scispace - formally typeset
Search or ask a question

Showing papers on "Substrate (chemistry) published in 2008"


Journal ArticleDOI
TL;DR: Results demonstrated viable approach for utilization of this huge biomass by solid-state fermentation for the production of industrial enzymes due to low cost and abundant availability of cake during biodiesel production.

252 citations


Journal ArticleDOI
TL;DR: Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements and the development of an enzyme assay inside picoliter microdroplets.
Abstract: We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.

191 citations


Journal ArticleDOI
TL;DR: Various phenoxazine, phenothiazine, phenazine, indophenol and bipyridilium derivatives were tested for their effectiveness as redox mediators in microbial fuel cells containing Alcaligenes eutrophus, Bacillus subtilis, Escherichia coli, or Proteus vulgaris as the active biological agent, and glucose or succinate as the oxidisable substrate.
Abstract: Various phenoxazine, phenothiazine, phenazine, indophenol and bipyridilium derivatives were tested for their effectiveness as redox mediators in microbial fuel cells containing Alcaligenes eutrophus, Bacillus subtilis, Escherichia coli, or Proteus vulgaris as the active biological agent, and glucose or succinate as the oxidisable substrate. A ferricyanide-Pt cathode was used. The open-circuit cell e.m.f.′s increased in the order of increasing negative formal redox potentials at pH 7(E7m) of the redox compounds. Several of the redox agents worked well as mediators, maintaining steady currents over several hours, and thionine was found to be particularly effective in maintaining relatively high cell voltages when current was drawn from the cell. A number of the compounds tested did not function well, either because they were incompletely or slowly reduced by the microorganisms or because of their instability. P. vulgaris, with thionine as mediator and glucose as substrate, showed the best performance in a fuel cell. This system was examined in some detail under various conditions of external load to establish the effects of organism concentration, mediator concentration, and substrate addition. Coulombic outputs from these cells were calculated by integration of the current-time plots. Coulombic yields of 30–60% were obtained, on the basis of (theoretical) complete oxidation of added substrate to CO2 and water.

184 citations


Journal ArticleDOI
TL;DR: The extradiol aromatic ring-cleaving dioxygenases activate molecular oxygen by binding both O(2) and the catecholic substrate to a reduced active site metal, generally Fe(II).

169 citations


Journal ArticleDOI
TL;DR: The extension of the high selectivity and muM sensitivity of the tandem assay principle has allowed for the accurate measurement of D-lysine enantiomeric excesses of up to 99.98 %, as only the L-enantiomer is accepted by the enzyme as a substrate and is converted to the product that is responsible for the observed fluorescence signal.
Abstract: The coupling of an enzymatic transformation with dynamic host-guest exchange allows the unselective binding of macrocycles to be used for highly selective analyte sensing. The resulting supramolecular tandem enzyme assays require the enzymatic substrate and its corresponding product to differ significantly in their affinity for macrocycles, for example, cation receptors, and to show a differential propensity to displace a fluorescent dye from its host-guest complex. The enzymatic transformation results in a concomitant dye displacement that can be accurately followed by optical spectroscopy, specifically fluorescence. By exploiting this label-free continuous enzyme assay principle with the fluorescent dye Dapoxyl and the macrocyclic host cucurbit[7]uril, a multiparameter sensor array has been designed, which is capable of detecting the presence of amino acids (e.g. histidine, arginine, lysine, and tyrosine) and their decarboxylases. Only in the presence of both, the particular amino acid and the corresponding decarboxylase, is the amine or diamine product formed. These products are more highly positively charged than the substrate, have a higher affinity for the macrocycle and, therefore, displace the dye from the complex. The extension of the high selectivity and muM sensitivity of the tandem assay principle has also allowed for the accurate measurement of D-lysine enantiomeric excesses of up to 99.98 %, as only the L-enantiomer is accepted by the enzyme as a substrate and is converted to the product that is responsible for the observed fluorescence signal.

