Home About us Contact
|
Home About us Contact | ||
|
|
||
Enzymatic Reaction (enzymatic + reaction)
Selected AbstractsChemInform Abstract: Synthesis of Galactofuranosides by Regioselective Ring Opening of a 1,4-Anhydrogalactopyranose Derivative: A Possible Chemical Model for an Unprecedented Enzymatic Reaction.CHEMINFORM, Issue 6 2001Jose Kovensky Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] A Wide Range of Strategies Yields New Enzymatic Reactions and ProcessesADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 7-8 2005Chi-Huey Wong No abstract is available for this article. [source] Microfluidic tectonics platform: A colorimetric, disposable botulinum toxin enzyme-linked immunosorbent assay systemELECTROPHORESIS, Issue 10-11 2004Jaisree Moorthy Abstract A fabrication platform for realizing integrated microfluidic devices is discussed. The platform allows for creating specific microsystems for multistep assays in an ad hoc manner as the components that perform the assay steps can be created at any location inside the device via in situ fabrication. The platform was utilized to create a prototype microsystem for detecting botulinum neurotoxin directly from whole blood. Process steps such as sample preparation by filtration, mixing and incubation with reagents was carried out on the device. Various microfluidic components such as channel network, valves and porous filter were fabricated from prepolymer mixture consisting of monomer, cross-linker and a photoinitiator. For detection of the toxoid, biotinylated antibodies were immobilized on streptavidin-functionalized agarose gel beads. The gel beads were introduced into the device and were used as readouts. Enzymatic reaction between alkaline phosphatase (on secondary antibody) and substrate produced an insoluble, colored precipitate that coated the beads thus making the readout visible to the naked eye. Clinically relevant amounts of the toxin can be detected from whole blood using the portable enzyme-linked immunosorbent assay (ELISA) system. Multiple layers can be realized for effective space utilization and creating a three-dimensional (3-D) chaotic mixer. In addition, external materials such as membranes can be incorporated into the device as components. Individual components that were necessary to perform these steps were characterized, and their mutual compatibility is also discussed. [source] Changes of Volatile Compounds, Lactic Acid Bacteria, pH, and Headspace Gases in Kimchi, a Traditional Korean Fermented Vegetable ProductJOURNAL OF FOOD SCIENCE, Issue 3 2003J.H. Kang ABSTRACT Headspace volatiles of Kimchi stored at 5 °C increased over a 7 d period by 20.0% and then decreased from 7 to 27 d by 8.3%. Forty volatile compounds including 18 sulfur compounds were identified in Kimchi. Lactic acid bacteria in Kimchi increased from 3.1 to 4.5 (1 × 108cfu/mL) over a 17-d period and decreased by 40% from 17 to 27 d. As the storage time increased from 2 to 27 d, pH decreased from 4.3 to 3.8, headspace oxygen decreased from 14.3 to 1.3%, and headspace carbon dioxide increased from 27.7 to 45.3%. Enzymatic reactions and chemical oxidations in Kimchi explained the changes of volatile compounds, lactic acid bacteria, pH, headspace oxygen and carbon dioxide. [source] An analysis of all the relevant facts and arguments indicates that enzyme catalysis does not involve large contributions from nuclear tunnelingJOURNAL OF PHYSICAL ORGANIC CHEMISTRY, Issue 7 2010Shina C. L. Kamerlin Abstract Enzymatic reactions are crucial toward controlling and performing most life processes, and, as such, understanding how they really work has both fundamental and practical importance. Thus, one of the major current challenges of biophysics involves understanding the origin of the enormous catalytic power of enzymes, an issue that is still not widely understood and remains controversial within the scientific community. Several proposals have been put forth to try to explain the origin of enzyme catalysis, one of which is the idea that enzyme catalysis involves special factors such as nuclear quantum mechanical (NQM) effects, and, in particular, nuclear tunneling. Here, we will discuss both the factors for and against this proposition, and demonstrate that an analysis of all the relevant facts and arguments seems to establish that enzyme catalysis does not involve large contributions from nuclear tunneling. Copyright © 2010 John Wiley & Sons, Ltd. [source] Fabrication of a Sensitive Cholesterol Biosensor Based on Cobalt-oxide Nanostructures Electrodeposited onto Glassy Carbon