Showing papers on "Substrate (chemistry) published in 2012"

Understanding and controlling the substrate effect on graphene electron-transfer chemistry via reactivity imprint lithography

Abstract: The chemical modification of graphene is important for its use in many applications. Now it is shown that the reactivity of graphene towards covalent modification varies widely depending on its underlying support substrate, and that the substrate can be patterned to induce spatial control of chemical reactions in graphene.

Porous Pt-Ni-P Composite Nanotube Arrays: Highly Electroactive and Durable Catalysts for Methanol Electrooxidation

Abstract: Porous Pt-Ni-P composite nanotube arrays (NTAs) on a conductive substrate in good solid contact are successfully synthesized via template-assisted electrodeposition and show high electrochemical activity and long-term stability for methanol electrooxidation. Hollow nanotubular structures, porous nanostructures, and synergistic electronic effects of various elements contribute to the high electrocatalytic performance of porous Pt-Ni-P composite NTA electrocatalysts.

Design of biomimetic catalysts by molecular imprinting in synthetic polymers: the role of transition state stabilization.

Abstract: The impressive efficiency and selectivity of biological catalysts has engendered a long-standing effort to understand the details of enzyme action. It is widely accepted that enzymes accelerate reactions through their steric and electronic complementarity to the reactants in the rate-determining transition states. Thus, tight binding to the transition state of a reactant (rather than to the corresponding substrate) lowers the activation energy of the reaction, providing strong catalytic activity. Debates concerning the fundamentals of enzyme catalysis continue, however, and non-natural enzyme mimics offer important additional insight in this area. Molecular structures that mimic enzymes through the design of a predetermined binding site that stabilizes the transition state of a desired reaction are invaluable in this regard. Catalytic antibodies, which can be quite active when raised against stable transition state analogues of the corresponding reaction, represent particularly successful examples. Recent...

Dehydrogenase Activity in the Soil Environment

Abstract: The most common laboratory procedure used for DHA determination is the method developed by Casida et al. (1964). According this method, specific dyes such as the triphenyltetrazolium chloride (TTC), that can specify the flow of electrons are useful indicators of electron transport system (ETS) activity. By the reduction of colorless, water soluble substrate (TTC) by dehydrogenases present in the soil environment, an insoluble product with red color (triphenylformazan-TPF) is formed. TPF can be easily quantified calorimetrically at the range of visible light (485 nm). This test however, reflected positive answer only at neutral range of pH and in presence of calcium carbonate for buffering soil system. Briefly, if the red colors of soil samples prepared for spectrophotometer analyses are more intensive, the measured level of DHA is higher. Consequently, soil samples without red colors or those with light red colors are characterized by lower DHA values.

Assembly of graphene oxide-enzyme conjugates through hydrophobic interaction.

Abstract: Biochemical and biomedical applications of graphene oxide (GO) critically rely on the interaction of biomolecules with it. It has been previously reported that the biological activity of the GO-enzyme conjugate decreases due to electrostatic interaction between the enzymes and GO. Herein, the immobilization of horseradish peroxidase (HRP) and oxalate oxidase (OxOx) on chemically reduced graphene oxide (CRGO) are reported. The enzymes can be adsorbed onto CRGO directly with a tenfold higher enzyme loading than that on GO, and maximum enzyme loadings reach 1.3 and 12 mg mg(-1) for HRP and OxOx, respectively. Significantly, the more CRGO is reduced, the higher the enzyme loading. The CRGO-HRP conjugates also exhibit higher enzyme activity and stability than GO-HRP. Excellent properties of the CRGO-enzyme conjugates are attributed to hydrophobic interaction between the enzymes and the CRGO. The hydrophobic interaction mode of the CRGO-enzyme conjugates can be applied to other hydrophobic proteins, and thus could dramatically improve the performance of immobilized proteins. The results indicate that CRGO is a potential substrate for efficient enzyme immobilization, and is an ideal candidate as a macromolecule carrier and biosensor.

NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions.

Abstract: Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu2+ (Kd = 55 nM; from isothermal titration calorimetry) and higher preference for Cu1+ (Kd ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu2+ of 275 mV using a thermodynamic cycle). Strong binding of Cu1+ was also reflected in a reduction in the pKa values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a 2H/1H exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates.

