Showing papers on "Substrate (chemistry) published in 2007"

Substrate pretreatment: the key to effective enzymatic hydrolysis of lignocellulosics?

Abstract: Although the structure and function of cellulase systems continue to be the subject of intense research, it is widely acknowledged that the rate and extent of the cellulolytic hydrolysis of lignocellulosic substrates is influenced not only by the effectiveness of the enzymes but also by the chemical, physical and morphological characteristics of the heterogeneous lignocellulosic substrates. Although strategies such as site-directed mutagenesis or directed evolution have been successfully employed to improve cellulase properties such as binding affinity, catalytic activity and thermostability, complementary goals that we and other groups have studied have been the determination of which substrate characteristics are responsible for limiting hydrolysis and the development of pretreatment methods that maximize substrate accessibility to the cellulase complex. Over the last few years we have looked at the various lignocellulosic substrate characteristics at the fiber, fibril and microfibril level that have been modified during pretreatment and subsequent hydrolysis. The initial characteristics of the woody biomass and the effect of subsequent pretreatment play a significant role on the development of substrate properties, which in turn govern the efficacy of enzymatic hydrolysis. Focusing particularly on steam pretreatment, this review examines the influence that pretreatment conditions have on substrate characteristics such as lignin and hemicellulose content, crystallinity, degree of polymerization and specific surface, and the resulting implications for effective hydrolysis by cellulases.

Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter

Abstract: Secondary transporters are integral membrane proteins that catalyse the movement of substrate molecules across the lipid bilayer by coupling substrate transport to one or more ion gradients, thereby providing a mechanism for the concentrative uptake of substrates. Here we describe crystallographic and thermodynamic studies of Glt(Ph), a sodium (Na+)-coupled aspartate transporter, defining sites for aspartate, two sodium ions and d,l-threo-beta-benzyloxyaspartate, an inhibitor. We further show that helical hairpin 2 is the extracellular gate that controls access of substrate and ions to the internal binding sites. At least two sodium ions bind in close proximity to the substrate and these sodium-binding sites, together with the sodium-binding sites in another sodium-coupled transporter, LeuT, define an unwound alpha-helix as the central element of the ion-binding motif, a motif well suited to the binding of sodium and to participation in conformational changes that accompany ion binding and unbinding during the transport cycle.

Structural basis for substrate loading in bacterial RNA polymerase

Abstract: The mechanism of substrate loading in multisubunit RNA polymerase is crucial for understanding the general principles of transcription yet remains hotly debated Here we report the 30-A resolution structures of the Thermus thermophilus elongation complex (EC) with a non-hydrolysable substrate analogue, adenosine-5′-[(α,β)-methyleno]-triphosphate (AMPcPP), and with AMPcPP plus the inhibitor streptolydigin In the EC/AMPcPP structure, the substrate binds to the active (‘insertion’) site closed through refolding of the trigger loop (TL) into two α-helices In contrast, the EC/AMPcPP/streptolydigin structure reveals an inactive (‘preinsertion’) substrate configuration stabilized by streptolydigin-induced displacement of the TL Our structural and biochemical data suggest that refolding of the TL is vital for catalysis and have three main implications First, despite differences in the details, the two-step preinsertion/insertion mechanism of substrate loading may be universal for all RNA polymerases Second, freezing of the preinsertion state is an attractive target for the design of novel antibiotics Last, the TL emerges as a prominent target whose refolding can be modulated by regulatory factors Two complementary papers this week focus on the structure and function of bacterial RNA polymerase In the first, the enzyme is bound to the DNA template and RNA product, to give a close-up of the transcription elongation complex The structure reveals details of the DNA-to-RNA transcription process, vital to all living cells In the second paper, the RNA polymerase elongation complex is pictured bound to various substrate analogues and to an antibiotic, revealing the mechanism of substrate loading and antibiotic inhibition Comparisons between the structures of human and bacteria RNA polymerase should aid in drug design: RNA polymerase is a target of antibiotics, including rifamycin and its derivatives Crystal structures of bacterial RNA polymerase elongation complexes bound to NTP substrate analogues with an antibiotic, revealing the mechanism of substrate loading and antibiotic inhibition