168 citations


01 Jun 2008
TL;DR: It was found that endothelial progenitor cells (EPCs) responded to ridge-groove grating of 1200 nm in period and 600 nm in depth through alignment, elongation, reduced proliferation, and enhanced migration, and an in vitro Matrigel assay led to enhanced capillary tube formation and organization.
Abstract: Vascular engineering remains a key thrust in advancing the field of tissue engineering of highly vascularized, complex, metabolic organs. A wide variety of strategies have been employed to control the formation of organized vascular structures in vitro and in vivo. Some of these methods include, but are not limited to, controlled growth factor delivery,[1] filamentous scaffold geometry,[2] protein micropatterning,[3] and enhanced scaffold biomaterials.[4] Many of these approaches are motivated by biomimicry of the in vivo microenvironment. Extracellular matrix (ECM) proteins, both in vitro and in vivo, provide mammalian cells with biophysical cues including specific surface chemistry and rich three-dimensional surface topography[5] with features on the nanometer length scale.[6] ECM substrates provide chemical and physical external cues that dictate a variety of cell responses. Therefore, it is not only the milieu of soluble, diffusible factors, but also the adhesive, mechanical interactions with scaffolding materials, both natural and synthetic, that control select cell functions including cell attachment, migration, proliferation, differentiation, and regulation of genes.[7–9] We hypothesized that physical features on nanofabricated substrates could promote the organization of endothelial cell lineages into well-defined vascular structures in vitro by inducing the contact guidance phenomenon, which is known to affect the morphology of endothelial cells.[10–12]We found that endothelial progenitor cells (EPCs) responded to ridge-groove grating of 1200 nm in period and 600 nm in depth through alignment, elongation, reduced proliferation, and enhanced migration. Although endothelial- specific markers were not significantly altered, EPCs cultured on substrate nanotopography formed supercellular band structures after 6 d. Furthermore, an in vitro Matrigel assay led to enhanced capillary tube formation and organization.

166 citations


Journal ArticleDOI
TL;DR: In this article, nanosized titanium dioxide (TiO 2 ) powders were used to connect substrate and hydroxyapatite (HA) in order to reduce the HA decomposition due to ion migration from the metal substrate into the HA.
Abstract: Nanosized hydroxyapatite (HA) powders were prepared by a chemical precipitation method and electrophoretically deposited on Ti6Al4V substrates. The powders were calcined before the deposition process in order to obtain crack-free coating surfaces. As an inner layer between Ti6Al4V substrate and HA coating, nanosized titanium dioxide (TiO 2 ) powders were deposited, using different coating voltages, in order to connect substrate and HA tightly. Moreover, this layer is considered to be acting as a diffusion barrier, reducing the HA decomposition due to ion migration from the metal substrate into the HA. After the sintering stage, adhesion strengths of coatings were measured by shear testing, phase changes were studied by X-ray diffraction, and coating morphology was analyzed through scanning electron microscopy observations. Results showed that usage of the TiO 2 inner layer prevented HA decomposition. Furthermore, decreasing the voltage used in TiO 2 deposition resulted in crack-free surfaces and increased adhesion strength of the overall coating.

158 citations


Patent
03 Sep 2008
TL;DR: In this article, a method of depositing a SiN x C y liner on a porous low thermal conductivity (low-k) substrate by plasma-enhanced atomic layer deposition (PE-ALD) was proposed.
Abstract: A method of depositing a SiN x C y liner on a porous low thermal conductivity (low-k) substrate by plasma-enhanced atomic layer deposition (PE-ALD), which includes forming a SiN x C y liner on a surface of a low-k substrate having pores on a surface thereon, in which the low-k substrate is repeatedly exposed to a aminosilane-based precursor and a plasma selected from nitrogen, hydrogen, oxygen, helium, and combinations thereof until a thickness of the liner is obtained, and wherein the liner is prevented from penetrating inside the pores of a surface of the substrate. A porous low thermal conductivity substrate having a SiN x C y liner formed thereon by the method is also disclosed.