ElectrodeELECTROANALYSIS, Issue 24 2009Abdollah Salimi Abstract Electrodeposited cobalt oxide (CoOx) nanomaterials are not only used for immobilization of cholesterol oxidase (ChOx) but also as electron transfer mediator for oxidation of H2O2 generated in the enzymatic reaction. Voltammetry and flow injection analysis (FIA) were used for determination of cholesterol. FIA determination of cholesterol with biosensors yielded a calibration curve with the following characteristics: linear range up to 50,,M, sensitivity of 43.5,nA ,M,1 cm,2 and detection limit of 4.2,,M. The apparent Michaelis-Menten constant and the response time of the biosensor are 0.49,mM and 15,s, respectively. This biosensor also exhibits good stability, reproducibility and long life time. [source] Detection of C Reactive Protein (CRP) in Serum by an Electrochemical Aptamer-Based Sandwich AssayELECTROANALYSIS, Issue 11 2009Sonia Centi Abstract A disposable electrochemical assay involving magnetic particles and carbon-based screen-printed electrodes (SPCEs) was developed for the detection of C Reactive Protein (CRP). CRP is a plasma protein and is among the most expressed proteins in acute phase inflammation cases, being a known biomarker for inflammatory states. The assay was based on a sandwich format in which a RNA aptamer was coupled to a monoclonal antibody and alkaline phosphatase (AP) was used as enzymatic label. After the sandwich assay, the modified magnetic beads were captured by a magnet on the surface of a graphite working electrode and the electrochemical detection was thus achieved through the addition of the AP substrate (,-naphthyl-phosphate) and ,-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry (DPV). The parameters influencing the different steps of the assay were optimized in order to reach the best sensitivity and specificity. With the optimized conditions, the assay was applied to the analysis of CRP free serum and serum samples. [source] Photo-Induced Electron Transfer Between Photosystem 2 via Cross-linked Redox HydrogelsELECTROANALYSIS, Issue 10 2008Adrian Badura Abstract Photosystem 2 (PS2) that catalyses light driven water splitting in photosynthesis was ,wired' to electrode surfaces via osmium-containing redox polymers based on poly(vinyl)imidazol. The redox polymer hydrogel worked as both immobilization matrix and electron acceptor for the enzyme. Upon illumination, the enzymatic reaction could be switched on and a catalytic current was observed at the electrode. The catalytic current is directly dependent on the intensity of light used for the excitation of PS2. A typical current density of 45,,A cm,2 at a light intensity of 2.65,mW cm,2 could be demonstrated with a significantly improved operational stability. [source] Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substratesELECTROPHORESIS, Issue 12 2006Jamshed Iqbal Abstract Fast and convenient CE assays were developed for the screening of adenosine kinase,(AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260,nm. An MEKC method using borate buffer (pH,9.5) containing 100,mM SDS (method,A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH,7.5 or 8.5) was used and a constant current (95,,A) was applied (method,B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10,min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method,C). After hydrodynamic injection of a plug of reaction buffer (20,mM Tris-HCl, 0.2,mM MgCl2, pH,7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1,mM,ATP, 100,,M adenosine, and 20,,M,UMP as an internal standard,(I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5,kV separation voltage (negative polarity) for 0.20,min to let the plugs interpenetrate. The voltage was turned off for 5,min (zero-potential amplification) and again turned on at a constant current of ,60,,A to elute the products within 7,min. The method employing a polyacrylamide-coated capillary of 20,cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose,response curves and calculated Ki values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay. [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] Electrophoretically mediated reaction of glycosidases at a nanoliter scaleELECTROPHORESIS, Issue 6 2003Yoshimi Kanie Abstract We have investigated electrophoretically mediated microanalysis (EMMA) for the assay of a native glyco-enzyme. As a representative of this class of enzyme, ,-glucosidase was selected, and the reaction was analyzed. Our EMMA was based on the plug-plug interaction of enzyme and substrate plugs, which is essential to reduce quantities