was examined using 14 C- or tritium-labelled thymidine. The methods used to measure the cell cycle parameters were described: labelled-mitosis curve method, double-labelling and continuous labelling methods, proliferation coefficient measurement. Cell kinetics were shown to allow an interpretation of radiobiological data. Finally the practical value of cell kinetics research was shown [fr

  8. Kinetic theory and transport phenomena

    CERN Document Server

    Soto, Rodrigo


    This textbook presents kinetic theory, which is a systematic approach to describing nonequilibrium systems. The text is balanced between the fundamental concepts of kinetic theory (irreversibility, transport processes, separation of time scales, conservations, coarse graining, distribution functions, etc.) and the results and predictions of the theory, where the relevant properties of different systems are computed. The book is organised in thematic chapters where different paradigmatic systems are studied. The specific features of these systems are described, building and analysing the appropriate kinetic equations. Specifically, the book considers the classical transport of charges, the dynamics of classical gases, Brownian motion, plasmas, and self-gravitating systems, quantum gases, the electronic transport in solids and, finally, semiconductors. Besides these systems that are studied in detail, concepts are applied to some modern examples including the quark–gluon plasma, the motion of bacterial suspen...

  9. Zymography methods for visualizing hydrolytic enzymes


    Vandooren, Jennifer; Geurts, Nathalie; Martens, Erik; Van den Steen, Philippe E.; Opdenakker, Ghislain


    Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful., but often misinterpreted, tool. yielding information on potential. hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tis...

  10. Biomedical Applications of Enzymes From Marine Actinobacteria. (United States)

    Kamala, K; Sivaperumal, P

    Marine microbial enzyme technologies have progressed significantly in the last few decades for different applications. Among the various microorganisms, marine actinobacterial enzymes have significant active properties, which could allow them to be biocatalysts with tremendous bioactive metabolites. Moreover, marine actinobacteria have been considered as biofactories, since their enzymes fulfill biomedical and industrial needs. In this chapter, the marine actinobacteria and their enzymes' uses in biological activities and biomedical applications are described. © 2017 Elsevier Inc. All rights reserved.

  11. Visualization of enzyme activities inside earthworm biopores by in situ soil zymography (United States)

    Thu Duyen Hoang, Thi; Razavi, Bahar. S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov


    Earthworms can strongly activate microorganisms, increase microbial and enzyme activities and consequently the turnover of native soil organic matter. In extremely dynamic microhabitats and hotspots as biopores made by earthworms, the in situ enzyme activities are a footprint of complex biotic interactions. The effect of earthworms on the alteration of enzyme activities inside biopores and the difference between bio-pores and earthworm-free soil was visualized by in situ soil zymography (Spohn and Kuzyakov, 2014). For the first time, we prepared quantitative imaging of enzyme activities in biopores. Furthermore, we developed the zymography technique by direct application of a substrate saturated membrane to the soil to obtain better spatial resolution. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). Simultaneously, maize seed was sown in the soil. Control soil box with maize and without earthworm was prepared in the same way. After two weeks when bio-pore systems were formed by earthworm, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine aminopeptidase) and phosphatase. Followed by non-destructive zymography, biopore samples and control soil were destructively collected to assay enzyme kinetics by fluorogenically labeled substrates method. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. These differences were further confirmed by fluorimetric microplate enzyme assay detected significant difference of Vmax in four above mentioned enzymes. Vmax of β-glucosidase, chitinase, xylanase and phosphatase in biopores is 68%, 108%, 50% and 49% higher than that of control soil. However, no difference in cellobiohydrolase and leucine aminopeptidase kinetics between biopores and control soil were detected. This indicated little effect of earthworms on protein and cellulose transformation in soil

  12. Kinetics and equilibria of cyanide binding to prostaglandin H synthase. (United States)

    MacDonald, I D; Dunford, H B


    Cyanide binding to prostaglandin H (PGH) synthase results in a spectral shift in the Soret region. This shift was exploited to determine equilibrium and kinetic parameters of the cyanide binding process. At pH 8.0, ionic strength 0.22 M, 4 degrees C, the cyanide dissociation constant, determined from equilibrium experiments, is (65 +/- 10) microM. The binding rate constant is (2.8 +/- 0.2) x 10(3) M-1 s-1, and the dissociation rate constant is zero within experimental error. Through a kinetic study of the binding process as a function of pH, from pH 3.96 to 8.00, it was possible to determine the pKa of a heme-linked acid group on the enzyme of 4.15 +/- 0.10 with citrate buffer. An apparent pKa of 4.75 +/- 0.03 was determined with acetate buffer; this different value is attributed to complexation of the enzyme with one of the components of the acetate buffer.

  13. Selected readings in chemical kinetics

    CERN Document Server

    Back, Margaret H


    Selected Readings in Chemical Kinetics covers excerpts from 12 papers in the field of general and gas-phase kinetics. The book discusses papers on the laws of connexion between the conditions of a chemical change and its amount; on the reaction velocity of the inversion of the cane sugar by acids; and the calculation in absolute measure of velocity constants and equilibrium constants in gaseous systems. The text then tackles papers on simple gas reactions; on the absolute rate of reactions in condensed phases; on the radiation theory of chemical action; and on the theory of unimolecular reacti

  14. Chemical kinetics and reaction dynamics

    CERN Document Server

    Houston, Paul L


    This text teaches the principles underlying modern chemical kinetics in a clear, direct fashion, using several examples to enhance basic understanding. It features solutions to selected problems, with separate sections and appendices that cover more technical applications.Each chapter is self-contained and features an introduction that identifies its basic goals, their significance, and a general plan for their achievement. This text's important aims are to demonstrate that the basic kinetic principles are essential to the solution of modern chemical problems, and to show how the underlying qu

  15. Chemical kinetics and combustion modeling

    Energy Technology Data Exchange (ETDEWEB)

    Miller, J.A. [Sandia National Laboratories, Livermore, CA (United States)


    The goal of this program is to gain qualitative insight into how pollutants are formed in combustion systems and to develop quantitative mathematical models to predict their formation rates. The approach is an integrated one, combining low-pressure flame experiments, chemical kinetics modeling, theory, and kinetics experiments to gain as clear a picture as possible of the process in question. These efforts are focused on problems involved with the nitrogen chemistry of combustion systems and on the formation of soot and PAH in flames.