152 citations


Patent
14 Oct 2008
TL;DR: In this paper, a method of forming a single-metal film on a substrate by plasma ALD is described, which involves contacting a surface of a substrate with a β-diketone metal complex in a gas phase, exposing the molecule-attached surface to a nitrogen-hydrogen mixed plasma, and repeating the above steps, thereby accumulating atomic layers to form a single metal film on the substrate.
Abstract: A method of forming a single-metal film on a substrate by plasma ALD includes: contacting a surface of a substrate with a β-diketone metal complex in a gas phase; exposing molecule-attached surface to a nitrogen-hydrogen mixed plasma; and repeating the above steps, thereby accumulating atomic layers to form a single-metal film on the substrate.

136 citations


Journal ArticleDOI
TL;DR: The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved.
Abstract: Glycerol is an important byproduct of bioethanol and biodiesel production processes This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose Both reactors operated at a pH of 8 and a dilution rate of 01 h−1 Glycerol was mainly converted into ethanol and formate When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations Glucose fermentation mainly generated acetate, ethanol and butyrate At higher substrate concentrations, acetate and ethanol were the dominant products Co-fermentations of glucose–glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively) The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved Biotechnol Biotechnol Bioeng 2008;100: 1088–1098 © 2008 Wiley Periodicals, Inc

118 citations


Journal ArticleDOI
Else K. Bünemann1
TL;DR: In this paper, the methodological aspects of enzyme additions are discussed using examples from studies in which enzymes from the phosphorus cycle were used, and a meta-analysis performed for various soil extracts and water samples revealed that the majority of studies (75th percentile) indicate availability of organic phosphorus to enzymatic hydrolysis of up to 60%, with crude phytase preparations showing the lowest substrate specificity and greatest release of phosphorus.
Abstract: Potential enzyme activities in soil and water samples are measured by addition of an excess amount of suitable substrate and subsequent determination of product release. If the approach is reversed and an excess of enzyme is added, substrate availability becomes rate-limiting and the maximum release of product indicates the availability of a given substrate in a sample. This approach has been used in a range of studies using phosphatase enzyme additions to soil, manure and sediment extracts, soil suspensions, and lake and sea water ( n = 41). Significantly fewer studies have used enzymes from the carbon, nitrogen and sulfur cycles ( n = 14). In this review, the methodological aspects of enzyme additions are discussed using examples from studies in which enzymes from the phosphorus cycle were used. A meta-analysis performed for various soil extracts and water samples revealed that the majority of studies (75th percentile) indicate availability of organic phosphorus to enzymatic hydrolysis of up to 60%, with crude phytase preparations showing the lowest substrate specificity and greatest release of phosphorus. Compared to addition of enzymes from the phosphorus cycle, lower substrate degradation was generally achieved by addition of enzymes from the carbon, nitrogen and sulfur cycles to soil suspensions and soil organic matter extracts. Enzyme additions can be a valuable tool in process research, provided all the necessary controls are included and assay conditions optimized to ensure that the reaction reaches completion. Recommendations for the development of a standard protocol are made.