of materials. Furthermore, we have addressed the problem of incompatibility of the enzymatic reaction and separation of the reactants. As a result, EMMA of native glycosidase was achieved with a reaction volume of ,,20 nL and the Michaelis constant was estimated according to the Lineweaver-Burk plot. The current method may have advantages over traditional assay methods, especially in terms of the amount of enzyme (ng order) and substrate (pmol order) required for a reaction*. [source] Electroenzymatic Synthesis of Chiral SulfoxidesENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 2 2006C. Kohlmann Abstract Chloroperoxidase (CPO) from Caldariomyces fumago (E.C.,1.11.1.10) is able to enantioselectively oxidize various sulfides to the corresponding (R)-enantiomer of the sulfoxides. For these oxidations the enzyme requires an oxidant. Most commonly, tert -butyl hydroperoxide (TBHP) and hydrogen peroxide are used. As it is known that these oxidants inactivate the enzyme, the enzymatic reaction was combined with the electrochemical in situ generation of hydrogen peroxide. As substrates for this combination of an enzymatic and an electrochemical reaction methyl p-tolyl sulfide, 1-methoxy-4-(methylthio)benzene and N-MOC- L -methionine methyl ester were used to carry out batch experiments. [source] Side chain specificity of ADP-ribosylation by a sirtuinFEBS JOURNAL, Issue 23 2009Kamau Fahie Endogenous mono-ADP-ribosylation in eukaryotes is involved in regulating protein synthesis, signal transduction, cytoskeletal integrity, and cell proliferation, although few cellular ADP-ribosyltransferases have been identified. The sirtuins constitute a highly conserved family of protein deacetylases, and several family members have also been reported to perform protein ADP-ribosylation. We characterized the ADP-ribosylation reaction of the nuclear sirtuin homolog Trypanosoma brucei SIR2-related protein 1 (TbSIR2RP1) on both acetylated and unacetylated substrates. We demonstrated that an acetylated substrate is not required for ADP-ribosylation to occur, indicating that the reaction performed by TbSIR2RP1 is a genuine enzymatic reaction and not a side reaction of deacetylation. Biochemical and MS data showed that arginine is the major ADP-ribose acceptor for unacetylated substrates, whereas arginine does not appear to be the major ADP-ribose acceptor in reactions with acetylated histone H1.1. We performed combined ab initio quantum mechanical/molecular mechanical molecular dynamics simulations, which indicated that sirtuin ADP-ribosylation at arginine is energetically feasible, and involves a concerted mechanism with a highly dissociative transition state. In comparison with the corresponding nicotinamide cleavage in the deacetylation reaction, the simulations suggest that sirtuin ADP-ribosylation would be several orders slower but less sensitive to nicotinamide inhibition, which is consistent with experimental results. These results suggest that TbSIR2RP1 can perform ADP-ribosylation using two distinct mechanisms, depending on whether or not the substrate is acetylated. Structured digital abstract ,,MINT-7288298: TbSIR2 (uniprotkb:O96670) adp ribosylates (MI:0557) histone H1.1 (uniprotkb:Q02539) by enzymatic studies (MI:0415) ,,MINT-7288305, MINT-7288325, MINT-7288338, MINT-7288352, MINT-7288370, MINT-7288395, MINT-7288412: TbSIR2 (uniprotkb:O96670) adp ribosylates (MI:0557) histone H1.1 (uniprotkb:P02253) by enzymatic studies (MI:0415) ,,MINT-7288385: TbSIR2 (uniprotkb:O96670) deacetylates (MI:0197) histone H1.1 (uniprotkb:Q02539) by deacetylase assay (MI:0406) ,,MINT-7288424: hADPRH (uniprotkb:P54922) cleaves (MI:0194) histone H1.1 (uniprotkb:Q02539) by enzymatic studies (MI:0415) [source] Molecular dynamics simulations of solvated UDP,glucose in interaction with Mg2+ cationsFEBS JOURNAL, Issue 20 2001Pavla Petrová ,Glycosyltransferases are key enzymes involved in biosynthesis of oligosaccharides. Nucleotide-sugars, the glycosyltransferase substrates, serve as activated donors of sugar residues during the enzymatic reaction Although very little is known about the catalytic mechanism of these enzymes, it appears that the catalytic activity in most glycosyltransferases is dependent upon the presence of a divalent cation, for example Mn2+ or Mg2+. It is not known whether the ion is bound to the enzyme before its interaction with the substrate, or if it binds the substrate before the enzymatic reaction to modify its conformation to fit better the active site of the enzyme. We have inspected the latter possibility by running