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Model Enzyme (model + enzyme)
Selected AbstractsCinnamic Acid Esters as Potent Inhibitors of Fungal 17,-Hydroxysteroid Dehydrogenase , A Model Enzyme of the Short-Chain Dehydrogenase/Reductase SuperfamilyCHEMINFORM, Issue 46 2004Stanislav Gobec Abstract For Abstract see ChemInform Abstract in Full Text. [source] Amperometric Glucose Biosensors Based on Glassy Carbon and SWCNT-Modified Glassy Carbon ElectrodesELECTROANALYSIS, Issue 1 2008Irene Carpani Abstract Different carbonaceous materials, such as single-walled carbon nanotubes (SWCNTs) and glassy carbon submitted to an electrochemical activation at +1.80,V (vs. SCE) for 900,s, have been used with the aim of comparing their performances in the development of enzyme electrodes. Commercial SWCNTs have been pretreated with 2.2,M HNO3 for 20,h prior to use. The utility of activated GC as promising material for amperometric oxidase-based biosensors has been confirmed. With glucose oxidase (GOx) as a model enzyme, glucose was efficiently detected up to 1 mM without the use of a mediator. Both electrodes operated in stirred solutions of 0.1,M phosphate buffer (pH,5.5), containing dissolved oxygen, at a potential of ,0.40,V vs. SCE. Although the performances of the two carbonaceous materials were comparable, the biosensors based on activated GC were characterized by a practically unchanged response 40 days after the fabrication, a better signal to noise ratio, and a little worse sensitivity. In addition, the preparation procedure of such biosensors was more simple, rapid and reproducible. [source] Electrophoretically mediated microanalysis with partial filling technique and indirect or direct detection as a tool for inhibition studies of enzymatic reactionELECTROPHORESIS, Issue 7-8 2004Magdaléna Telnarová Abstract The inhibition of the model enzyme, haloalkane dehalogenase from Sphingomonas paucimobilis, was investigated by a combination of electrophoretically mediated microanalysis with a partial filling technique, followed by indirect or direct detection. In this setup, part of the capillary is filled with a buffer suitable for the enzymatic reaction (20 mM glycine buffer, pH 8.6) whereas the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. Two different background electrolytes and corresponding detection approaches were used to show the versatility of the developed method. The inhibition effect of 1,2-dichloroethane on the dehalogenation of brominated substrate 1-bromobutane was studied by means of 10 mM chromate , 0.1 mM cetyltrimethylammonium bromide (pH 9.2) in combination with indirect detection or 20 mM ,-alanine , hydrochloric acid (pH 3.5) in combination with direct detection. The method was used to estimate the inhibition constant KI (0.44 mM by indirect detection and 0.63 mM by of direct detection) and to determine the inhibition type. Compared to spectrophotometric and other discontinuous assays, the method is rapid, can be automated, and requires only small amount of reagents that is especially important in the case of enzymes and inhibitors. [source] Induced SER-Activity in Nanostructured Ag,Silica,Au Supports via Long-Range Plasmon CouplingADVANCED FUNCTIONAL MATERIALS, Issue 12 2010Jiu-Ju Feng Abstract A novel Ag,silica,Au hybrid device is developed that displays a long-range plasmon transfer of Ag to Au leading to enhanced Raman scattering of molecules largely separated from the optically excited Ag surface. A nanoscopically rough Ag surface is coated by a silica spacer of variable thickness from ,1 to 21,nm and a thin Au film of ,25,nm thickness. The outer Au surface is further functionalized by a self-assembled monolayer (SAM) for electrostatic binding of the heme protein cytochrome c (Cyt c) that serves as a Raman probe and model enzyme. High-quality surface-enhanced resonance Raman (SERR) spectra are obtained with 413,nm excitation, demonstrating that the enhancement results exclusively from excitation of Ag surface plasmons. The enhancement factor is estimated to be 2,×,104,8,×,103 for a separation of Cyt c from the Ag surface by 28,47,nm, corresponding to an attenuation of the enhancement by a factor of only 2,6 compared to Cyt c adsorbed directly on a SAM-coated Ag electrode. Upon immobilization of Cyt c on the functionalized Ag,silica,Au device, the native structure and