Probing the chemistry of thioredoxin catalysis with force

Abstract: Thioredoxins are enzymes that catalyse disulphide bond reduction in all living organisms. Although catalysis is thought to proceed through a substitution nucleophilic bimolecular (S(N)2) reaction, the role of the enzyme in modulating this chemical reaction is unknown. Here, using single-molecule force-clamp spectroscopy, we investigate the catalytic mechanism of Escherichia coli thioredoxin (Trx). We applied mechanical force in the range of 25-600 pN to a disulphide bond substrate and monitored the reduction of these bonds by individual enzymes. We detected two alternative forms of the catalytic reaction, the first requiring a reorientation of the substrate disulphide bond, causing a shortening of the substrate polypeptide by 0.79 +/- 0.09 A (+/- s.e.m.), and the second elongating the substrate disulphide bond by 0.17 +/- 0.02 A (+/- s.e.m.). These results support the view that the Trx active site regulates the geometry of the participating sulphur atoms with sub-angstrom precision to achieve efficient catalysis. Our results indicate that substrate conformational changes may be important in the regulation of Trx activity under conditions of oxidative stress and mechanical injury, such as those experienced in cardiovascular disease. Furthermore, single-molecule atomic force microscopy techniques, as shown here, can probe dynamic rearrangements within an enzyme's active site during catalysis that cannot be resolved with any other current structural biological technique.

Electron and carbon balances in microbial fuel cells reveal temporary bacterial storage behavior during electricity generation.

Abstract: Microbial fuel cells (MFCs) are emerging as a novel technology with a great potential to reduce the costs of wastewater treatment. Their most studied application is organic carbon removal. One of the parameters commonly used to quantify the performance of these cells is the Coulombic efficiency, i.e., the electron recovery as electricity from the removed substrate. However, the "inefficiencies" of the process have never been fully identified. This study presents a method that uses the combination of electrochemical monitoring, chemical analysis, and a titration and off-gas analysis (TOGA) sensor to identify and quantify the sources of electron loss. The method was used successfully to close electron, carbon, and proton balances in acetate and glucose fed microbial fuel cells. The method revealed that in the case that a substrate is loaded as pulses carbon is stored inside the cells during initial high substrate conditions and consumed during starvation, with up to 57% of the current being generated after depletion of the external carbon source. Nile blue staining of biomass samples revealed lipophilic inclusions during high substrate conditions, thus confirming the storage of polymeric material in the bacterial cells. The method also allows for indirect measurement of growth yields, which ranged from 0 to 0.54 g biomass-C formed per g substrate-C used, depending on the type of substrate and the external resistance of the circuit.

Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8-substrate complex.

Abstract: Histone deacetylases (HDACs)—an enzyme family that deacetylates histones and non-histone proteins—are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 Å resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor–HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.

Surface Modification of Stretched TiO2 Nanotubes for Solid-State Dye-Sensitized Solar Cells

Abstract: Straight-stranded anatase TiO2 nanotubes were produced by anodic oxidation on a pure titanium substrate in an aqueous solution containing a 0.45 wt % NaF electrolyte (pH 4.3 fixed). The average length of the TiO2 nanotubes was approximately 3 μm, which had an effect on the level of dye adsorption in the dye-sensitized solar cells. The anodic TiO2 nanotubes were applied as a working electrode in a solid-state dye-sensitized solar cell. An approximately 1 nm ZnO shell was coated on the TiO2 nanotube to improve the open-circuit voltage (Voc) and conversion efficiency of the solar cell, and to retard any back reaction. Although the Voc and short-circuit current (Jsc) of the cell were improved, there was a low fill factor as a result of the formation of a thick TiO2 barrier layer in the anodic TiO2/Ti substrate. A parameter on the degradation of fill factor (37%) is related to the formation of a thick TiO2 barrier layer in the anodic TiO2/Ti substrate interface. A hydrogen peroxide treatment was performed in a...