Journal ArticleDOI
TL;DR: The efficiency of oxidation was found to increase with the decrease in redox potential of the substrates, and the Marcus reorganisation energy for electron transfer to the T1 copper site was determined.
Abstract: Laccases catalyze the one-electron oxidation of a broad range of substrates coupled to the 4 electron reduction of O2 to H2O. Phenols are typical substrates, because their redox potentials (ranging from 0.5 to 1.0 V vs. NHE) are low enough to allow electron abstraction by the T1 Cu(II) that, although a relatively modest oxidant (in the 0.4–0.8 V range), is the electron-acceptor in laccases. The present study comparatively investigated the oxidation performances of Trametes villosa and Myceliophthora thermophila laccases, two enzymes markedly differing in redox potential (0.79 and 0.46 V). The oxidation efficiency and kinetic constants of laccase-catalyzed conversion of putative substrates were determined. Hammett plots related to the oxidation of substituted phenols by the two laccases, in combination with the kinetic isotope effect determination, confirmed a rate-determining electron transfer from the substrate to the enzyme. The efficiency of oxidation was found to increase with the decrease in redox potential of the substrates, and the Marcus reorganisation energy for electron transfer to the T1 copper site was determined. Steric hindrance to substrate docking was inferred because some of the phenols and anilines investigated, despite possessing a redox potential compatible with one-electron abstraction, were scarcely oxidised. A threshold value of steric hindrance of the substrate, allowed for fitting into the active site of T. villosa laccase, was extrapolated from structural information provided by X-ray analysis of T. versicolor lac3B, sharing an identity of 99% at the protein level, thus enabling us to assess the relative contribution of steric and redox properties of a substrate in determining its susceptibility to laccase oxidation. The inferred structural threshold is compatible with the distance between two phenylalanine residues that mark the entrance to the active site. Interaction of the substrate with other residues of the active site is commented on.

Journal ArticleDOI
TL;DR: The extent of isotopic fractionation between the carbon substrate and the products of M. barkeri was dependent on the substrate type and availability, and the 13 C content of lipids varied with substrate availability in some cases, but did not show patterns that could be used to identify the growth substrate of methanogens in natural environments.

Journal ArticleDOI
TL;DR: An alkaline protease secreting Haloalkaliphilic bacterium was isolated from the Saurashtra Coast in Western India and the study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance.
Abstract: An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8–13, optimally at 9–11. It was stable with 0–4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 °C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25–80 °C (optimum at 50 °C), the purified enzyme had temperature optimum at 37 °C, which shifted to 80 °C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K cat). The enzyme was also calcium dependent and with 2 mM Ca+2, the activity reached to maximum at 50 °C. The crude enzyme was highly thermostable (37–90 °C); however, the purified enzyme lost its stability above 50 °C and its half life was enhanced by 30 and sevenfold at 60 °C with 1 M NaCl and 50 mM Ca+2, respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.

Journal ArticleDOI
TL;DR: Evaluated transition state binding energies obtained from interactions between low molecular weight metal ion complexes or high molecular weight protein catalysts and the phosphate group of bound substrate, which show a high affinity for oxyphosphorane transition state dianions and a stable methyl phosphate transition state analogue.
Abstract: Catalysis is an important process in chemistry and enzymology. The rate acceleration for any catalyzed reaction is the difference between the activation barriers for the uncatalyzed (Delta G(HO)(#)) and catalyzed (Delta G(Me)(#)) reactions, which corresponds to the binding energy (Delta G(S)(#) = Delta G(Me)(#)-Delta G(HO)(#)) for transfer of the reaction transition state from solution to the catalyst. This transition state binding energy is a fundamental descriptor of catalyzed reactions, and its evaluation is necessary for an understanding of any and all catalytic processes. We have evaluated the transition state binding energies obtained from interactions between low molecular weight metal ion complexes or high molecular weight protein catalysts and the phosphate group of bound substrate. Work on catalysis by small molecules is exemplified by studies on the mechanism of action of Zn2(1)(H2O). A binding energy of Delta G(S)(#) = -9.6 kcal/mol was determined for Zn2(1)(H2O)-catalyzed cleavage of the RNA analogue HpPNP. The pH-rate profile for this cleavage reaction showed that there is optimal catalytic activity at high pH, where the catalyst is in the basic form [Zn2(1)(HO-)]. However, it was also shown that the active form of the catalyst is Zn2(1)(H2O) and that this recognizes the C2-oxygen-ionized substrate in the cleavage reaction. The active catalyst Zn2(1)(H2O) shows a high affinity for oxyphosphorane transition state dianions and a stable methyl phosphate transition state analogue, compared with the affinity for phosphate monoanion substrates. The transition state binding energies, Delta G(S)(#), for cleavage of HpPNP catalyzed by a variety of Zn2+ and Eu3+ metal ion complexes reflect the increase in the catalytic activity with increasing total positive charge at the catalyst. These values of Delta G(S)(#) are affected by interactions between the metal ion and its ligands, but these effects are small in comparison with Delta G(S)(#) observed for catalysis by free metal ions, where the ligands are water. Enzymes are unique in having evolved mechanisms to effectively utilize binding interactions with nonreacting fragments of the substrate in stabilization of the reaction transition state. Orotidine 5'-monophosphate decarboxylase, alpha-glycerol phosphate dehydrogenase, and triosephosphate isomerase catalyze dissimilar decarboxylation, hydride transfer, and proton transfer reactions, respectively. Each enzyme derives ca. 12 kcal/mol of transition state stabilization from protein interactions with the nonreacting phosphate group, which is larger than the highest approximately 10 kcal/mol transition state stabilization that we have determined for small-molecule catalysis of phosphate diester cleavage in water. Each of these enzymes catalyze the slow reaction of a truncated substrate that lacks the phosphate group, and in each case, the reaction of the truncated substrate is strongly activated by the allosteric binding of the second substrate "piece" phosphite dianion, HPO3(2-). We propose a modular design for these enzymes with a classical active site that recognizes the reactive substrate fragment and a separate phosphodianion binding site. The second site is created, in part, by flexible protein loops that wrap around the substrate phosphodianion group and bury the substrate in an environment with an optimal local dielectric constant for the catalyzed reaction and with the most favorable positioning of the catalytic side chains. This design is easily generalized to a wide variety of enzyme-catalyzed reactions.