  16. Deuteration kinetics of the graphene

    Energy Technology Data Exchange (ETDEWEB)

    Nefedov, Alexei; Woell, Christof [KIT, Leopoldshafen (Germany); Paris, Alessio; Calliari, Lucia [FBK-CMM, Trento (Italy); Verbitskiy, Nikolay [MSU, Moscow (Russian Federation); University of Vienna, Vienna (Austria); Wang, Ying; Irle, Stephan [Nagoya University, Nagoya (Japan); Fedorov, Alexander [IFW Dresden, Dresden (Germany); St. Petersburg University, St. Petersburg (Russian Federation); Haberer, Danny; Knupfer, Martin; Buechner, Bernd [IFW Dresden, Dresden (Germany); Oetzelt, Martin [BESSY II, Berlin (Germany); Petaccia, Luca [Elettra, Trieste (Italy); Usachov, Dmitry [St. Petersburg University, St. Petersburg (Russian Federation); Vyalikh, Denis [St. Petersburg University, St. Petersburg (Russian Federation); TU Dresden, Dresden (Germany); Sagdev, Hermann [MPI fuer Polymerforschung, Mainz (Germany); Yashina, Lada [MSU, Moscow (Russian Federation); Grueneis, Alexander [IFW Dresden, Dresden (Germany); University of Vienna, Vienna (Austria)


    The kinetics of the hydrogenation/deuteration reaction of graphene was studied by time-dependent x-ray photoemission spectroscopy (XPS). The graphene layer was then exposed to hydrogen or deuterium atomic gas beams, obtained by thermal cracking in a tungsten capillary at T=3000 K. After each step XPS of the C1s line was performed in order to measure H/C and D/C ratios. We have observed a strong kinetic isotope effect for the hydrogenation/deuteration reaction leading to substantially faster adsorption and higher maximum D/C ratios as compared to H/C (D/C 35% vs. H/C 25%).

  17. Cellulolytic enzyme compositions and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Iyer, Prashant; Gaspar, Armindo Ribiero; Croonenberghs, James; Binder, Thomas P.


    The present invention relates enzyme composition comprising a cellulolytic preparation and an acetylxylan esterase (AXE); and the used of cellulolytic enzyme compositions for hydrolyzing acetylated cellulosic material. Finally the invention also relates to processes of producing fermentation products from acetylated cellulosic materials using a cellulolytic enzyme composition of the invention.

  18. Immobilization of Enzymes in Polymer Supports. (United States)

    Conlon, Hugh D.; Walt, David R.


    Two experiments in which an enzyme is immobilized onto a polymeric support are described. The experiments (which also demonstrate two different polymer preparations) involve: (1) entrapping an enzyme in an acrylamide polymer; and (2) reacting the amino groups on the enzyme's (esterase) lysine residues with an activated polymer. (JN)

  19. Purification and characterization of extracellular amylolytic enzyme ...

    African Journals Online (AJOL)

    In the present study, the amylase enzyme producing potential of four different Aspergillus species was analyzed. The extracted amylase enzyme was purified by diethyl amino ethyl (DEAE) cellulose and Sephadex G-50 column chromatography and the enzyme activity was measured by using synthetic substrate starch.

  20. Activation of interfacial enzymes at membrane surfaces

    DEFF Research Database (Denmark)

    Mouritsen, Ole G.; Andresen, Thomas Lars; Halperin, Avi


    A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A2 (s...


    NARCIS (Netherlands)

    Andela, C.; Feijen, Jan; Dillissen, Marc


    New enzyme granules are provided with improved properties. The granules are based on core particles having a good pore size and pore size distribution to allow an enzyme solution to enter into the particle. Accordingly, the core material comprises the enzyme in liquid form, thus eliminating the

  2. Vlasov simulations of Kinetic Alfven Waves at proton kinetic scales

    NARCIS (Netherlands)

    C.L. Vasconez; F. Valentini (Francesco); E. Camporeale (Enrico); P. Veltri


    htmlabstractKinetic Alfv ́en waves represent an important subject in space plasma physics, since they are thought to play a crucial role in the development of the turbulent energy cascade in the solar wind plasma at short wavelengths (of the order of the proton inertial length d p and beyond). A

  3. Enhancement of photoassimilate utilization by manipulation of starch regulatory enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Okita, Thomas W. [Washington State Univ., Pullman, WA (United States)


    maturation to a starch granule. Although Pho1 catalyzes a reversible reaction, our DoE supported studies clearly demonstrated that the kinetic properties of this enzyme strongly favor synthesis of starch and that these catalytic properties are independent of the L80 peptide, a structural domain that is absent in phosphorylases from other organisms. Interesting expression of a Pho1 lacking the L80 peptide enhanced plant growth and seed yields, suggesting that Pho1 has a second function in controlling growth. Overall, results from these biochemical and physiological studies have increased our fundamental understanding on how these important starch regulatory enzymes operate at the molecular level and in planta, which will collectively aid in efforts to increase the utilization of higher plants as a renewable source of energy.