redox properties are preserved as demonstrated by time- and potential-dependent SERR spectroscopy. [source] Fabrication of poly(ethylene glycol)-based hydrogels entrapping enzyme-immobilized silica nanoparticlesPOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 7 2010Eunji Jang Abstract In this study, we immobilized enzymes by combining covalent surface immobilization and hydrogel entrapment. A model enzyme, glucose oxidase (GOX), was first covalently immobilized on the surface of silica nanoparticles (SNPs) via 3-aminopropyltriethoxysilane (APTES), and the resultant SNP-immobilized enzyme was physically entrapped within photopolymerized hydrogels prepared from two different molecular weights (MWs) (575 and 8000,Da) of poly(ethylene glycol)(PEG). The hydrogel entrapment resulted in a decrease in reaction rate and an increase in apparent Km of SNP-immobilized GOX, but these negative effects could be minimized by using hydrogel with a higher MW PEG, which provides higher water content and larger mesh size. The catalytic rate of the PEG 8000 hydrogel was about ten times faster than that of the PEG 575 hydrogel because of enhanced mass transfer. Long-term stability test demonstrated that SNP-immobilized GOX entrapped within hydrogel maintained more than 60% of its initial activity after a week, whereas non-entrapped SNP-immobilized GOX and entrapped GOX without SNP immobilization maintained less than 20% of their initial activity. Incorporation of SNPs into hydrogel enhanced the mechanical strength of the hydrogel six-fold relative to bare hydrogels. Finally, a hydrogel microarray entrapping SNP-immobilized GOX was fabricated using photolithography and successfully used for quantitative glucose detection. Copyright © 2009 John Wiley & Sons, Ltd. [source] Biocatalytic hydrogels by template polymerizationPOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 5 2008H. El-Sherif Abstract Novel ionizable hydrogels were prepared from poly(acrylic acid) and dimethylaminoethyl methacrylate monomer employing template polymerization technique as an alternative to traditional physical and chemical crosslinking. The mode of interaction, as proved by Fourier Transform Infrared Spectroscopy (FTIR), was multiple H-bonding between the tertiary amino group of the monomer and the carboxylic groups of the polymer. The hydrogels represented suitable matrices for enzyme immobilization. The effect of varying the polymer,monomer molar ratio on the swelling kinetics and parameters was investigated. The dynamic swelling isotherm exhibited a Fickian mode of penetrant sorption and a plateau that increases with the amino group content. A polymer complex of molar ratio (polymer:monomer) 0.5:0.8 had a weight swelling ratio of 10 and 7 at pHs 3 and 8, respectively. The proven pH sensitivity together with the amphoteric character of these hydrogels make them good candidates for another bioapplication such as oral delivery systems of therapeutic peptides and proteins. The structural integrity of the hydrogels was proved by their swelling reversibility. , -Galactosidase, as an acidic model enzyme, was immobilized covalently on the synthesized hydrogels. The maximum enzyme velocity (Vmax) was enhanced to 19,µmol/min/mg, for polycomplex of molar ratio 0.5:0.8, compared with 3.2,µmol/min/mg for the free enzyme. Copyright © 2008 John Wiley & Sons, Ltd. [source] Improvement of student understanding of how kinetic data facilitates the determination of amino acid catalytic function through an alkaline phosphatase structure/mechanism bioinformatics exercise,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2008Sandra K. Grunwald Abstract 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 developed and implemented. In this exercise, students examine the structure of alkaline phosphatase using the free, on-line bioinformatics protein-modeling program Protein Explorer. Specifically, students examine the active site residues of alkaline phosphatase and propose functions for these residues. Furthermore, by examining the mechanism of alkaline phosphatase and by using the published kinetic data, students propose specific roles for several active-site residues. Paired t -test analysis of pre- versus postexercise assessment data shows that the completion of the exercise improves student's ability to use kinetic data correctly thereby determining