Journal ArticleDOI
TL;DR: Good agreement was found between experimental data and calculated values using the Michaelis-Menten mechanism, with a total average relative deviation of 2.1%.
Abstract: The activity and stability of commercial laccase (DeniLite base) in three different water soluble ionic liquids (ILs) (1-ethyl-3-methylimidazolium 2-(2-methoxyethoxy) ethylsulfate, [emim][[MDEGSO4], 1-ethyl-3-methylimidazolium ethylsulfate, [emim][EtSO4], and 1-ethyl-3-methylimidazolium methanesulfonate, [emim][MeSO3]) have been studied and compared to that in two organic solvents (acetonitrile and dimethyl sulfoxide). Initial enzyme activities were similar among the ILs if the same conditions were used. A high reduction on initial enzyme activity was found with acidic pH (5.0). The effect of pH and solvent concentration on enzyme stability were investigated in more detail for 1 week. The enzyme maintained a high stability at pH 9.0 for all ILs tested. [emim][MDEGSO4] was the most promising IL for laccase with an activity loss of about 10% after 7 days of incubation. The kinetic studies in the presence of ABTS as substrate allowed to calculate the Michaelis- Menten parameters. Good agreement was found between experimental data and calculated values using the Michaelis-Menten mechanism, with a total average relative deviation of 2.1%.

Journal ArticleDOI
TL;DR: In this article, the optimal conditions for enzymatic hydrolysis of brewer's spent grain were identified as 100rpm, 45 FPU/g and 2% w/v substrate, under which a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved.
Abstract: Brewer’s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 °C to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 23 full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer’s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.

Journal ArticleDOI
TL;DR: The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation as discussed by the authors.
Abstract: Choline oxidase catalyzes the oxidation of choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Ne2 and FAD C8M atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a Kd value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a kcat/KO2 ...

Patent
Bin Xia1, Ashutosh Misra1
21 Feb 2008
TL;DR: In this article, a ruthenium-based precursor is used to form a film on a substrate in a semiconductor manufacturing process, which is then used to produce a substrate.
Abstract: Methods for forming a film on a substrate in a semiconductor manufacturing process. A reaction chamber a substrate in the chamber are provided. A ruthenium based precursor, which includes ruthenium tetroxide dissolved in a mixture of at least two non-flammable fluorinated solvents, is provided and a ruthenium containing film is produced on the substrate.