  4. A new, sensitive method for enzyme kinetic studies of scarce glucosides

    Czech Academy of Sciences Publication Activity Database

    Mazura, Pavel; Fohlerová, Radka; Brzobohatý, Břetislav; Kiran, N.S.; Janda, L.


    Roč. 68, č. 1 (2006), s. 55-63 ISSN 0165-022X R&D Projects: GA ČR(CZ) GA203/02/0865 Institutional research plan: CEZ:AV0Z50040507 Keywords : Amplex Ultra Red reagent * beta-glucosidase * zeatin-O-glukozid Subject RIV: BO - Biophysics Impact factor: 1.403, year: 2006

  5. Aqueous solutions that model the cytosol : studies on polarity, chemical reactivity and enzyme kinetics

    NARCIS (Netherlands)

    Asaad, N.; den Otter, M.J.; Engberts, J.B.F.N.


    Concentrated solutions of a series of organic compounds have been prepared and the effects of these solutes on the properties of the solvent system assessed as a function of their concentration and nature. Polarity, as measured by Reichardt's E-T(30) probe, exhibits a linear variation with both

  6. Problems Students Experience with Inquiry Processes in the Study of Enzyme Kinetics (United States)

    Ferrés Gurt, Concepció; Marbà Tallada, Anna


    This case study describes a classroom-based questionnaire that was carried out with a group of 36 high school students (17-18 years old) in Catalonia. The aim was to examine the usefulness of questionnaires focused on scientific inquiry, both to evaluate students' inquiry abilities and for their potential as tools to improve the understanding of…

  7. An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors (United States)

    Wendelsdorf, Katherine V.; Song, Zhuo; Cao, Yang; Samuels, David C.


    Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication. PMID:19132079

  8. Enzyme structure and interaction with inhibitors

    International Nuclear Information System (INIS)

    London, R.E.


    This article reviews some of the results of studies on the 13 C-labeled enzyme dihydrofolate reductase (DHFR). Nuclear magnetic resonance (NMR) techniques are used in combination with isotopic labeling to learn about the structure and dynamics of this enzyme. 13 C-labeling is used for the purpose of studying enzyme/substrate and enzyme/inhibitor interactions. A second set of studies with DHFR was designed to investigate the basis for the high affinity between the inhibitor methotrexate and DHFR. The label was placed on the inhibitor, rather than the enzyme

  9. Characterization of CobB kinetics and inhibition by nicotinamide.

    Directory of Open Access Journals (Sweden)

    Julia Gallego-Jara

    Full Text Available Lysine acetylation has emerged as a global protein regulation system in all domains of life. Sirtuins, or Sir2-like enzymes, are a family of histone deacetylases characterized by their employing NAD+ as a co-substrate. Sirtuins can deacetylate several acetylated proteins, but a consensus substrate recognition sequence has not yet been established. Product inhibition of many eukaryotic sirtuins by nicotinamide and its analogues has been studied in vitro due to their potential role as anticancer agents. In this work, the kinetics of CobB, the main Escherichia coli deacetylase, have been characterized. To our knowledge, this is the first kinetic characterization of a sirtuin employing a fully acetylated and natively folded protein as a substrate. CobB deacetylated several acetyl-CoA synthetase acetylated lysines with a single kinetic rate. In addition, in vitro nicotinamide inhibition of CobB has been characterized, and the intracellular nicotinamide concentrations have been determined under different growth conditions. The results suggest that nicotinamide can act as a CobB regulator in vivo. A nicotinamidase deletion strain was thus phenotypically characterized, and it behaved similarly to the ΔcobB strain. The results of this work demonstrate the potential regulatory role of the nicotinamide metabolite in vivo.

  10. Characterization of CobB kinetics and inhibition by nicotinamide. (United States)

    Gallego-Jara, Julia; Écija Conesa, Ana; de Diego Puente, Teresa; Lozano Terol, Gema; Cánovas Díaz, Manuel


    Lysine acetylation has emerged as a global protein regulation system in all domains of life. Sirtuins, or Sir2-like enzymes, are a family of histone deacetylases characterized by their employing NAD+ as a co-substrate. Sirtuins can deacetylate several acetylated proteins, but a consensus substrate recognition sequence has not yet been established. Product inhibition of many eukaryotic sirtuins by nicotinamide and its analogues has been studied in vitro due to their potential role as anticancer agents. In this work, the kinetics of CobB, the main Escherichia coli deacetylase, have been characterized. To our knowledge, this is the first kinetic characterization of a sirtuin employing a fully acetylated and natively folded protein as a substrate. CobB deacetylated several acetyl-CoA synthetase acetylated lysines with a single kinetic rate. In addition, in vitro nicotinamide inhibition of CobB has been characterized, and the intracellular nicotinamide concentrations have been determined under different growth conditions. The results suggest that nicotinamide can act as a CobB regulator in vivo. A nicotinamidase deletion strain was thus phenotypically characterized, and it behaved similarly to the ΔcobB strain. The results of this work demonstrate the potential regulatory role of the nicotinamide metabolite in vivo.

  11. Periodic and stochastic thermal modulation of protein folding kinetics. (United States)

    Platkov, Max; Gruebele, Martin


    Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.