Effects of Substrate Loading on Enzymatic Hydrolysis and Viscosity of Pretreated Barley Straw

Abstract: In this study, the applicability of a “fed-batch” strategy, that is, sequential loading of substrate or substrate plus enzymes during enzymatic hydrolysis was evaluated for hydrolysis of steam-pretreated barley straw. The specific aims were to achieve hydrolysis of high substrate levels, low viscosity during hydrolysis, and high glucose concentrations. An enzyme system comprising Celluclast and Novozyme 188, a commercial cellulase product derived from Trichoderma reesei and a β-glucosidase derived from Aspergillus niger, respectively, was used for the enzymatic hydrolysis. The highest final glucose concentration, 78 g/l, after 72 h of reaction, was obtained with an initial, full substrate loading of 15% dry matter weight/weight (w/w DM). Conversely, the glucose yields, in grams per gram of DM, were highest at lower substrate concentrations, with the highest glucose yield being 0.53 g/g DM for the reaction with a substrate loading of 5% w/w DM after 72 h. The reactions subjected to gradual loading of substrate or substrate plus enzymes to increase the substrate levels from 5 to 15% w/w DM, consistently provided lower concentrations of glucose after 72 h of reaction; however, the initial rates of conversion varied in the different reactions. Rapid cellulose degradation was accompanied by rapid decreases in viscosity before addition of extra substrate, but when extra substrate or substrate plus enzymes were added, the viscosities of the slurries increased and the hydrolytic efficiencies decreased temporarily.

Structure of fungal fatty acid synthase and implications for iterative substrate shuttling.

Abstract: We report crystal structures of the 2.6-megadalton α6β6 heterododecameric fatty acid synthase from Thermomyces lanuginosus at 3.1 angstrom resolution. The α and β polypeptide chains form the six catalytic domains required for fatty acid synthesis and numerous expansion segments responsible for extensive intersubunit connections. Detailed views of all active sites provide insights into substrate specificities and catalytic mechanisms and reveal their unique characteristics, which are due to the integration into the multienzyme. The mode of acyl carrier protein attachment in the reaction chamber, together with the spatial distribution of active sites, suggests that iterative substrate shuttling is achieved by a relatively restricted circular motion of the carrier domain in the multifunctional enzyme.

Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase.

Abstract: Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-Å resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate l-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the l-stereospecificity. A second, possibly allosteric, l-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety of l-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.

Metal organic chemical vapor deposition equipment

Abstract: Metal organic chemical vapor deposition equipment is metal organic chemical vapor deposition equipment for forming a film on a substrate by using a reactant gas, and includes a susceptor heating the substrate and having a holding surface for holding the substrate, and a flow channel for introducing the reactant gas to the substrate. The susceptor is rotatable with the holding surface kept facing an inner portion of the flow channel, and a height of the flow channel along a flow direction of the reactant gas is kept constant from a position to a position, and is monotonically decreased from the position to the downstream side. It is thereby possible to improve film formation efficiency while allowing the formed film to have a uniform thickness.

Thermodynamic parameters of enzymes in grassland soils from Galicia, NW Spain.

Abstract: The thermodynamic parameters of the enzymes catalase, dehydrogenase, casein-protease, α -N-benzoyl- l -argininamide (BAA)-protease, urease, Carboxymethyl (CM)-cellulase, invertase, β -glucosidase and arylsulphatase, were investigated in grassland soils from a European temperate-humid zone (Galicia, NW Spain). The effect of temperature on enzyme activity was determined at 5, 18, 27, 37, 57 and 70 °C. The temperature-dependence of the rate of substrate hydrolysis varied depending on the enzyme and soil. In general, the soil containing the least amount of organic matter (OM) showed the lowest enzyme activity for all temperatures and enzymes, whereas soils with similar OM contents showed similar levels of activity for the entire temperature range. Temperature had a noteworthy effect on the activity of oxidoreductases. Product formation in the reaction catalyzed by dehydrogenase increased with increasing temperature until 70 °C, which was attributed to chemical reduction of iodonitrotetrazolium violet (INT) at high temperatures. Catalase activity was not affected above 37 °C, which may be explained either by non-enzymatic decomposition of hydrogen peroxide or by the fact that catalase has reached kinetic perfection, and is therefore not saturated with substrate. The Arrhenius equation was used to determine the activation energy ( E a ) and the temperature coefficient ( Q 10 ) for all enzymes. The values of E a and Q 10 for each enzyme differed among soils, although in general the differences were small, especially for those enzymes that act on substrates of low molecular weight. In terms of the values of E a and Q 10 and the differences established among soils, the results obtained for those enzymes that act on substrates of high molecular weight differed most from those corresponding to the other enzymes. Thus the lowest E a and Q 10 values corresponded to BAA-protease, and the highest values to CM-cellulase and casein-protease. Except for catalase in one of the soils, the values of E a and Q 10 for the oxidoreductases were similar to those of most of the hydrolases. In general, the effect of temperature appeared to be more dependent on the type of enzyme than on the characteristics of the soil.