four 2-ns molecular dynamics trajectories on fully solvated UDP-glucose in the presence of Mg2+ ions. Our results indicate that the divalent cation interacts strongly with the nucleotide-sugar in solution, and that it can alter its conformational behavior. It is also shown that a conformation of the pyrophosphate moiety that results in an eclipsed or almost eclipsed orientation of two of the oxygen atoms, and which is found in protein interacting with a nucleotide di- or tri-phosphate X-ray data, is energetically favored. The results are also discussed in light of existing NMR data, and are found to be in a good agreement with them. [source] Oxidation by mushroom tyrosinase of monophenols generating slightly unstable o -quinonesFEBS JOURNAL, Issue 19 2000Lorena G. Fenoll Tyrosinase can act on monophenols because of the mixture of mettyrosinase (Em) and oxytyrosinase (Eox) that exists in the native form of the enzyme. The latter form is active on monophenols although the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex as the products of the enzymatic reaction, the o -quinones, are unstable and continue evolving to generate o -diphenols in the medium. In the case of substrates such as 4-methoxyphenol, 4-ethoxyphenol and 4- tert -butylphenol, tyrosinase generates o -quinones which become unstable with small constants of approximately < 10,3 s,1. The system evolves from an initial steady state, reached when t,0, through a transition state towards a final steady state, which is never reached because the substrate is largely consumed. The mechanisms proposed to explain the enzyme's action can be differentiated by the kinetics of the first steady state. The results suggest that tyrosinase hydroxylates monophenols to o -diphenols, generating an intermediate Em -diphenol in the process, which may oxidize the o -diphenol or release it directly into the medium. In the case of o -quinone formation, its slow instability generates o -diphenol which activates the enzymatic system yielding parabolic time recordings. [source] Enzyme-mediated sulfide production for the reconstitution of [2Fe,2S] clusters into apo-biotin synthase of Escherichia coliFEBS JOURNAL, Issue 9 2000Sulfide transfer from cysteine to biotin We previously showed that biotin synthase in which the (Fe,S) cluster was labelled with 34S by reconstitution donates 34S to biotin [B. Tse Sum Bui, D. Florentin, F. Fournier, O. Ploux, A. Méjean & A. Marquet (1998) FEBS Lett. 440, 226,230]. We therefore proposed that the source of sulfur was very likely the (Fe,S) centre. This depletion of sulfur from the cluster during enzymatic reaction could explain the absence of turnover of the enzyme which means that to restore a catalytic activity, the clusters have to be regenerated. In this report, we show that the NifS protein from Azotobacter vinelandii and C-DES from Synechocystis as well as rhodanese from bovine liver can mobilize the sulfur, respectively, from cysteine and thiosulfate for the formation of a [2Fe,2S] cluster in the apoprotein of Escherichia coli biotin synthase. The reconstituted enzymes were as active as the native enzyme. When [35S]cysteine was used during the reconstitution experiments in the presence of NifS, labelled (Fe35S) biotin synthase was obtained. This enzyme produced [35S]biotin, confirming the results obtained with the 34S-reconstituted enzyme. NifS was also effective in mobilizing selenium from selenocystine to produce an (Fe,Se) cluster. However, though NifS could efficiently reconstitute holobiotin synthase from the apoform, starting from cysteine, these two effectors had no significant effect on the turnover of the enzyme in the in vitro assay. [source] Chill injury in the eggs of the migratory locust, Locusta migratoria (Orthoptera: Acrididae): the time-temperature relationship with high-temperature interruptionINSECT SCIENCE, Issue 3 2005XIAO-HONG JING Abstract Mortality of the overwintering egg of the migratory locust, Locusta migratoria L., was attributed to chill injury because of its occurrence well above the egg's super cooling point. In this study, two parameters, upper limit of chill injury zone (ULCIZ) and sum of the injurious temperature (SIT), were used to examine the locust egg's cold hardiness. The value of ULCIZ for the locust egg is 1.06 ± 0.54°C, and the SIT is -329.7 (hour · degree). The superoxide dismutase (SOD) and catalase (CAT) activities changed dramatically after cold stress, indicating that oxygen and hydroxide free radicals are probably efficiently detoxified at low temperatures. It was suggested that the nature