  12. Quantum tunneling observed without its characteristic large kinetic isotope effects. (United States)

    Hama, Tetsuya; Ueta, Hirokazu; Kouchi, Akira; Watanabe, Naoki


    Classical transition-state theory is fundamental to describing chemical kinetics; however, quantum tunneling is also important in explaining the unexpectedly large reaction efficiencies observed in many chemical systems. Tunneling is often indicated by anomalously large kinetic isotope effects (KIEs), because a particle's ability to tunnel decreases significantly with its increasing mass. Here we experimentally demonstrate that cold hydrogen (H) and deuterium (D) atoms can add to solid benzene by tunneling; however, the observed H/D KIE was very small (1-1.5) despite the large intrinsic H/D KIE of tunneling (≳ 100). This strong reduction is due to the chemical kinetics being controlled not by tunneling but by the surface diffusion of the H/D atoms, a process not greatly affected by the isotope type. Because tunneling need not be accompanied by a large KIE in surface and interfacial chemical systems, it might be overlooked in other systems such as aerosols or enzymes. Our results suggest that surface tunneling reactions on interstellar dust may contribute to the deuteration of interstellar aromatic and aliphatic hydrocarbons, which could represent a major source of the deuterium enrichment observed in carbonaceous meteorites and interplanetary dust particles. These findings could improve our understanding of interstellar physicochemical processes, including those during the formation of the solar system.

  13. Integrated stoichiometric, thermodynamic and kinetic modelling of steady state metabolism. (United States)

    Fleming, R M T; Thiele, I; Provan, G; Nasheuer, H P


    The quantitative analysis of biochemical reactions and metabolites is at frontier of biological sciences. The recent availability of high-throughput technology data sets in biology has paved the way for new modelling approaches at various levels of complexity including the metabolome of a cell or an organism. Understanding the metabolism of a single cell and multi-cell organism will provide the knowledge for the rational design of growth conditions to produce commercially valuable reagents in biotechnology. Here, we demonstrate how equations representing steady state mass conservation, energy conservation, the second law of thermodynamics, and reversible enzyme kinetics can be formulated as a single system of linear equalities and inequalities, in addition to linear equalities on exponential variables. Even though the feasible set is non-convex, the reformulation is exact and amenable to large-scale numerical analysis, a prerequisite for computationally feasible genome scale modelling. Integrating flux, concentration and kinetic variables in a unified constraint-based formulation is aimed at increasing the quantitative predictive capacity of flux balance analysis. Incorporation of experimental and theoretical bounds on thermodynamic and kinetic variables ensures that the predicted steady state fluxes are both thermodynamically and biochemically feasible. The resulting in silico predictions are tested against fluxomic data for central metabolism in Escherichia coli and compare favourably with in silico prediction by flux balance analysis. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  14. Elements of plasma kinetic theory

    International Nuclear Information System (INIS)

    Guasp, J.


    The physical foundations of plasma kinetic equations are exposed inside a series of seminars on plasma and fusion physics. The Vlasov and collisional equations with its application range have been discussed. The momenta equations for the macroscopic magnitudes and the more usual approximations have been obtained: two fluid equations for cold and warm plasmas, magnetohydrodynamic equations and the double-adiabatic theory. (author)

  15. Kinetic studies on leucite precursors

    Czech Academy of Sciences Publication Activity Database

    Mrázová, M.; Kloužková, A.; Kohoutková, Martina


    Roč. 7, č. 2 (2009), s. 205-210 ISSN 1895-1066 R&D Projects: GA MPO 2A-1TP1/063 Institutional research plan: CEZ:AV0Z40320502 Keywords : leucite * crystallization kinetics * hydrothermal Subject RIV: JH - Ceramics, Fire-Resistant Materials and Glass Impact factor: 1.065, year: 2009

  16. Some parameters of radionuclide kinetics

    International Nuclear Information System (INIS)

    Prokof'ev, O.N.; Smirnov, V.A.; Belen'kij, E.I.


    Numerical values of the rates of radionuclide absorption into, and elimination from, bovine organs were determined. Kinetic rate constants of radionuclides such as 89 Sr, 99 Mo, 131 I, 132 Tl, and 140 Be were calculated. The calculations were done for muscle, liver, and kidney

  17. Solving Simple Kinetics without Integrals (United States)

    de la Pen~a, Lisandro Herna´ndez


    The solution of simple kinetic equations is analyzed without referencing any topic from differential equations or integral calculus. Guided by the physical meaning of the rate equation, a systematic procedure is used to generate an approximate solution that converges uniformly to the exact solution in the case of zero, first, and second order…

  18. Robustness Analysis of Kinetic Structures

    DEFF Research Database (Denmark)

    Kirkegaard, Poul Henning; Sørensen, John Dalsgaard


    Kinetic structures in architecture follows a new trend which is emerging in responsive architecture coined by Nicholas Negroponte when he proposed that architecture may benefit from the integration of computing power into built spaces and structures, and that better performing, more rational...

  19. Kinetics of Bio-Reactions

    DEFF Research Database (Denmark)

    Villadsen, John


    his chapter predicts the specific rates of reaction by means of a mathematical expression, the kinetics of the reaction. This expression can be derived through a mechanistic interpretation of an enzymatically catalyzed reaction, but it is essentially of empirical nature for cell reactions. The mo...

  20. Resonance transport and kinetic entropy

    International Nuclear Information System (INIS)

    Ivanov, Yu.B.; Knoll, J.; Voskresensky, D.N.