Journal ArticleDOI
TL;DR: Amino groups containing magnetic beads were used in covalent immobilization of the enzyme "chloroperoxidase (CPO)" which is one of a few enzymes that can catalyse the peroxide dependent oxidation of a wide spectrum of organic and inorganic compounds as mentioned in this paper.

Journal ArticleDOI
TL;DR: Two mutant enzymes showed significant changes in their substrate specificity such that they catalyzed transamination of a broad range of aliphatic amines without losing the original activities toward aromatic amines and enantioselectivity.
Abstract: Substrate specificity of the omega-aminotransferase obtained from Vibrio fluvialis (omega-ATVf) was rationally redesigned for the kinetic resolution of aliphatic chiral amines. omega-ATVf showed unique substrate specificity toward aromatic amines with a high enantioselectivity (E > 100) for (S)-enantiomers. However, the substrate specificity of this enzyme was much narrower toward aliphatic amines. To overcome the narrow substrate specificity toward aliphatic amines, we redesigned the substrate specificity of omega-ATVf using homology modeling and the substrate structure- activity relationship. The homology model and the substrate structure-activity relationship showed that the active site of omega-ATVf consists of one large substrate-binding site and another small substrate-binding site. The key determinant in the small substrate-binding site was D25, whose role was expected to mask R415 and to generate the electrostatic repulsion with the substrate's alpha-carboxylate group. In the large substrate-binding site, R256 was predicted to recognize the alpha-carboxylate group of substrate thus obeying the dual substrate recognition mechanism of aminotransferase subgroup II enzymes. Among the several amino acid residues in the large substrate-binding site, W57 and W147, with their bulky side chains, were expected to restrict the recognition of aliphatic amines. Two mutant enzymes, W57G and W147G, showed significant changes in their substrate specificity such that they catalyzed transamination of a broad range of aliphatic amines without losing the original activities toward aromatic amines and enantioselectivity.

Journal ArticleDOI
TL;DR: In this paper, the structure and morphology of double hydroxide (LDH) films were investigated by X-ray diffraction (XRD), attenuated total reflectance-Fourier transform infrared (ATR-FT-IR), and scanning electron microscopy (SEM).

Journal ArticleDOI
TL;DR: In this article, the effect of substrate concentration ranging from 0 to 300 g/L on fermentative hydrogen production by mixed cultures was investigated in batch tests using glucose as a substrate.
Abstract: The effect of substrate concentration ranging from 0 to 300 g/L on fermentative hydrogen production by mixed cultures was investigated in batch tests using glucose as substrate. The experimental results showed that, at 35 and initial pH 7.0, during the fermentative hydrogen production, the hydrogen °C production potential and hydrogen production rate increased with increasing substrate concentration from 0 to 25 g/L. The maximal hydrogen production potential of 426.8 mL and maximal hydrogen production rate of 15.1 mL/h were obtained at the substrate concentration of 25 g/L. The maximal hydrogen yield and the maximal substrate degradation efficiency were respectively 384.3 mL/g glucose and 97.6%, at the substrate concentration of 2 g/L. The modified Logistic model could be used to describe the progress of cumulative hydrogen production in this study successfully. The Han-Levenspiel model could be used to describe the effect of substrate concentration on fermentative hydrogen production rate.