of chill injury in locust egg might be a complex of metabolic disorder and a non-proportional decrease in enzymatic reaction and transports, because the LDH activity at low temperature increased significantly and the ATPase activity decreased with prolonged duration of exposure to low temperatures. The results from high temperature interruption revealed that the high temperature intervals significantly increased the survival of locust eggs. [source] pKa optimized catalysis in serine proteinases, an ab initio study on the catalaytic HisINTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 11 2007Péter Hudáky Abstract First principle models of catalytic apparatus of enzymes can be used for studying stability as well as the atomic details of a catalytic mechanism. For example, the catalytic triad of chymotrypsin was recently investigated by using an ab initio geometry optimized (Hudáky and Perczel, Proteins: Struct Funct Genet, 2006, 62, 749) self-stabilizing molecule ensemble without the presence of the complete enzyme and substrate. Several parameters of the above catalytic reaction turned out to be the same within the model and the in vitro enzymatic reaction. Among the numerous parameters of the catalytic process geometrical changes of the catalytic histidine was investigated here and the variation of its pKa value was determined. A relatively large range, 3.5 unit, was determined as the variation of pKa as function of the conformational subspace available in serine proteases. Comparing PDB structures of the free and the complex enzymes it was shown, that histidine, after accepting the proton from the OH group of the catalytic serine, undergoes a minor conformational change accompanied by a 2.5 unit decrease in pKa. We conclude that the changes of pKa during catalysis are predominantly determined by the geometrical arrangement of the histidine moiety and this change serves as a significant driving force in the catalytic process. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2007 [source] Improvement of the catalytic performance of lignin peroxidase in reversed micellesJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 1 2008Jing Lan Abstract BACKGROUND: Anionic surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) had an inhibiting effect on lignin peroxidase (LiP). To improve the catalytic activity of LiP in an AOT reversed micelle in isooctane, nonionic surfactant polyoxyethylene lauryl ether (Brij30) was incorporated into the interfacial membrane. H2O2 played dual roles in the LiP-catalyzed oxidation of substrates. To obtain a sustainable high activity of LiP, a coupled enzymatic reaction, i.e. the glucose oxidase (GOD)-catalyzed oxidation of glucose was used as an H2O2 source. RESULTS: Owing to modification of the charge density of the interfacial membrane, the activity of LiP in an optimized AOT/Brij30 reversed micellar medium (,B (the molar percentage of Brij30) = 0.53, ,0 ([H2O]/([AOT] + [Brij30]) = 23, pH = 4.8) was 40 times that in a single AOT reversed micelle. Due to the controlled release of H2O2, the concentration of H2O2 in the mixed reversed micellar medium was maintained at a moderately high level throughout, which made the LiP-catalyzed oxidation of substrates proceed at a higher conversion rate than counterparts in which H2O2 was supplied externally in one batch at the beginning of the reaction. Decolourization of two waterless-soluble aromatic dyes (pyrogallol red and bromopyrogallol red) using LiP coupled with GOD in the medium also demonstrated that a higher decolourization percentage was obtained if H2O2 was supplied enzymatically. CONCLUSION: The proposed measures (both physicochemical and biochemical) were very effective, giving significant improvement in the catalytic performance of LiP in a single AOT reversed micelle in isooctane, which helped to degrade or transform hydrophobic aromatic compounds with LiP in reversed micelles more efficiently. Copyright © 2007 Society of Chemical Industry [source] Continuous flow isotope ratio mass spectrometry for the measurement of nanomole amounts of 13CO2 by a reverse isotope dilution methodJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2002J. Guitton Abstract A simple method for the determination of nanomole amounts of 13CO2 generated from an in vitro reaction is reported. The incubation medium contains a known amount of unlabeled sodium bicarbonate and the gaseous 13CO2 enriches the atmosphere upon which a measurement of the isotopic enrichment (13CO2/12CO2) is made corresponding to a reverse isotope dilution. The