    We continue the description of the dynamics of unstable particles within the real-time formulation of nonequilibrium field theory initiated in a previous paper . There we suggest to use Baym's PHI-functional method in order to achieve approximation schemes with 'built in' consistency with respect to conservation laws and thermodynamics even in the case of particles with finite damping width. Starting from Kadanoff-Baym equations we discuss a consistent first order gradient approach to transport which preserves the PHI-derivable properties. The validity conditions for the resulting quantum four-phase-space kinetic theory are discussed under the perspective to treat particles with broad damping widths. This non-equilibrium dynamics naturally includes all those quantum features already inherent in the corresponding equilibrium limit (e.g. Matsubara formalism) at the same level of PHI-derivable approximation. Various collision-term diagrams are discussed including those of higher order which lead to memory effects. As an important novel part we derive a generalized nonequilibrium expression for the kinetic entropy flow, which includes contributions from fluctuations and mass-width effects. In special cases an H-theorem is derived implying that the entropy can only increase with time. Memory effects in the kinetic terms provide contributions to the kinetic entropy flow that in the equilibrium limit recover the famous bosonic type T 3 lnT correction to the specific heat in the case of Fermi liquids like Helium-3

  1. Heat inactivation kinetics of Hypocrea orientalis β-glucosidase with enhanced thermal stability by glucose. (United States)

    Xu, Xin-Qi; Shi, Yan; Wu, Xiao-Bing; Zhan, Xi-Lan; Zhou, Han-Tao; Chen, Qing-Xi


    Thermal inactivation kinetics of Hypocrea orientalis β-glucosidase and effect of glucose on thermostability of the enzyme have been determined in this paper. Kinetic studies showed that the thermal inactivation was irreversible and first-order reaction. The microscopic rate constants for inactivation of free enzyme and substrate-enzyme complex were both determined, which suggested that substrates can protect β-glucosidase against thermal deactivation effectively. On the other hand, glucose was found to protect β-glucosidase from heat inactivation to remain almost whole activity below 70°C at 20mM concentration, whereas the apparent inactivation rate of BG decreased to be 0.3×10(-3)s(-1) in the presence of 5mM glucose, smaller than that of sugar-free enzyme (1.91×10(-3)s(-1)). The intrinsic fluorescence spectra results showed that glucose also had stabilizing effect on the conformation of BG against thermal denaturation. Docking simulation depicted the interaction mode between glucose and active residues of the enzyme to produce stabilizing effect. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Role of conformational dynamics in kinetics of an enzymatic cycle in a nonequilibrium steady state (United States)

    Min, Wei; Xie, X. Sunney; Bagchi, Biman


    Enzyme is a dynamic entity with diverse time scales, ranging from picoseconds to seconds or even longer. Here we develop a rate theory for enzyme catalysis that includes conformational dynamics as cycling on a two-dimensional (2D) reaction free energy surface involving an intrinsic reaction coordinate (X) and an enzyme conformational coordinate (Q). The validity of Michaelis-Menten (MM) equation, i.e., substrate concentration dependence of enzymatic velocity, is examined under a nonequilibrium steady state. Under certain conditions, the classic MM equation holds but with generalized microscopic interpretations of kinetic parameters. However, under other conditions, our rate theory predicts either positive (sigmoidal-like) or negative (biphasic-like) kinetic cooperativity due to the modified effective 2D reaction pathway on X-Q surface, which can explain non-MM dependence previously observed on many monomeric enzymes that involve slow or hysteretic conformational transitions. Furthermore, we find that a slow conformational relaxation during product release could retain the enzyme in a favorable configuration, such that enzymatic turnover is dynamically accelerated at high substrate concentrations. The effect of such conformation retainment in a nonequilibrium steady state is evaluated.

  3. Dynamic Modeling of Cell-Free Biochemical Networks Using Effective Kinetic Models

    Directory of Open Access Journals (Sweden)

    Joseph A. Wayman


    Full Text Available Cell-free systems offer many advantages for the study, manipulation and modeling of metabolism compared to in vivo processes. Many of the challenges confronting genome-scale kinetic modeling can potentially be overcome in a cell-free system. For example, there is no complex transcriptional regulation to consider, transient metabolic measurements are easier to obtain, and we no longer have to consider cell growth. Thus, cell-free operation holds several significant advantages for model development, identification and validation. Theoretically, genome-scale cell-free kinetic models may be possible for industrially important organisms, such as E. coli, if a simple, tractable framework for integrating allosteric regulation with enzyme kinetics can be formulated. Toward this unmet need, we present an effective biochemical network modeling framework for building dynamic cell-free metabolic models. The key innovation of our approach is the integration of simple effective rules encoding complex allosteric regulation with traditional kinetic pathway modeling. We tested our approach by modeling the time evolution of several hypothetical cell-free metabolic networks. We found that simple effective rules, when integrated with traditional enzyme kinetic expressions, captured complex allosteric patterns such as ultrasensitivity or non-competitive inhibition in the absence of mechanistic information. Second, when integrated into network models, these rules captured classic regulatory patterns such as product-induced feedback inhibition. Lastly, we showed, at least for the network architectures considered here, that we could simultaneously estimate kinetic parameters and allosteric connectivity from synthetic data starting from an unbiased collection of possible allosteric structures using particle swarm optimization. However, when starting with an initial population that was heavily enriched with incorrect structures, our particle swarm approach could converge

  4. k-OptForce: integrating kinetics with flux balance analysis for strain design.