Journal ArticleDOI
TL;DR: The present complex structure provides the evidence of an increased stability of the structure upon interaction with the substrate and allows identification of an N‐terminal pyrrolidonecarboxylic acid in PPA.
Abstract: The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) that was soaked with the substrate maltopentaose showed electron density corresponding to two independent carbohydrate recognition sites on the surface of the molecule. Both binding sites are distinct from the active site described in detail in our previous high-resolution study of a complex between PPA and a carbohydrate inhibitor (Qian M, Buisson G, Duee E, Haser H, Payan F, 1994, Biochemistry 33:6284-6294). One of the binding sites previously identified in a 5-A-resolution electron density map, lies at a distance of 20 A from the active site cleft and can accommodate two glucose units. The second affinity site for sugar units is located close to the calcium binding site. The crystal structure of the maltopentaose complex was refined at 2.1 A resolution, to an R-factor of 17.5%, with an RMS deviation in bond distances of 0.007 A. The model includes all 496 residues of the enzyme, 1 calcium ion, 1 chloride ion, 425 water molecules, and 3 bound sugar rings. The binding sites are characterized and described in detail. The present complex structure provides the evidence of an increased stability of the structure upon interaction with the substrate and allows identification of an N-terminal pyrrolidonecarboxylic acid in PPA.

Journal ArticleDOI
TL;DR: A quantitative index of promiscuity (I) that can be calculated from the catalytic efficiencies of an enzyme toward a defined set of substrates is defined and a weighted promiscuit index (J) that accounts for patterns of similarity and dissimilarity among the substrates in the set is defined.
Abstract: Catalytic promiscuity is a widespread, but poorly understood, phenomenon among enzymes with particular relevance to the evolution of new functions, drug metabolism, and in vitro biocatalyst engineering. However, there is at present no way to quantitatively measure or compare this important parameter of enzyme function. Here we define a quantitative index of promiscuity (I) that can be calculated from the catalytic efficiencies of an enzyme toward a defined set of substrates. A weighted promiscuity index (J) that accounts for patterns of similarity and dissimilarity among the substrates in the set is also defined. Promiscuity indices were calculated for three different enzyme classes: eight serine and cysteine proteases, two glutathione S-transferase (GST) isoforms, and three cytochrome P450 (CYP) isoforms. The proteases ranged from completely specific (granzyme B, J = 0.00) to highly promiscuous (cruzain, J = 0.83). The four drug-metabolizing enzymes studied (GST A1-1 and the CYP isoforms) were highly promiscuous, with J values between 0.72 and 0.92; GST A4-4, involved in the clearance of lipid peroxidation products, is moderately promiscuous (J = 0.37). Promiscuity indices also allowed for studies of correlation between substrate promiscuity and an enzyme's activity toward its most-favored substrate, for each of the three enzyme classes.

Journal ArticleDOI
TL;DR: Wrightin, a new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria by cation-exchange chromatography, and complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serinerotease class.
Abstract: Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.

Journal ArticleDOI
TL;DR: This is the first report of endo-α-GalNAcases EngEF and EngPA acting on Core 3 in addition to Core 1 O-glycans, and there were no significant differences in transglycosylation activities when Galβ1,3GalNAcα1pNP or GlcNAcβ1
Abstract: In an effort to identify novel endo-alpha-N-acetylgalactosaminidases (endo-alpha-GalNAcases), four potential genes were cloned. Three of the expressed proteins EngEF from Enterococcus faecalis, EngPA from Propionibacterium acnes, and EngCP from Clostridium perfringens were purified and characterized. Their substrate specificity was investigated and compared to the commercially available endo-alpha-GalNAcases from Streptococcus pneumoniae (EngSP) and Alcaligenes sp. (EngAL). All enzymes were incubated with various synthetic substrates, and natural glycoproteins and the released sugars were detected by colorimetric assay and thin layer chromatography analysis. The Core 1 disaccharide Gal beta 1,3GalNAc alpha 1pNP was the most rapidly hydrolyzed substrate by all enzymes tested. EngEF exhibited the highest k(cat) for this substrate. EngEF and EngPA were also able to fully hydrolyze the Core 3 disaccharide GlcNAc beta 1,3GalNAc alpha 1pNP. This is the first report of endo-alpha-GalNAcases EngEF and EngPA acting on Core 3 in addition to Core 1 O-glycans. Interestingly, there were no significant differences in transglycosylation activities when Gal beta 1,3GalNAc alpha 1pNP or GlcNAc beta 1,3GalNAc alpha 1pNP was incubated with various 1-alkanols in the presence of the endo-alpha-GalNAcases tested in this work.