Recycling cellulases during the hydrolysis of steam exploded and ethanol pretreated Lodgepole pine.

Abstract: Recycling of cellulases is one way of reducing the high cost of enzymes during the bioconversion process. The effects of surfactant addition on enzymatic hydrolysis and the potential recycling of cellulases were studied during the hydrolysis of steam exploded Lodgepole pine (SELP) and ethanol pretreated Lodgepole pine (EPLP). Three cellulase preparations (Celluclast, Spezyme CP, and MSUBC) were evaluated to determine their hydrolysis efficiencies over multiple rounds of recycling. The surfactant, Tween 80, significantly increased the yield from 63% to 86% during the hydrolysis of the SELP substrate. The addition of surfactant to the hydrolysis of the EPLP substrate increased the free enzymes in the supernatant from 71% of the initial protein to 96%. Based on the Langmuir adsorption constants, cellulases (Celluclast and Spezyme CP) from Trichoderma reesei showed a higher affinity (3.48 mL/mg and 3.17 mL/mg) for the EPLP substrate than did the Penicillium enzyme (0.62 mg/mg). The Trichoderma reesei enzyme was used in four successive rounds of enzyme recycling using surfactant addition and readsorption onto fresh substrates during the hydrolysis of EPLP. In contrast, the Penicillium-derived enzyme preparation (MSUBC) could only be recycled once. When the same recycling strategy was carried out using the SELP substrate, the hydrolysis yield declined during each enzyme recycling round. These results suggested that the higher lignin content of the SELP substrate, and the low affinity of cellulases for the SELP substrate limited enzyme recycling by readsorption onto fresh substrates.

Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization

Abstract: Polyacrylonitrile (PAN) nanofibers could be fabricated by electrospinning with fiber diameter in the range of 150–300 nm, providing huge surface area for enzyme immobilization and catalytic reactions. Lipase from Candida rugosa was covalently immobilized onto PAN nanofibers by amidination reaction. Aggregates of enzyme molecules were found on nanofiber surface from field emission scanning electron microscopy and covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After 5 min activation and 60 min reaction with enzyme-containing solution, the protein loading efficiency was quantitative and the activity retention of the immobilized lipase was 81% that of free enzyme. The mechanical strength of the NFM improved after lipase immobilization where tensile stress at break and Young's modulus were almost doubled. The immobilized lipase retained >95% of its initial activity when stored in buffer at 30 °C for 20 days, whereas free lipase lost 80% of its initial activity. The immobilized lipase still retained 70% of its specific activity after 10 repeated batches of reaction. This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters.

Deposition by adsorption under an electrical field

Abstract: A method for depositing a material by adsorption onto a substrate, includes a step of exposing the substrate to a precursor molecule in the gaseous phase These precursor molecules present a non-zero dipole moment An electrical field is applied during the substrate exposing step to cause a reactive branch of the precursor molecules to adsorb into the surface of the substrate in a manner such that the precursor molecules have essentially a same orientation Next, the substrate is exposed to reagent molecules in the gaseous phase which react with the adsorbed precursor molecules so that organic branches of the adsorbed precursor molecules other than the reactive organic branch are replaced by elements of the reagent molecules This process results in the formation of a monoatomic layer

Protein arginine methyltransferase 1: positively charged residues in substrate peptides distal to the site of methylation are important for substrate binding and catalysis.

Abstract: Protein arginine methyltransferases (PRMTs) are a group of eukaryotic enzymes that catalyze the methylation of Arg residues in a variety of proteins (e.g., histones H3 and H4), and their activities influence a wide range of cellular processes, including cell growth, RNA splicing, differentiation, and transcriptional regulation. Dysregulation of these enzymes has been linked to heart disease and cancer, suggesting this enzyme family as a novel therapeutic target. To aid the development of PRMT inhibitors, we characterized the substrate specificity of both the rat and human PRMT1 orthologues using histone based peptide substrates. N- and C-terminal truncations to identify a minimal peptide substrate indicate that long-range interactions between enzyme and substrate are important for high rates of substrate capture. The importance of these long-range interactions to substrate capture were confirmed by "mutagenesis" experiments on a minimal peptide substrate. Inhibition studies on S-adenosyl-homocysteine, thioadenosine, methylthioadenosine, homocysteine, and sinefungin suggest that potent and selective bisubstrate analogue inhibitor(s) for PRMT1 can be developed by linking a histone based peptide substrate to homocysteine or sinefungin. Additionally, we present evidence that PRMT1 utilizes a partially processive mechanism to dimethylate its substrates.