Nanoscale Graphene Oxide (nGO) as Artificial Receptors: Implications for Biomolecular Interactions and Sensing

Abstract: The role of conventional graphene-oxide in biosensing has been limited to that of a quenching substrate or signal transducer due to size inconsistencies and poor supramolecular response. We overcame these issues by using nanoscale GOs (nGO) as artificial receptors. Unlike conventional GO, nGOs are sheets with near uniform lateral dimension of 20 nm. Due to its nanoscale architecture, its supramolecular response was enhanced, with demonstrated improvements in biomacromolecular affinities. This rendered their surface capable of detecting unknown proteins with cognizance not seen with conventional GOs. Different proteins at 100 and 10 nM concentrations revealed consistent patterns that are quantitatively differentiable by linear discriminant analysis. Identification of 48 unknowns in both concentrations demonstrated a >95% success rate. The 10 nM detection represents a 10-fold improvement over analogous arrays. This demonstrates for the first time that the supramolecular chemistry of GO is highly size depend...

Au@Pt core/shell nanorods with peroxidase- and ascorbate oxidase-like activities for improved detection of glucose

Abstract: Au nanorods @ Pt nanodots core/shell nanostructures, prepared by the Au nanorods (NRs)-mediated growth, exhibit dual functional enzyme-like (peroxidase and oxidase-like) activities. From the viewpoint of enzyme mimics, the relationship between apparent enzyme kinetic parameters and nanomaterials structure is investigated in order to rationally design the catalytic activity. Using peroxidase-like properties of the Au@Pt NRs, the determination of hydrogen peroxide (H2O2) was demonstrated with a limit of detection (LOD) of 4.5 × 10−5 M and a linear range of 4.5 × 10−5–1 × 10−3 M using o-phenylenediamine (OPD) as chromogenic substrate. Furthermore, in combination with highly specific reactions provided by natural enzymes, selective detections of glucose and lipophilic cholesterol were demonstrated with similar LODs and linear ranges. Additionally, owing to the specific oxidase-like activity of the Au@Pt NRs (ascorbate oxidase), interference of ascorbic acid in the detection of glucose could be eliminated. In conclusion, considering the flexibility in the design of nanomatererials, there is a lot of space to improve their activity and explore their potential applications, especially in relatively harsh conditions.

Facilitated substrate channeling in a self-assembled trifunctional enzyme complex.