Journal ArticleDOI
TL;DR: The microbial degradation of organic substrates often exhibits a fractionation of stable isotopes which leads to an enrichment of the heavier isotope in the remaining substrate, and changes in observed isotope fractionation might occur while contaminant concentration decreases along a flow path.
Abstract: The microbial degradation of organic substrates often exhibits a fractionation of stable isotopes which leads to an enrichment of the heavier isotope in the remaining substrate. The use of this effect to quantify the amount of biodegraded substrate in contaminated aquifers requires that the isotope fractionation factor is constant in time and space. In many natural and engineered systems the bioavailable concentration at the location of the enzymes differs from the average bulk concentration of the substrate. When enzymatically driven substrate degradation is coupled to a preceding transport step controlling the bioavailability of the substrate, the observed isotope fractionation becomes a function of the bulk substrate concentration. The sensitivity of the observed isotope fractionation factor toward such substrate concentration changes depends on the ratio of bulk substrate concentration and Michaelis-Menten constant and on the ratio between the specific affinity of the microorganisms toward the substrate and the first order rate constant of the bioavailability limiting transport process. Highest sensitivities toward substrate concentration were found for combinations of high substrate concentration with low substrate bioavailability (i.e., high ratios of substrate concentration and Michaelis-Menten constant, and high ratios of specific affinity and transport rate constant). As a consequence, changes in concentration and isotopic composition of a bioavailability limited substrate in batch experiments should not exhibit a linear relation in a Rayleigh plot, and the slope of the Rayleigh plot should show a decreasing trend with concentration decrease. When using isotope fractionation to quantify biodegradation along groundwater flow paths, changes in observed isotope fractionation might occur while contaminant concentration decreases along a flow path.

Patent
Lee Chen1
21 Mar 2008
TL;DR: In this paper, a chemical processing system and a method of using the chemical process to treat a substrate with a mono-energetic space-charge neutralized neutral beam-activated chemical process is described.
Abstract: A chemical processing system and a method of using the chemical processing system to treat a substrate with a mono-energetic space-charge neutralized neutral beam-activated chemical process is described. The chemical processing system comprises a first plasma chamber for forming a first plasma at a first plasma potential, and a second plasma chamber for forming a second plasma at a second plasma potential greater than the first plasma potential, wherein the second plasma is formed using electron flux from the first plasma. Further, the chemical processing system comprises a substrate holder configured to position a substrate in the second plasma chamber.

Journal ArticleDOI
TL;DR: Results of a series of density functional theory studies on enzyme active site models of nitric oxide synthase (NOS) indicate that compound II is the actual oxidant in NOS enzymes that performs the hydroxylation reaction of arginine, which is in sharp contrast with the cytochromes P450 where compound II was shown to be a sluggish oxidant.
Abstract: We present here results of a series of density functional theory (DFT) studies on enzyme active site models of nitric oxide synthase (NOS) and address the key steps in the catalytic cycle whereby the substrate (l-arginine) is hydroxylated to Nω-hydroxo-arginine. It has been proposed that the mechanism follows a cytochrome P450-type catalytic cycle; however, our calculations find an alternative low energy pathway whereby the bound l-arginine substrate has two important functions in the catalytic cycle, namely first as a proton donor and later as the substrate in the reaction mechanism. Thus, the DFT studies show that the oxo-iron active species (compound I) cannot abstract a proton and neither a hydrogen atom from protonated l-arginine due to the strength of the N−H bonds of the substrate. However, the hydroxylation of neutral arginine by compound I and its one electron reduced form (compound II) requires much lower barriers and is highly exothermic. Detailed analysis of proton transfer mechanisms shows th...