Showing papers on "Substrate (chemistry) published in 2005"

Fast Carbon−Carbon Bond Formation by a Promiscuous Lipase

Abstract: Lipase B from Candida antarctica was redesigned to catalyze the promiscuous reaction of carbon−carbon bond formation. Mutation of the catalytic serine to alanine afforded a mutant that catalyzed Michael additions of 1,3-dicarbonyls to α,β-unsaturated carbonyl compounds at high specific rates, such as 4000 s-1. The enzyme-catalyzed Michael addition reaction followed saturation kinetics and showed substrate inhibition. The designed enzyme showed high rate enhancements with a catalytic proficiency higher than 108, which is on the same level as that observed for enzymes with native substrates.

Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae.

Abstract: We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C4-C6 and C3-C8 chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.

Methods for nucleic acid amplification and sequence determination

Abstract: The invention provides methods for sequencing a nucleic acid comprising conducting rolling circle amplification on a circular nucleic acid template, wherein the resulting amplicon is optionally anchored to a substrate in an individually optically resolvable manner, and performing a sequencing reaction.

Single-enzyme kinetics of CALB-catalyzed hydrolysis

Abstract: Real-time measurement of the catalysis and substrate kinetics of a single-enzyme hydrolysis reaction is demonstrated with confocal fluorescence microscopy (CFM; see picture, green = CFM beam). A single lipase is shown to have a broad range of conformations; each conformation contributes to the overall enzymatic activity, an observation that is often masked by ensemble measurements.

Method for silicon based dielectric chemical vapor deposition

Abstract: Embodiments of the invention generally provide a method for depositing silicon-containing films. In one embodiment, a method for depositing silicon-containing material film (434) on a substrate (122) includes flowing a nitrogen and carbon containing chemical into a deposition chamber (100), flowing a silicon-containing source chemical having silicon-nitrogen bonds into the processing chamber (100), and heating the substrate (122) disposed in the chamber to a temperature less than about 550 degrees Celsius. In another embodiment, the silicon containing chemical is trisilylamine and the nitrogen and carbon containing chemical is (CH3)3-N.

Method for forming a high density dielectric film by chemical vapor deposition

Abstract: A method for forming a high density dielectric film by chemical vapor deposition. The method comprises: (a) a substrate is provided in a processing chamber; (b) a first gas is introduced into the processing chamber with a first pressure and adsorbed on the substrate, wherein the first gas comprises silicon-containing or carbon-containing gas; (c) the first gas is stopped, and the first pressure is lowered to a second pressure; (d) a second gas is introduced into the processing chamber with a third pressure, and forced to react with the first gas absorbed on the substrate and remained in the processing chamber, wherein the second gas comprises oxidizer or reduction agent; (e) the steps (b)˜(d) are repeated until a high density dielectric film is formed on the substrate.

Atomic Resolution Structures of Resting-State, Substrate- and Product-Complexed Cu-Nitrite Reductase Provide Insight Into Catalytic Mechanism

Abstract: Copper-containing nitrite reductases catalyze the reduction of nitrite to nitric oxide (NO), a key step in denitrification that results in the loss of terrestrial nitrogen to the atmosphere. They are found in a wide variety of denitrifying bacteria and fungi of different physiology from a range of soil and aquatic ecosystems. Structural analysis of potential intermediates in the catalytic cycle is an important goal in understanding enzyme mechanism. Using "crystal harvesting" and substrate-soaking techniques, we have determined atomic resolution structures of four forms of the green Cu-nitrite reductase, from the soil bacterium Achromobacter cycloclastes. These structures are the resting state of the enzyme at 0.9 A, two species exhibiting different conformations of nitrite bound at the catalytic type 2 Cu, one of which is stable and also has NO present, at 1.10 A and 1.15 A, and a stable form with the product NO bound side-on to the catalytic type 2 Cu, at 1.12 A resolution. These structures provide incisive insights into the initial binding of substrate, its repositioning before catalysis, bond breakage (O-NO), and the formation of a stable NO adduct.