    Directory of Open Access Journals (Sweden)

    Anupam Chowdhury


    Full Text Available Computational strain design protocols aim at the system-wide identification of intervention strategies for the enhanced production of biochemicals in microorganisms. Existing approaches relying solely on stoichiometry and rudimentary constraint-based regulation overlook the effects of metabolite concentrations and substrate-level enzyme regulation while identifying metabolic interventions. In this paper, we introduce k-OptForce, which integrates the available kinetic descriptions of metabolic steps with stoichiometric models to sharpen the prediction of intervention strategies for improving the bio-production of a chemical of interest. It enables identification of a minimal set of interventions comprised of both enzymatic parameter changes (for reactions with available kinetics and reaction flux changes (for reactions with only stoichiometric information. Application of k-OptForce to the overproduction of L-serine in E. coli and triacetic acid lactone (TAL in S. cerevisiae revealed that the identified interventions tend to cause less dramatic rearrangements of the flux distribution so as not to violate concentration bounds. In some cases the incorporation of kinetic information leads to the need for additional interventions as kinetic expressions render stoichiometry-only derived interventions infeasible by violating concentration bounds, whereas in other cases the kinetic expressions impart flux changes that favor the overproduction of the target product thereby requiring fewer direct interventions. A sensitivity analysis on metabolite concentrations shows that the required number of interventions can be significantly affected by changing the imposed bounds on metabolite concentrations. Furthermore, k-OptForce was capable of finding non-intuitive interventions aiming at alleviating the substrate-level inhibition of key enzymes in order to enhance the flux towards the product of interest, which cannot be captured by stoichiometry-alone analysis

  5. Applications of Microbial Enzymes in Food Industry

    Directory of Open Access Journals (Sweden)

    Binod Parameswaran


    Full Text Available The use of enzymes or microorganisms in food preparations is an age-old process. With the advancement of technology, novel enzymes with wide range of applications and specificity have been developed and new application areas are still being explored. Microorganisms such as bacteria, yeast and fungi and their enzymes are widely used in several food preparations for improving the taste and texture and they offer huge economic benefits to industries. Microbial enzymes are the preferred source to plants or animals due to several advantages such as easy, cost-effective and consistent production. The present review discusses the recent advancement in enzyme technology for food industries. A comprehensive list of enzymes used in food processing, the microbial source of these enzymes and the wide range of their application are discussed.

  6. DNA-Based Enzyme Reactors and Systems

    Directory of Open Access Journals (Sweden)

    Veikko Linko


    Full Text Available During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

  7. Using enzyme folding to explore the mechanism of therapeutic touch: a feasibility study. (United States)

    Strickland, Mallory L; Boylan, Helen M


    The goal of this research is to design a novel model using protein folding to study Therapeutic Touch, a noncontact form of energy manipulation healing. Presented is a feasibility study suggesting that the denaturation path of ribonuclease A may be a useful model to study the energy exchange underlying therapeutic touch. The folding of ribonuclease A serves as a controlled energy-requiring system in which energy manipulation can be measured by the degree of folding achieved. A kinetic assay and fluorescence spectroscopy are used to assess the enzyme-folding state. The data suggest that the kinetic assay is a useful means of assessing the degree of refolding, and specifically, the enzyme function. However, fluorescence spectroscopy was not shown to be an effective measurement of enzyme structure for the purposes of this work. More research is needed to assess the underlying mechanism of therapeutic touch to complement the existing studies. An enzyme-folding model may provide a useful means of studying the energy exchange in therapeutic touch.

  8. Determination of enzyme-substrate dissociation rates by dynamic isotope exchange enhancement experiments

    International Nuclear Information System (INIS)

    Kim, S.C.; Raushel, F.M.


    A new method for the determination of dissociation rates of enzyme-substrate complexes has been developed. The rate of exchange of a labeled product back into the substrate is measured during catalysis of the forward reaction when the forward reaction is kept far from equilibrium by the enzymatic removal of the nonexchanging product. The ratio of the exchange rate and the net rate for product formation is then determined at various concentrations of the exchanging product. A plot of this ratio is a diagnostic indication of the kinetic mechanism and the relative rates of product dissociation from the binary and ternary enzyme complexes. This technique has been applied to the reaction catalyzed by bovine liver argininosuccinate lyase. The ratio for the rate of exchange of fumarate into argininosuccinate and the net rate for product formation was found to increase with the concentration of fumarate but to reach a limit of 3.3. The ratio of rates was half-maximal at 36 mM fumarate. The data have been interpreted to indicate the argininosuccinate lyase has a random kinetic mechanism. The calculated lower limit for the rate of release of arginine from the enzyme-fumarate-arginine complex is 0.35 times as fast as the Vmax in the reverse direction. The rate of release of arginine from the enzyme-arginine binary complex is 210 times faster than Vmax in the reverse direction

  9. Hide unhairing and characterization of commercial enzymes used in leather manufacture

    Directory of Open Access Journals (Sweden)

    A Dettmer


    Full Text Available The enzymatic treatment of hides in tannery processes is a promising technology. However, the reaction kinetics of commercial enzymes available to the leather industry are not fully understood and their activities have been mainly determined with model proteins such as casein as substrate, which are not of direct relevance for cattle hides. Therefore, it is important to determine their activities on collagen and keratin, the main proteins of skin, in order to use these enzymes in leather processing. This work describes the study of five proteases, used commercially in tanneries, to assess their ability to act upon collagen and keratin and to determine their unhairing. Results showed that all commercial enzymes tested had more activity on collagen than on keratin. Unhairing was also tested and four out of the five enzymes tested showed some unhairing activity. Optima of the temperature and pH of the enzymes were very similar for all five enzymes, with maximal activities around 55ºC and pH 9 to 12, respectively.