Abstract: Most cascade enzymes in metabolic pathways are spatially held together by noncovalent protein–protein interactions. The formation of a cascade enzyme complex often allows the product of one enzyme to be transferred to an adjacent enzyme where it acts as the substrate, thereby resulting in an enhanced reaction rate, because reaching equilibrium in the cytoplasm is not required; this mechanism is called substrate channeling. In nature, most intracellular enzyme complexes are dynamic so that they may be dissociated or associated, thereby resulting in forestallment of substrate competition among different pathways, regulation of metabolic fluxes, mitigation of metabolite inhibition, and circumvention of unfavorable equilibrium and kinetics. The simplest way to facilitate substrate channeling between cascade enzymes is the construction of fusion proteins, but substrate channeling in fusion proteins might not take place. The assembly of numerous enzymes and/or co-enzymes in vitro is called cascade enzyme biocatalysis and has been proposed for the implementation of complicated bioconversion that microbes and chemical catalysts cannot do, such as hydrogen production from cellulosic materials and water with high yield. Inspired by natural enzyme complexes (e.g., metabolons, which are complexes of sequential enzymes of a metabolic pathway), the construction of static rather than dynamic enzyme complexes could be an important approach to accelerating reaction rates among cascade enzymes and to avoiding the regulation of enzyme–enzyme interactions. For example, Wilner et al. linked glucose oxidase and horseradish peroxidase by DNA scaffolds of different lengths, resulting in reaction rates that were enhanced by 20–30-fold. However, DNA scaffolds may be too costly for scale-up as compared to protein scaffolds. Minteer and co-workers demonstrated that chemical cross-linking of proteins within the mitochondria of Saccharomyces cerevisiae resulted in significant increases of the power output in enzymatic fuel cells. But chemical covalent linking often impairs enzyme activity so that it may not be applied to most intracellular enzymes. Herein we demonstrate a general approach for constructing a static self-assembled enzyme complex by using the highaffinity interaction between cohesin and dockerin modules, which occur in natural extracellular complexed cellulase systems, called cellulosomes. Cohesin domains are part of the natural scaffoldin protein of the cellulosome, which is crucial to the construction of the cellulase complex by binding to enzymes carrying dockerin domains. Bayer et al. proposed to construct designed enzyme complexes by utilizing speciesspecific dockerins and cohesins, which can bind tightly in these complexes at a molar ratio of 1:1. Later, several synthetic mini-cellulosomes containing various extracellular glycoside hydrolases were constructed. However, no one attempted to construct an enzyme complex containing cascade enzymes from a metabolic pathway by using dockerins and cohesins and investigated its potential applications in cascade enzyme biocatalysis. Triosephosphate isomerase (TIM, EC 5.3.1.1), aldolase (ALD, EC 4.1.2.13), and fructose 1,6-bisphosphatase (FBP, EC3.1.3.11) are cascade enzymes in the glycolysis and gluconeogenesis pathways. TIM catalyzes the reversible conversion of glycer-aldehyde-3-phosphate (G3P) to dihydroxy-acetone phosphate (DHAP). ALD catalyzes the reversible aldol condensation of G3P and DHAP to fructose-1,6bisphosphate (F16P). FBP catalyzes the irreversible conversion of F16P to fructose-6-phosphate (F6P; Scheme 1). Previous studies reported that substrate channeling existed in dynamic metabolons of enzymes such as TIM, ALD, or FBP. Three dockerin-free proteins: Thermus thermophilus HB27 TIM (TTC0581) as well as the Thermotoga maritima ALD (TM0273) and FBP (TM1415) were expressed in E. coli and purified to homogeneity by using nickel–nitrilotriacetate (Ni–NTA) resin or a self-cleaving intein. However, a mixture of these three enzymes did not form a putative enzyme complex, as examined by affinity electrophoresis (data not shown). The synthetic static three-enzyme complex was assembled in vitro through a synthetic trifunctional scaffoldin containing a family 3 cellulose-binding module (CBM3) at the N terminus followed by three different types of cohesins from the Clostridium thermocellum ATCC 27405 CipA, Clostridium cellulovorans ATCC 35296 CbpA, and Ruminococcus flavefaciens ScaB (cohesins CTCoh, CCCoh, and RFCoh, [*] Dr. C. You, S. Myung, Y.-H. P. Zhang Biological Systems Engineering Department Virginia Tech, 304 Seitz Hall Blacksburg, VA 24061 (USA) E-mail: ypzhang@vt.edu Homepage: http://www.sugarcar.com

A theoretical model of C- and N-acquiring exoenzyme activities, which balances microbial demands during decomposition

Abstract: We developed an Extracellular EnZYme model (EEZY) of decomposition that produces two separate pools of C- and N-acquiring enzymes, that in turn hydrolyze two qualitatively different substrates, one containing only C (e.g., cellulose) and the other containing both C and N (e.g., chitin or protein). Hence, this model approximates the actions of commonly measured indicator enzymes s-1,4-glucosidase and s-1,4-N-acetylglucosaminidase (or leucine aminopeptidase) as they hydrolyze cellulose and chitin (or protein), respectively. EEZY provides an analytical solution to the allocation of these two enzymes, which in turn release C and N from the two substrates to maximize microbial growth. Model behaviors were both qualitatively and quantitatively consistent with patterns of litter decay generated by other decomposition models. However, EEZY demonstrated greater sensitivity to the C:N of individual substrate pools in addition to responding to factors directly affecting enzyme activity. Output approximated field observations of extracellular enzyme activities from studies of terrestrial soils, aquatic sediments, freshwater biofilm and plankton communities. Although EEZY is largely a theoretical model, simulated C- and N-acquiring enzyme activities approximated a 1:1 ratio, consistent with the bulk of these field observations, only when the N-containing substrate had a C:N ratio similar to commonly occurring substrates (e.g., proteins or chitin). This result supported the emerging view of the stoichiometry of extracellular enzyme activities from an environmental context, which suggests that a relatively narrow range of microbial C:N, carbon use efficiency and soil/sediment organic matter C:N across ecosystems explains the tendency towards this 1:1 ratio of enzyme activities associated with C- and N-acquisition. Sensitivity analyses indicated that simulated extracellular enzyme activity was most responsive to variations in carbon use efficiency of microorganisms, although kinetic characteristics of enzymes also had significant impacts. Thus EEZY provides a quantitative framework in which to interpret mechanisms underlying empirical patterns of extracellular enzyme activity.

Mechanism of the rhodium(III)-catalyzed arylation of imines via C-H bond functionalization: inhibition by substrate.