Stochastic inhibitor release and binding from single-enzyme molecules

Abstract: Inhibition kinetics of single-beta-galactosidase molecules with the slow-binding inhibitor d-galactal have been characterized by segregating individual enzyme molecules in an array of 50,000 ultra small reaction containers and observing substrate turnover changes with fluorescence microscopy. Inhibited and active states of beta-galactosidase could be clearly distinguished, and the large array size provided very good statistics. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics. Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, which are accompanied by conformational changes of the enzyme's catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk-reaction kinetics.

Articles having localized molecules disposed thereon and methods of producing same

Abstract: Methods of producing substrates having selected active chemical regions by employing elements of the substrates in assisting the localization of active chemical groups in desired regions of the substrate. The methods may include optical, chemical and/or mechanical processes for the deposition, removal, activation and/or deactivation of chemical groups in selected regions of the substrate to provide selective active regions of the substrate.

Heat treatment method, heat treatment apparatus and substrate processing apparatus

Abstract: Disclosed is a heat treatment unit 4 serving as a heat treatment apparatus, which includes a chamber 42 for containing a wafer W on which a low dielectric constant interlayer insulating film is formed, a formic acid supply device 44 for supplying gaseous formic acid into the chamber 42 , and a heater 43 for heating the wafer W in the chamber 42 which is supplied with formic acid by the formic acid supply device 44.

Surface plasmon resonance based fiber-optic sensor for the detection of pesticide

Abstract: Fabrication and characterization of a surface plasmon resonance based fiber-optic sensor for the detection of organophosphate pesticide, chlorphyrifos, have been reported. The probe is prepared by immobilizing acetylcholinesterase (AChE) enzyme on the silver coated core of a plastic-cladded silica (PCS) fiber. The detection is based on the principle of competitive binding of the pesticide (acting as inhibitor) for the substrate (acetlythiocholine iodide) to the enzyme AChE. The spectral interrogation method has been used to characterize the sensor. It has been observed that the SPR wavelength decreases with the increase in the concentration of the pesticide for the fixed concentration of substrate in the fluid around the probe. Further, the effect of the concentration of the substrate in the fluid on the resonance wavelength has been studied. The sensitivity, detection accuracy, reproducibility and stability of the sensor have been also determined. It has been found that the sensitivity decreases with the increase in the concentration of the pesticide while reverse is the case for detection accuracy.

Kinetic modeling of the oxidation of p-hydroxybenzoic acid by Fenton's reagent: implications of the role of quinones in the redox cycling of iron.

Abstract: As Fenton (and Fenton-like) chemistry is increasingly implicated in a variety of areas and applications, an understanding of the mechanism and rates governing the system becomes relevant for a growing number of disciplines and purposes. In this work a kinetic model capable of describing species concentrations measured experimentally during the Fenton-mediated oxidation of p-hydroxybenzoic acid (pHBA) is presented and discussed. Experiments were conducted in the dark at low pH using reagent and substrate concentrations ranging from 100 μM to 2 mM. Analysis of the experimental and modeling results reveals that redox reactions between Fe and quinone or quinone-like compounds are essential for the model to qualitatively predict species concentration profiles observed in the laboratory. The quinone and quinone-like compounds generated as byproducts during the oxidation of pHBA act as reducing agents toward Fe(III), thereby assisting the redox cycling of Fe and increasing degradation of the target substrate. Th...

Effect of substrate-induced strains on the spontaneous polarization of epitaxial BiFeO3 thin films

Abstract: A single-domain thermodynamic theory is employed to predict the spontaneous polarizations of (001)c, (101)c, and (111)c oriented epitaxial BiFeO3 thin films grown on dissimilar substrates. The effects of various substrate-induced strains on the spontaneous polarization were studied. The dependences of the spontaneous polarization on film orientations and the types of substrate-induced strains were analyzed.