  10. The Secretion and Action of Brush Border Enzymes in the Mammalian Small Intestine. (United States)

    Hooton, Diane; Lentle, Roger; Monro, John; Wickham, Martin; Simpson, Robert


    Microvilli are conventionally regarded as an extension of the small intestinal absorptive surface, but they are also, as latterly discovered, a launching pad for brush border digestive enzymes. Recent work has demonstrated that motor elements of the microvillus cytoskeleton operate to displace the apical membrane toward the apex of the microvillus, where it vesiculates and is shed into the periapical space. Catalytically active brush border digestive enzymes remain incorporated within the membranes of these vesicles, which shifts the site of BB digestion from the surface of the enterocyte to the periapical space. This process enables nutrient hydrolysis to occur adjacent to the membrane in a pre-absorptive step. The characterization of BB digestive enzymes is influenced by the way in which these enzymes are anchored to the apical membranes of microvilli, their subsequent shedding in membrane vesicles, and their differing susceptibilities to cleavage from the component membranes. In addition, the presence of active intracellular components of these enzymes complicates their quantitative assay and the elucidation of their dynamics. This review summarizes the ontogeny and regulation of BB digestive enzymes and what is known of their kinetics and their action in the peripheral and axial regions of the small intestinal lumen.

  11. Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme. (United States)

    Watkins, Daniel W; Jenkins, Jonathan M X; Grayson, Katie J; Wood, Nicola; Steventon, Jack W; Le Vay, Kristian K; Goodwin, Matthew I; Mullen, Anna S; Bailey, Henry J; Crump, Matthew P; MacMillan, Fraser; Mulholland, Adrian J; Cameron, Gus; Sessions, Richard B; Mann, Stephen; Anderson, J L Ross


    Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 . The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.Catalytic mechanisms of enzymes are well understood, but achieving diverse reaction chemistries in re-engineered proteins can be difficult. Here the authors show a highly efficient and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 .

  12. Recovery of Extracellular Lipolytic Enzymes from Macrophomina phaseolina by Foam Fractionation with Air

    Directory of Open Access Journals (Sweden)

    Claudia Schinke


    Full Text Available Macrophomina phaseolina was cultivated in complex and simple media for the production of extracellular lipolytic enzymes. Culture supernatants were batch foam fractionated for the recovery of these enzymes, and column design and operation included the use of P 2 frit (porosity 40 to 100 μm, air as sparging gas at variable flow rates, and Triton X-100 added at the beginning or gradually in aliquots. Samples taken at intervals showed the progress of the kinetic and the efficiency parameters. Best results were obtained with the simple medium supernatant by combining the stepwise addition of small amounts of the surfactant with the variation of the air flow rates along the separation. Inert proteins were foamed out first, and the subsequent foamate was enriched in the enzymes, showing estimated activity recovery (R, enrichment ratio (E, and purification factor (P of 45%, 34.7, and 2.9, respectively. Lipases were present in the enriched foamate.

  13. Recycle bioreactor for bioethanol production from wheat starch. 1. Cold enzyme hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Lang, X.; Hill, G.A.; MacDonald, D.G. [Department of Chemical Engineering, Saskatchewan (Canada)


    A 5 L membrane bioreactor system has been designed and operated at low temperature to hydrolyze starch granules directly to sugars using barley {alpha}-amylase. The system includes a temperature and pH controlled, well-mixed bioreactor; microfilters to separate and recycle granules; and ultrafilters to separate and recycle enzyme molecules. Operation in batch mode demonstrated similar kinetics and low productivity observed earlier in shake flasks, whereas continuous flow operation was not successful due to enzyme inhibition and degradation. Sequential batch mode operation, involving filtration after each batch hydrolysis, produced optimum productivity measured at 0.16 grams of starch granules hydrolyzed per gram of enzyme per hour for more than 100 hours of operation. (author)

  14. The Structural and Functional Coordination of Glycolytic Enzymes in Muscle: Evidence of a Metabolon?

    Directory of Open Access Journals (Sweden)

    Lynda Menard


    Full Text Available Metabolism sustains life through enzyme-catalyzed chemical reactions within the cells of all organisms. The coupling of catalytic function to the structural organization of enzymes contributes to the kinetic optimization important to tissue-specific and whole-body function. This coupling is of paramount importance in the role that muscle plays in the success of Animalia. The structure and function of glycolytic enzyme complexes in anaerobic metabolism have long been regarded as a major regulatory element necessary for muscle activity and whole-body homeostasis. While the details of this complex remain to be elucidated through in vivo studies, this review will touch on recent studies that suggest the existence of such a complex and its structure. A potential model for glycolytic complexes and related subcomplexes is introduced.

  15. Mechanism-based Inhibitors of the Human Sirtuin 5 Deacylase: Structure-Activity Relationship, Biostructural, and Kinetic Insight

    DEFF Research Database (Denmark)

    Rajabi, Nima; Auth, Marina; Troelsen, Kathrin Rentzius


    to date. We provide rationalization of the mode of binding by solving co-crystal structures of selected inhibitors in complex with both human and zebrafish SIRT5, which provide insight for future optimization of inhibitors with more "drug-like" properties. Importantly, enzyme kinetic evaluation revealed...

  16. Kinetic characterization of Aspergillus niger chitinase CfcI using a HPAEC-PAD method for native chitin oligosaccharides

    NARCIS (Netherlands)

    van Munster, Jolanda M.; Sanders, Peter; ten Kate, Geralt A.; Dijkhuizen, Lubbert; van der Maarel, Marc J. E. C.