Abstract: Rh(III)-catalyzed arylation of imines provides a new method for C–C bond formation while simultaneously introducing an α-branched amine as a functional group. This detailed mechanistic study provides insights for the rational future development of this new reaction. Relevant intermediate Rh(III) complexes have been isolated and characterized, and their reactivities in stoichiometric reactions with relevant substrates have been monitored. The reaction was found to be first order in the catalyst resting state and inverse first order in the C–H activation substrate.

Mechanistic Rationalization of Unusual Kinetics in Pd-Catalyzed C–H Olefination

Abstract: Detailed kinetic studies and novel graphical manipulations of reaction progress data in Pd(II)-catalyzed olefinations in the presence of mono-N-protected amino acid ligands reveal anomalous concentration dependences (zero order in o-CF3-phenylacetic acid concentration, zero order in oxygen pressure, and negative orders in both olefin and product concentrations), leaving the catalyst concentration as the sole positive driving force in the reaction. NMR spectroscopic studies support the proposal that rate inhibition by the olefinic substrate and product is caused by formation of reversible off-cycle reservoirs that remove catalyst from the active cycle. NMR studies comparing the interaction between the catalyst and substrate in the presence and absence of the ligand suggest that weak coordination of the ligand to Pd prevents formation of an inactive mixed acetate species. A fuller understanding of these features may lead to the design of more efficient Pd(II) catalysts for this potentially powerful C–H func...

Cost-Effective Filter Materials Coated with Silver Nanoparticles for the Removal of Pathogenic Bacteria in Groundwater

Abstract: The contamination of groundwater sources by pathogenic bacteria poses a public health concern to communities who depend totally on this water supply. In the present study, potentially low-cost filter materials coated with silver nanoparticles were developed for the disinfection of groundwater. Silver nanoparticles were deposited on zeolite, sand, fibreglass, anion and cation resin substrates in various concentrations (0.01 mM, 0.03 mM, 0.05 mM and 0.1 mM) of AgNO3. These substrates were characterised by SEM, EDS, TEM, particle size distribution and XRD analyses. In the first phase, the five substrates coated with various concentrations of AgNO3 were tested against E. coli spiked in synthetic water to determine the best loading concentration that could remove pathogenic bacteria completely from test water. The results revealed that all filters were able to decrease the concentration of E. coli from synthetic water, with a higher removal efficiency achieved at 0.1 mM (21–100%) and a lower efficiency at 0.01 mM (7–50%) concentrations. The cation resin-silver nanoparticle filter was found to remove this pathogenic bacterium at the highest rate, namely 100%. In the second phase, only the best performing concentration of 0.1 mM was considered and tested against presumptive E. coli, S. typhimurium, S. dysenteriae and V. cholerae from groundwater. The results revealed the highest bacteria removal efficiency by the Ag/cation resin filter with complete (100%) removal of all targeted bacteria and the lowest by the Ag/zeolite filter with an 8% to 67% removal rate. This study therefore suggests that the filter system with Ag/cation resin substrate can be used as a potential alternative cost-effective filter for the disinfection of groundwater and production of safe drinking water.

Substrate Selectivity of (tBu-Allyl)Co(CO)3 during Thermal Atomic Layer Deposition of Cobalt

Abstract: Tertbutylallylcobalttricarbonyl (tBu-AllylCo(CO)3) is shown to have strong substrate selectivity during atomic layer deposition of metallic cobalt. The interaction of tBu-AllylCo(CO)3 with SiO2 surfaces, where hydroxyl groups would normally provide more active reaction sites for nucleation during typical ALD processes, is thermodynamically disfavored, resulting in no chemical reaction on the surface at a deposition temperature of 140 °C. On the other hand, the precursor reacts strongly with H-terminated Si surfaces (H/Si), depositing ∼1 ML of cobalt after the first pulse by forming Si–Co metallic bonds. This remarkable substrate selectivity of tBu-AllylCo(CO)3 is due to an ALD nucleation reaction process paralleling a catalytic hydrogenation, which requires a coreactant that acts as a hydrogen donor rather than a source of bare protons. The chemical specificity demonstrated in this work suggests a new paradigm for developing selective ALD precursors. Namely, selectivity can be achieved by tailoring the li...

Enantioselective Brønsted base catalysis with chiral cyclopropenimines.