quantification of the 13CO2 was performed by gas chromatography/isotope ratio mass spectrometry. This assay was validated in terms of linearity, accuracy and precision using three different substrates which produce 13CO2 either by enzymatic reaction [13C]urea, sodium [13C]formate) or by chemical reaction (sodium [13C]bicarbonate). Four calibration curves were tested for each 13C-labeled substrate, allowing the quantification of 13CO2 from 25 pmol to 150 nmol. The dynamics of the assay were obtained as a function of the quantity of unlabeled sodium bicarbonate added to each sample. Copyright © 2001 John Wiley & Sons, Ltd. [source] Simulation and optimization of supercritical fluid purification of phytosterol estersAICHE JOURNAL, Issue 4 2009Tiziana Fornari Abstract Supercritical carbon dioxide extraction to separate phytosterol esters from fatty acid esters and tocopherols was simulated and optimized using the group contribution equation of state. Experimental extraction data at 328 K, pressures ranging from 200 to 280 bar and solvent-to-feed ratio around 25, was employed to verify the performance of the thermodynamic model. The raw material is the product obtained after a two-step enzymatic reaction carried out on soybean oil deodorizer distillates, and contains mainly fatty-acid ethyl esters, tocopherols and phytosterol esters. The extraction process was simulated using model substances to represent the complex multicomponent feed material. Nonlinear programming techniques were applied to find out optimal process conditions for a steady-state countercurrent process with partial reflux of the extract. The process optimization procedure predicts that a product with 94.2 wt % of phytosterol ester purity and 80% yield could be achieved. © 2009 American Institute of Chemical Engineers AIChE J, 2009 [source] Lipase-Catalyzed Ring-Opening Polymerization of Molecularly Pure Cyclic Oligomers for Use in Synthesis and Chemical Recycling of Aliphatic PolyestersMACROMOLECULAR BIOSCIENCE, Issue 6 2008Asato Kondo Abstract The lipase-catalyzed ROP of molecularly pure cyclic oligomers with a definite degree of oligomerization is analyzed with respect to the molecular weights of the resulting polymers and certain kinetic parameters of the enzymatic reaction. Cyclic BA dimers, trimers, and tetramers polymerize faster than the equivalent monomer; however, the latter produces PBA of significantly higher molecular weight. The reason is that the ring opening of the cyclic monomer is slow, leading to a lower initiator concentration than that produced by the cyclic BA dimer and trimer. Similarly, the cyclic BS dimer produces PBS of higher molecular weight than that obtained from the cyclic BS trimer. [source] Light-driven Hydrogen Production by a Hybrid Complex of a [NiFe]-Hydrogenase and the Cyanobacterial Photosystem IPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2006Masaki Ihara ABSTRACT In order to generate renewable and clean fuels, increasing efforts are focused on the exploitation of photosynthetic microorganisms for the production of molecular hydrogen from water and light. In this study we engineered a ,hard-wired' protein complex consisting of a hydrogenase and photosystem I (hydrogenase-PSI complex) as a direct light-to-hydrogen conversion system. The key component was an artificial fusion protein composed of the membrane-bound [NiFe] hydrogenase from the ,-proteobacterium Ralstonia eutropha H16 and the peripheral PSI subunit PsaE of the cyanobacterium Thermosy-nechococcus elongatus. The resulting hydrogenase-PsaE fusion protein associated with PsaE-free PSI spontaneously, thereby forming a hydrogenase-PSI complex as confirmed by sucrosegradient ultracentrifuge and immunoblot analysis. The hydrogenase-PSI complex displayed light-driven hydrogen production at a rate of 0.58 ,mol H2· mg chlorophyll,1· h,1. The complex maintained its accessibility to the native electron acceptor ferredoxin. This study provides the first example of a light-driven enzymatic reaction by an artificial complex between a redox enzyme and photosystem I and represents an important step on the way to design a photosynthetic organism that efficiently converts solar energy and water into hydrogen. [source] Capacitive electrolyte,insulator,semiconductor structures functionalised with a polyelectrolyte/enzyme multilayer: New strategy for enhanced field-effect biosensingPHYSICA STATUS SOLIDI (A) APPLICATIONS AND MATERIALS SCIENCE, Issue 4 2010Maryam H. Abouzar Abstract A novel strategy for enhanced field-effect biosensing using capacitive electrolyte,insulator,semiconductor (EIS) structures functionalised with pH-responsive weak polyelectrolyte/enzyme or dendrimer/enzyme multilayers is presented. The feasibility of the proposed approach is exemplarily demonstrated by realising a penicillin biosensor based on a capacitive p-Si,SiO2 EIS structure functionalised with a poly(allylamine hydrochloride) (PAH)/penicillinase and a poly(amidoamine) dendrimer/penicillinase multilayer. The developed sensors response to changes in both the local pH value near the gate surface and the charge of macromolecules induced via enzymatic reaction, resulting in a higher sensitivity. For comparison, an EIS penicillin biosensor with adsorptively immobilised penicillinase has been also studied. The highest penicillin sensitivity of 100,mV/dec has been observed for the EIS sensor functionalised with the PAH/penicillinase multilayer. The lower and upper detection limit was around 20,µM and 10,mM, respectively. In addition, an incorporation of enzymes in a multilayer prepared by layer-by-layer technique provides a larger amount of immobilised enzymes per sensor area, reduces enzyme leaching effects and thus, enhances the biosensor lifetime (the loss of penicillin sensitivity after 2,months was 10,12%). [source] Multistep filling of porous silicon with conductive polymer by electropolymerizationPHYSICA STATUS SOLIDI (A) APPLICATIONS AND MATERIALS SCIENCE, Issue 6 2009Kazuhiro Fukami Abstract The filling of porous silicon with polypyrrole by electropolymerization was investigated. The filling with polypyrrole proceeded preferentially along the porous silicon wall, leading to the formation of tubular structures. By repeating the porosification, the pore filling and the additional porosification, through-tubes of polypyrrole were formed in macropores. The technique to form through-tube was also applied to medium-sized pores. A double layer with polypyrrole was produced by the repetition of porosification and pore filling twice. The immobilization of glucose oxidase was performed by electropolymerization in an aqueous solution containing glucose oxidase and pyrrole. Glucose oxidase was immobilized physically in the polypyrrole film. In the double layer, the sensitivity of glucose oxidase was measured by electrochemical oxidation of hydrogen peroxide, which was produced by the enzymatic reaction of glucose oxidase to gluconolactone. When glucose oxidase was immobilized in the upper layer, glucose was detected sensitively. On the other hand, when glucose oxidase was immobilized in the lower layer, the sensing current showed a slow and a low response. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Comparative Biochemistry of Eumelanogenesis and the Protective Roles of Phenoloxidase and Melanin in InsectsPIGMENT CELL & MELANOMA RESEARCH, Issue 1 2002Manickam Sugumaran The phenolic biopolymer eumelanin is an important skin pigment found throughout the animal kingdom. The enzyme, tyrosinase, initiates melanogenesis in mammals. The biogenesis is assisted by a number of mammalian protein factors including dopachrome tautomerase and 5,6-dihydroxyindole-2-carboxylate oxidase. Invertebrates, such as insects, employ phenoloxidase and dopachrome (decarboxylating) isomerase for melanin biosynthesis. Recently generated molecular biological and biochemical data indicate that tyrosinase and phenoloxidase are distinctly different enzymes in spite of possessing both monophenol monooxygenase activity as well as o -diphenoloxidase activity. Similarly, insect dopachrome isomerase also differs significantly from its mammalian counterpart in several of its properties including the nature of the enzymatic reaction. In addition, there are considerable differences in the eumelanogenic pathways of these two animal groups that include the utility of substrates, use of dihydroxyindoles and the nature of eumelanin pigment. Thus, the biochemistry and molecular biology of melanogenesis in mammals and insects are significantly different. The advantages of generating different eumelanin pigments and intermediates by the insects are discussed. [source] X-ray diffraction structure of a cell-wall invertase from Arabidopsis thalianaACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2006Maureen Verhaest Cell-wall invertases play crucial roles during plant development. They hydrolyse sucrose into its fructose and glucose subunits by cleavage of the ,1,,2 glycosidic bond. Here, the structure of the Arabidopsis thaliana cell-wall invertase 1 (AtcwINV1; gene accession code At3g13790) is described at a resolution of 2.15,Ĺ. The structure