Mechanism of Taurine: α-Ketoglutarate Dioxygenase (TauD) from Escherichia coli

Abstract: The iron(II)- and α-ketoglutarate-dependent dioxygenases comprise enzymes that catalyze a variety of important reactions in biology, including steps in the biosynthesis of collagen and antibiotics, the degradation of xenobiotics, the repair of alkylated DNA, and the sensing of oxygen and response to hypoxia. In these reactions, the reductive activation of oxygen is coupled to hydroxylation of the substrate and decarboxylation of the co-substrate, α-ketoglutarate. It is believed that a single, conserved mechanistic pathway for formation of a high-valent iron intermediate that attacks the substrate is operant in all members of this family. Application

An alcohol acyl transferase from apple (cv. Royal Gala), MpAAT1, produces esters involved in apple fruit flavor

Abstract: Apple flavor is characterized by combinations of ester compounds, which increase markedly during fruit ripening. The final step in ester biosynthesis is catalyzed by alcohol acyl transferases (AATs) that use coenzyme A (CoA) donors together with alcohol acceptors as substrates. The gene MpAAT1, which produces a predicted protein containing features of other plant acyl transferases, was isolated from Malus pumila (cv. Royal Gala). The MpAAT1 gene is expressed in leaves, flowers and fruit of apple. The recombinant enzyme can utilize a range of alcohol substrates from short to medium straight chain (C3-C10), branched chain, aromatic and terpene alcohols. The enzyme can also utilize a range of short to medium chain CoAs. The binding of the alcohol substrate is rate limiting compared with the binding of the CoA substrate. Among different alcohol substrates there is more variation in turnover compared with K(m) values. MpAAT1 is capable of producing many esters found in Royal Gala fruit, including hexyl esters, butyl acetate and 2-methylbutyl acetate. Of these, MpAAT1 prefers to produce the hexyl esters of C3, C6 and C8 CoAs. For the acetate esters, however, MpAAT1 preference depends upon substrate concentration. At low concentrations of alcohol substrate the enzyme prefers utilizing the 2-methylbutanol over hexanol and butanol, while at high concentrations of substrate hexanol can be used at a greater rate than 2-methylbutanol and butanol. Such kinetic characteristics of AATs may therefore be another important factor in understanding how the distinct flavor profiles of different fruit are produced during ripening.

Conservation of mus-ms enzyme motions in the apo- and substrate-mimicked state.

Abstract: Solution NMR spin-relaxation experiments were used to compare μs−ms dynamics in RNase A in the apo form and as complexed to the substrate-mimic, pTppAp. The crystal structure of the RNase A/pTppAp complex was determined and demonstrates that this ligand binds at the active site and utilizes established substrate binding sites in its interaction with RNase A. Relaxation-compensated CPMG experiments identify flexible residues in and around the active site in both the apo and pTppAp-bound enzyme. Quantitative analysis of the NMR spin-relaxation dispersion curves show that the time scale of motion in RNase A is unchanged when pTppAp binds and is similar to the time scale for the rate-determining step of the catalytic reaction. Temperature-dependent measurements provide an activation barrier for motion of 5.2 ± 1.0 kcal/mol and 4.5 ± 1.2 kcal/mol for the apo and pTppAp forms of RNase A, respectively. These data indicate very similar motion exists in the free and bound enzyme. Additionally, chemical shift data ...

Electrochemical detection of DNA hybridization using biometallization.

Abstract: We demonstrate the amplified detection of a target DNA based on the enzymatic deposition of silver. In this method, the target DNA and a biotinylated detection DNA probe hybridize to a capture DNA probe tethered onto a gold electrode. Neutravidin-conjugated alkaline phosphatase binds to the biotin of the detection probe on the electrode surface and converts the nonelectroactive substrate of the enzyme, p-aminophenyl phosphate, into the reducing agent, p-aminophenol. The latter, in turn, reduces metal ions in solutions leading to deposition of the metal onto the electrode surface and DNA backbone. This process, which we term biometallization, leads to a great enhancement in signal due to the accumulation of metallic silver by a catalytically generated enzyme product and, thus, the electrochemical amplification of a biochemically amplified signal. The anodic stripping current of enzymatically deposited silver provides a measure of the extent of hybridization of the target oligomers. This biometallization process is highly sensitive, detecting as little as 100 aM (10 zmol) of DNA. We also successfully applied this method to the sequence-selective discrimination between perfectly matched and mismatched target oligonucleotides including a single-base mismatched target.

Regulation of Cellulase Synthesis in Batch and Continuous Cultures of Clostridium thermocellum

Abstract: Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.

Immobilization of a catalytic DNA molecular beacon on Au for Pb(II) detection.