Abstract: Cyclopropenimines are shown to be a highly effective new class of enantioselective Bronsted base catalysts. A chiral 2,3-bis(dialkylamino)cyclopropenimine catalyzes the rapid Michael reaction of a glycine imine substrate with high levels of enantioselectivity. A preparative scale reaction to deliver 25 g of product is demonstrated, and a trivial large scale synthesis of the optimal catalyst is shown. In addition, the basicity of a 2,3-bis(dialkylamino)cyclopropenimine is measured for the first time and shown to be approximately equivalent to the P1-tBu phosphazene base. An X-ray crystal structure of the protonated catalyst is shown along with a proposed mechanistic and stereochemical rationale.

Synthesis of Oxazoles from Enamides via Phenyliodine Diacetate-Mediated Intramolecular Oxidative Cyclization

Abstract: A group of functionalized oxazoles were synthesized in moderate to good yields from enamides via phenyliodine diacetate (PIDA)-mediated intramolecular cyclization. The main advantageous features of the present method include its broad substrate scope and the heavy-metal-free characteristic of the oxidative carbon–oxygen bond formation process.

Validation of paper-based assay for rapid blood typing.

Abstract: We developed and validated a new paper-based assay for the detection of human blood type. Our method involves spotting a 3 μL blood sample on a paper surface where grouping antibodies have already been introduced. A thin film chromatograph tank was used to chromatographically elute the blood spot with 0.9% NaCl buffer for 10 min by capillary absorption. Agglutinated red blood cells (RBCs) were fixed on the paper substrate, resulting in a high optical density of the spot, with no visual trace in the buffer wicking path. Conversely, nonagglutinated RBCs could easily be eluted by the buffer and had low optical density of the spot and clearly visible trace of RBCs in the buffer wicking path. Different paper substrates had comparable ability to fix agglutinated blood, while a more porous substrate like Kleenex paper had enhanced ability to elute nonagglutinated blood. Using optimized conditions, a rapid assay for detection of blood groups was developed by spotting blood to antibodies absorbed to paper and elut...

Identification of residues defining phospholipid flippase substrate specificity of type IV P-type ATPases

Abstract: Type IV P-type ATPases (P4-ATPases) catalyze translocation of phospholipid across a membrane to establish an asymmetric bilayer structure with phosphatidylserine (PS) and phosphatidylethanolamine (PE) restricted to the cytosolic leaflet. The mechanism for how P4-ATPases recognize and flip phospholipid is unknown, and is described as the “giant substrate problem” because the canonical substrate binding pockets of homologous cation pumps are too small to accommodate a bulky phospholipid. Here, we identify residues that confer differences in substrate specificity between Drs2 and Dnf1, Saccharomyces cerevisiae P4-ATPases that preferentially flip PS and phosphatidylcholine (PC), respectively. Transplanting transmembrane segments 3 and 4 (TM3-4) of Drs2 into Dnf1 alters the substrate preference of Dnf1 from PC to PS. Acquisition of the PS substrate maps to a Tyr618Phe substitution in TM4 of Dnf1, representing the loss of a single hydroxyl group. The reciprocal Phe511Tyr substitution in Drs2 specifically abrogates PS recognition by this flippase causing PS exposure on the outer leaflet of the plasma membrane without disrupting PE asymmetry. TM3 and the adjoining lumenal loop contribute residues important for Dnf1 PC preference, including Phe587. Modeling of residues involved in substrate selection suggests a novel P-type ATPase transport pathway at the protein/lipid interface and a potential solution to the giant substrate problem.

A novel colorimetric determination of reduced glutathione in A549 cells based on Fe3O4 magnetic nanoparticles as peroxidase mimetics.

Abstract: In this paper, a novel and simple colorimetric method for the determination of reduced glutathione (GSH) based on Fe3O4 magnetic nanoparticles (MNPs) as peroxidase mimetics was developed. The Fe3O4 MNPs prepared via a coprecipitation method, which possess intrinsic peroxidase-like activity, were used as a catalyst in the color development reaction of a peroxidase substrate 2,2′-azino-bis(3-thylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) and H2O2. The existence of GSH can consume H2O2 and cause a color change of the reaction system which can be detected by the naked eye. Accordingly, the GSH can be detected by measuring the wastage of H2O2. A good linear relationship was obtained from 3.0 to 30.0 μM for GSH. Good recoveries ranging from 96.7 to 107% were obtained. Furthermore, it was used to detect GSH in A549 cells.