comprises an N-terminal fivefold ,-propeller domain followed by a C-terminal domain formed by two ,-sheets. The active site is positioned in the fivefold ,-propeller domain, containing the nucleophile Asp23 and the acid/base catalyst Glu203 of the double-displacement enzymatic reaction. The function of the C-terminal domain remains unknown. Unlike in other GH 32 family enzyme structures known to date, in AtcwINV1 the cleft formed between both domains is blocked by Asn299-linked carbohydrates. A preliminary site-directed mutagenesis experiment (Asn299Asp) removed the glycosyl chain but did not alter the activity profile of the enzyme. [source] Structural studies of glucose-6-phosphate and NADP+ binding to human glucose-6-phosphate dehydrogenaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2005Sheila Gover Human glucose-6-phosphate dehydrogenase (G6PD) is NADP+ -dependent and catalyses the first and rate-limiting step of the pentose phosphate shunt. Binary complexes of the human deletion mutant, ,G6PD, with glucose-6-phosphate and NADP+ have been crystallized and their structures solved to 2.9 and 2.5,Ĺ, respectively. The structures are compared with the previously determined structure of the Canton variant of human G6PD (G6PDCanton) in which NADP+ is bound at the structural site. Substrate binding in ,G6PD is shown to be very similar to that described previously in Leuconostoc mesenteroides G6PD. NADP+ binding at the coenzyme site is seen to be comparable to NADP+ binding in L. mesenteroides G6PD, although some differences arise as a result of sequence changes. The tetramer interface varies slightly among the human G6PD complexes, suggesting flexibility in the predominantly hydrophilic dimer,dimer interactions. In both complexes, Pro172 of the conserved peptide EKPxG is in the cis conformation; it is seen to be crucial for close approach of the substrate and coenzyme during the enzymatic reaction. Structural NADP+ binds in a very similar way in the ,G6PD,NADP+ complex and in G6PDCanton, while in the substrate complex the structural NADP+ has low occupancy and the C-terminal tail at the structural NADP+ site is disordered. The implications of possible interaction between the structural NADP+ and G6P are considered. [source] Structure and implications for the thermal stability of phosphopantetheine adenylyltransferase from Thermus thermophilusACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2004Phosphopantetheine adenylyltransferase Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyzes the rate-limiting step in coenzyme A (CoA) biosynthesis by transferring an adenylyl group from ATP to 4,-phosphopantetheine (Ppant), yielding 3,-dephospho-CoA (dPCoA). The crystal structure of PPAT from Thermus thermophilus HB8 (Tt PPAT) complexed with Ppant has been determined by the molecular-replacement method at 1.5,Ĺ resolution. The overall fold of the enzyme is almost the same as that of Escherichia coli PPAT, a hexamer having point group 32. The asymmetric unit of Tt PPAT contains a monomer and the crystallographic triad and dyad coincide with the threefold and twofold axes of the hexamer, respectively. Most of the important atoms surrounding the active site in E. coli PPAT are conserved in Tt PPAT, indicating similarities in their substrate binding and enzymatic reaction. The notable difference between E. coli PPAT and Tt PPAT is the simultaneous substrate recognition by all six subunits of Tt PPAT compared with substrate recognition by only three subunits in E. coli PPAT. Comparative analysis also revealed that the higher stability of Tt PPAT arises from stabilization of each subunit by hydrophobic effects, hydrogen bonds and entropic effects. [source] Crystallization and preliminary X-ray diffraction analysis of various enzyme,substrate complexes of isopropylmalate dehydrogenase from Thermus thermophilusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Angelo Merli The Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt -IPMDH) enzyme catalyses the penultimate step of the leucine-biosynthesis pathway. It converts (2R,3S)-3-isopropylmalate to (2S)-2-isopropyl-3-oxosuccinate in the presence of divalent Mg2+ or Mn2+ and with the help of NAD+. In order to elucidate the detailed structural and functional mode of the enzymatic reaction, crystals of Tt -IPMDH were grown in the presence of various combinations of substrate and/or cofactors. Here, the crystallization, data collection and preliminary crystallographic analyses of six such complexes are reported. [source] | |||||||||||||||||||||||||||||||||||||||||||