Abstract: A Pb(II)-specific DNAzyme fluorescent sensor has been modified with a thiol moiety in order to immobilize it on a Au surface. Self-assembly of the DNAzyme is accomplished by first adsorbing the single-thiolated enzyme strand (HS-17E-Dy) followed by adsorption of mercaptohexanol, which serves to displace any Au-N interactions and ensure that DNA is bound only through the Sheadgroup. The preformed self-assembled monolayer is then hybridized with the complementary fluorophorecontaining substrate strand (17DS-Fl). Upon reaction with Pb(II), the substrate strand is cleaved, releasing a fluorescent fragment for detection. Fluorescence intensity may be correlated with original Pb(II) concentration, and a linear calibration was obtained over nearly four decades: 10 IM g [Pb(II)] g 1 nM. The immobilized DNAzyme is a robust system; it may be regenerated after cleavage, allowing multiple sensing cycles. In addition, drying of fully assembled DNAzyme before reaction with Pb(II) does not significantly affect analytical performance. These results demonstrate that, in comparison with solutionbased schemes, immobilization of the DNAzyme sensor onto a Au surface lowers the detection limit (from 10 to 1 nM), maintains activity and specificity, and allows sensor regeneration and long-term storage. Realization of Pb(II) detection through an immobilized DNAzyme is the first important step toward creation of a stand-alone, portable Pb(II) detection device such as those immobilizing DNAzyme recognition motifs in the nanofluidic pores of a microfluidic-nanofluidic hybrid multilayer device. Lead has historically been, and remains today, a persistent contaminant in the environment. Environmentally available Pb may originate from lead-based paints, soil and dust generated from gasoline and industrial pursuits, and water from pipes with leadcontaining connectors or joints.1 Regulatory improvements have

Purification and characterization of laccase from the white rot fungus Trametes versicolor.

Abstract: Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50 degrees C. The Km value of the enzyme for substrate ABTS is 12.8 micrometer and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.

Optimized synthesis of lipase-catalyzed biodiesel by Novozym 435

Abstract: The ability of immobilized lipase from Candida antarctica (Novozym 435) to catalyze the alcoholysis of canola oil and methanol was investigated. Response surface methodology (RSM) and five–level–five–factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time, temperature, enzyme concentration, substrate molar ratio of methanol to canola oil, and added water content on percentage weight conversion of canola oil methyl ester by alcoholysis. Reaction temperature and enzyme concentration were the most important variables. High temperature and superabundant methanol inhibited the ability of Novozym 435 to catalyze the synthesis of biodiesel. Based on the analysis of ridge max, the optimum synthesis conditions were as follows: reaction time 12.4 h, temperature 38.0 °C, enzyme concentration 42.3%, substrate molar ratio 3.5:1, and added water 7.2%. The predicted value was 99.4% weight conversion, and the actual experimental value was 97.9% weight conversion. Copyright © 2004 Society of Chemical Industry

Dual substrate and reaction specificity in mouse serine racemase: identification of high-affinity dicarboxylate substrate and inhibitors and analysis of the beta-eliminase activity.

Abstract: Mouse serine racemase (mSR) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the biosynthesis of the N-methyl-d-aspartate receptor coagonist d-serine in the brain. Furthermore, mSR catalyzes beta-elimination of serine and l-serine-O-sulfate into pyruvate. The biological significance of this beta-elimination activity and the factors influencing mSR substrate and reaction specificity, which are crucial for prospective inhibitor design, are poorly understood. Using a bacterial expression system and ATP-agarose affinity chromatography, we have generated a pure and active recombinant mSR and investigated its substrate and reaction specificity in vitro by analyzing a systematic series of compounds derived from l-Ser and l-serine-O-sulfate. The analysis revealed several competitive inhibitors of serine racemization including glycine (K(I) = 1.63 mM), several dicarboxylic acids including malonate (K(I) = 0.077 mM), and l-erythro-3-hydroxyaspartate (K(I) = 0.049 mM). The latter compound represents the most effective inhibitor of SR reported to date. A simple inversion of the beta-carbon configuration of the compound yields an excellent beta-elimination substrate l-threo-3-hydroxyaspartate. Inhibition analysis indicates that racemization and beta-elimination activities of mSR reside at the same active site. While the racemization activity is specific to serine, the beta-elimination activity has a broader specificity for l-amino acids with a suitable leaving group at the beta-carbon and optimal spatial orientation of the alpha-carboxyl and leaving groups. The possible implications of our observations for inhibitor design, regulation of activity, and function of mSR are discussed.