a probable catalytic function for an active site amino acid. [source] Enzymatic stability of 2,-ethylcarbonate-linked paclitaxel in serum and conversion to paclitaxel by rabbit liver carboxylesterase for use in prodrug/enzyme therapyBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 5 2008Tadatoshi Tanino Abstract In prodrug/enzyme therapy for cancer, information on the sensitivity of hydrolytic enzymes to prodrug is required to reduce adverse effects of the parental drug and to find the activating enzyme. The aim of this study was to characterize the enzymatic stability of 2,-ethylcarbonate-linked paclitaxel (TAX-2,-Et) in the sera of several different species including humans. TAX-2,-Et disposition in serum was kinetically analysed using models with hydrolytic and/or degradation processes. To further evaluate the capability of liver carboxylesterases (CESs) in TAX-2,-Et hydrolysis, a CES isolated from rabbit liver (Ra-CES) was utilized as a model enzyme. Rat serum provided rapid enzymatic hydrolysis of TAX-2,-Et with a half-life of 4 min. The degradation of paclitaxel (TAX) (degradation rate constant, 0.16,h,1) was accompanied by the formation of an unknown compound. The conversion to TAX was almost completely inhibited by phenylmethyl sulfonylfluoride (PMSF) and bis(p-nitrophenyl) phosphate (BNPP). In human and rabbit sera, the degradation rate constant of TAX-2,-Et was 5.1,×,10,2 and 0.15,h,1, respectively, when excepting hydrolysis. The degradation products had the same molecular weight as TAX-2,-Et. The amount of TAX produced accounted for only 8,11% of the decrease in TAX-2,-Et after a 9 h exposure to rabbit or human serum. PMSF, but not BNPP, inhibited more than 90% of the TAX production in a 1.5,h incubation with human or rabbit serum. Ra-CES enzyme converted TAX-2,-Et to TAX with Vmax and Km of 74.7±13.8 nmol/min/mg protein and 8.8±2.8 µM, respectively. These results indicate that TAX-2,-Et is sensitive to serum CESs, but not cholinesterases. However, serum CESs show species-dependent hydrolysis of TAX-2,-Et. Although human serum allows the slow release of TAX, TAX-2,-Et is expected to reduce the side-effects of TAX. The Ra-CES enzyme is capable of hydrolysing TAX-2,-Et, which may be beneficial for the development of a TAX-2,-Et/enzyme therapy strategy for ovarian cancer. Copyright © 2008 John Wiley & Sons, Ltd. [source] Fourier transform infrared spectroscopy suggests unfolding of loop structures precedes complete unfolding of pig citrate synthaseBIOPOLYMERS, Issue 4 2003Feride Severcan Abstract Pig citrate synthase (PCS) can be used as a model enzyme to gain some insight into the structural basis of protein thermostability. The thermal unfolding characteristics of the specific secondary structure elements within PCS were monitored in detail by following changes in its amide I band components. The result of our study indicates that PCS undergoes irreversible thermal denaturation. Detailed analysis reveals that the different secondary structures display a multistep transition with a major and a minor transition at different temperatures and a very small initial transition at the same temperature (30°C). A plot of temperature-induced changes in 1H,2H exchange, the decrease in the absorbance of the ,-helical structures, and the increase in the absorbance of aggregated structures all have in common a multistep transition, the minor one centered at 45°C and the major one around 59°C. In contrast, a band that is tentatively assigned to loop structures displays these same minor and major transitions but at lower temperatures (39 and 52°C, respectively). The transition, which occurs at 39,45°C, is not associated with the appearance of aggregated structures. This transition may reflect a change in the tertiary structure of the protein. However, the final transition, which occurs at a higher temperature (52,59°C), reflects unfolding and aggregation of the polypeptide chains. The Fourier transform infrared (FTIR) analysis suggests that PCS has a thermolabile region that unfolds first, some 7°C below the main unfolding of the protein. We propose that this reflects the unfolding of the highly flexible loop segments, which in turn triggers the unfolding of the predominantly helical core structure of PCS. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 440,447, 2003 [source] Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobilityBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2003Hongfei Jia Abstract Nanoparticles provide an ideal remedy to the usually contradictory issues encountered in the optimization of immobilized enzymes: minimum diffusional limitation, maximum surface area per unit mass, and high effective enzyme loading. In addition to the promising performance features, the unique solution behaviors of the nanoparticles also point to a transitional region between the heterogeneous (with immobilized enzymes) and homogeneous (with soluble free enzymes) catalysis. The particle mobility, which is related to particle size and solution viscosity through Stokes-Einstein equation, may impact the reaction kinetics according to the collision theory. The mobility-activity relationship was examined through experimental studies and theoretical modeling in the present work. Polystyrene particles with diameters ranging from 110,1000 nm were prepared. A model enzyme, ,-chymotrypsin, was covalently attached to the nanoparticles up to 6.6 wt%. The collision theory model was found feasible in correlating the catalytic activities of particles to particle size and solution viscosity. Changes in the size of particles and the viscosity of reaction media, which all affect the mobility of the enzyme catalyst, evidently altered the intrinsic activity of the particle-attached enzyme. Compared to KM, kcat appeared to be less sensitive to particle size and viscosity. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng84: 406,414, 2003. [source] Microfabricated arrays of cylindrical wells facilitate single-molecule enzymology of ,-chymotrypsinBIOTECHNOLOGY PROGRESS, Issue 4 2009Angela Y. Chen Abstract Single-molecule enzymology allows scientists to examine the distributions of kinetic rates among members of a population. We describe a simple method for the analysis of single-molecule enzymatic kinetics and provide comparisons to ensemble-averaged kinetics. To isolate our model enzyme, ,-chymotrypsin, into single molecules, we use an array of cylindrical poly(dimethylsiloxane) wells 2 ,m in diameter and 1.35 ,m in height. Inside the wells, a protease assay with a profluorescent substrate detects ,-chymotrypsin activity. We hold the concentration of ,-chymotrypsin at 0.39 nM in a given well with an enzyme-to-substrate ratio of 1:6,666 molecules. Fluorescence emitted by the substrate is proportional to enzyme activity and detectable by a charge-coupled device. This method allows for the simultaneous real-time characterization of hundreds of individual enzymes. We analyze single-molecule kinetics by recording and observing their intensity trajectories over time. By testing our method with our current instruments, we confirm that our methodology is useful for the analysis of single enzymes for extracting static inhomogeneity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Enzyme-Carrying Polymeric Nanofibers Prepared via Electrospinning for Use as Unique BiocatalystsBIOTECHNOLOGY PROGRESS, Issue 5 2002Hongfei Jia Improvement of catalytic efficiency of immobilized enzymes via materials engineering was demonstrated through the preparation of bioactive nanofibers. Bioactive polystyrene (PS) nanofibers with a typical diameter of 120 nm were prepared and examined for catalytic efficiency for biotransformations. The nanofibers were produced by electrospinning functionalized PS, followed by the chemical attachment of a model enzyme, ,-chymotrypsin. The observed enzyme loading as determined by active site titration was up to 1.4% (wt/wt), corresponding to over 27.4% monolayer coverage of the external surface of nanofibers. The apparent hydrolytic activity of the nanofibrous enzyme in aqueous solutions was over 65% of that of the native enzyme, indicating a high catalytic efficiency as compared to other forms of immobilized enzymes. Furthermore, nanofibrous ,-chymotrypsin exhibited a much-improved nonaqueous activity that was over 3 orders of magnitude higher than that of its native counterpart suspended in organic solvents including hexane and isooctane. It appeared that the covalent binding also improved the enzyme's stability against structural denaturation, such that the half-life of the nanofibrous enzyme in methanol was 18-fold longer than that of the native enzyme. [source] | ||||||||||||||||