    The abundant polymer chitin can be degraded by chitinases (EC and beta-N-acetyl-hexosaminidases (EC to oligosaccharides and N-acetyl-glucosamine (GlcNAc) monomers. Kinetic characterization of these enzymes requires product quantification by an assay method with a low detection

  17. Modeling of pharmacokinetics of cocaine in human reveals the feasibility for development of enzyme therapies for drugs of abuse.

    Directory of Open Access Journals (Sweden)

    Fang Zheng

    Full Text Available A promising strategy for drug abuse treatment is to accelerate the drug metabolism by administration of a drug-metabolizing enzyme. The question is how effectively an enzyme can actually prevent the drug from entering brain and producing physiological effects. In the present study, we have developed a pharmacokinetic model through a combined use of in vitro kinetic parameters and positron emission tomography data in human to examine the effects of a cocaine-metabolizing enzyme in plasma on the time course of cocaine in plasma and brain of human. Without an exogenous enzyme, cocaine half-lives in both brain and plasma are almost linearly dependent on the initial cocaine concentration in plasma. The threshold concentration of cocaine in brain required to produce physiological effects has been estimated to be 0.22±0.07 µM, and the threshold area under the cocaine concentration versus time curve (AUC value in brain (denoted by AUC2(∞ required to produce physiological effects has been estimated to be 7.9±2.7 µM·min. It has been demonstrated that administration of a cocaine hydrolase/esterase (CocH/CocE can considerably decrease the cocaine half-lives in both brain and plasma, the peak cocaine concentration in brain, and the AUC2(∞. The estimated maximum cocaine plasma concentration which a given concentration of drug-metabolizing enzyme can effectively prevent from entering brain and producing physiological effects can be used to guide future preclinical/clinical studies on cocaine-metabolizing enzymes. Understanding of drug-metabolizing enzymes is key to the science of pharmacokinetics. The general insights into the effects of a drug-metabolizing enzyme on drug kinetics in human should be valuable also in future development of enzyme therapies for other drugs of abuse.

  18. Modeling of pharmacokinetics of cocaine in human reveals the feasibility for development of enzyme therapies for drugs of abuse. (United States)

    Zheng, Fang; Zhan, Chang-Guo


    A promising strategy for drug abuse treatment is to accelerate the drug metabolism by administration of a drug-metabolizing enzyme. The question is how effectively an enzyme can actually prevent the drug from entering brain and producing physiological effects. In the present study, we have developed a pharmacokinetic model through a combined use of in vitro kinetic parameters and positron emission tomography data in human to examine the effects of a cocaine-metabolizing enzyme in plasma on the time course of cocaine in plasma and brain of human. Without an exogenous enzyme, cocaine half-lives in both brain and plasma are almost linearly dependent on the initial cocaine concentration in plasma. The threshold concentration of cocaine in brain required to produce physiological effects has been estimated to be 0.22±0.07 µM, and the threshold area under the cocaine concentration versus time curve (AUC) value in brain (denoted by AUC2(∞)) required to produce physiological effects has been estimated to be 7.9±2.7 µM·min. It has been demonstrated that administration of a cocaine hydrolase/esterase (CocH/CocE) can considerably decrease the cocaine half-lives in both brain and plasma, the peak cocaine concentration in brain, and the AUC2(∞). The estimated maximum cocaine plasma concentration which a given concentration of drug-metabolizing enzyme can effectively prevent from entering brain and producing physiological effects can be used to guide future preclinical/clinical studies on cocaine-metabolizing enzymes. Understanding of drug-metabolizing enzymes is key to the science of pharmacokinetics. The general insights into the effects of a drug-metabolizing enzyme on drug kinetics in human should be valuable also in future development of enzyme therapies for other drugs of abuse.

  19. Electron flow in multicenter enzymes: theory, applications, and consequences on the natural design of redox chains. (United States)

    Léger, Christophe; Lederer, Florence; Guigliarelli, Bruno; Bertrand, Patrick


    In protein film voltammetry, a redox enzyme is directly connected to an electrode; in the presence of substrate and when the driving force provided by the electrode is appropriate, a current flow reveals the steady-state turnover. We show that, in the case of a multicenter enzyme, this signal reports on the energetics and kinetics of electron transfer (ET) along the redox chain that wires the active site to the electrode, and this provides a new strategy for studying intramolecular ET. We propose a model which takes into account all the enzyme's redox microstates, and we prove it useful to interpret data for various enzymes. Several general ideas emerge from this analysis. Considering the reversibility of ET is a requirement: the usual picture, where ET is depicted as a series of irreversible steps, is oversimplified and lacks the important features that we emphasize. We give justification to the concept of apparent reduction potential on the time scale of turnover and we explain how the value of this potential relates to the thermodynamic and kinetic properties of the system. When intramolecular ET does not limit turnover, the redox chain merely mediates the driving force provided by the electrode or the soluble redox partner, whereas when intramolecular ET is slow, the enzyme behaves as if its active active site had apparent redox properties which depend on the reduction potentials of the relays. This suggests an alternative to the idea that redox chains are optimized in terms of speed: evolutionary pressure may have resulted in slowing down intramolecular ET in order to tune the enzyme's "operating potential".

  20. Some Aspects of Extended Kinetic Equation

    Directory of Open Access Journals (Sweden)

    Dilip Kumar


    Full Text Available Motivated by the pathway model of Mathai introduced in 2005 [Linear Algebra and Its Applications, 396, 317–328] we extend the standard kinetic equations. Connection of the extended kinetic equation with fractional calculus operator is established. The solution of the general form of the fractional kinetic equation is obtained through Laplace transform. The results for the standard kinetic equation are obtained as the limiting case.