Showing papers on "Substrate (chemistry) published in 2001"

The depth of chemical time and the power of enzymes as catalysts.

Abstract: The fastest known reactions include reactions catalyzed by enzymes, but the rate enhancements that enzymes produce had not been fully appreciated until recently. In the absence of enzymes, these same reactions are among the slowest that have ever been measured, some with half-times approaching the age of the Earth. This difference provides a measure of the proficiencies of enzymes as catalysts and their relative susceptibilities to inhibition by transition-state analogue inhibitors. Thermodynamic comparisons between spontaneous and enzyme-catalyzed reactions, coupled with structural information, suggest that in addition to electrostatic and H-bonding interactions, the liberation of water molecules from an enzyme's active site into bulk solvent sometimes plays a prominent role in determining the relative binding affinities of the altered substrate in the ground state and transition state. These comparisons also indicate a high level of synergism in the action of binding determinants of both the substrate a...

Biohydrogen production as a function of ph and substrate concentration

Abstract: The conversion of organics in wastewaters into hydrogen gas could serve the dual role of renewable energy production and waste reduction. The chemical energy in a sucrose rich synthetic wastewater was recovered as hydrogen gas in this study. Using fractional factorial design batch experiments, the effect of varying pH (4.5-7.5) and substrate concentration (1.5-44.8 g COD/L) and their interaction on hydrogen gas production were tested. Mixed bacterial cultures obtained from a compost pile, a potato field, and a soybean field were heated to inhibit hydrogen-consuming methanogens and to enrich sporeforming, hydrogen-producing acidogens. It was determined that the highest rate (74.7 mL H2/(L*h)) of hydrogen production occurred at a pH of 5.5 and a substrate concentration of 7.5 g COD/Lwith a conversion efficiency of 38.9 mL H2/(g COD/L). The highest conversion efficiency was 46.6 mL H2/(g COD/L).

Guide to Cytochromes P450 : structure and function

Abstract: 1. Introduction 2. Evolution of the P450 superfamily 3. The P450 catalytic cycle 4. Substrate selectivity and metabolism 5. Regulation of P450 enzymes 6. The structures of cytochromes P450

Over-stabilization of Candida antarctica lipase B by ionic liquids in ester synthesis

Abstract: Four different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for butyl butyrate synthesis catalyzed by free Candida antarctica lipase B at 2% (v/v) water content and 50 °C. Lipase had enhanced synthetic activity in all ionic liquids in comparison with two organic solvents (hexane, and 1-butanol), the enhanced activity being related to the increase in polarity of ionic liquids. The continuous operation of lipase with all the assayed ionic liquids showed over-stabilization of the enzyme. The reuse of free lipase in 1-butyl-3-methylimidazolium hexafluorophosphate in continuous operation cycles showed a half-life time 2300 times greater than that observed when the enzyme was incubated in the absence of substrate (3.2 h), and a selectivity higher than 90%.

Stabilization of alpha-chymotrypsin by ionic liquids in transesterification reactions.

Abstract: Five different ionic liquids, based on dialkylimidazolium and quaternary ammonium cations associated with perfluorinated and bis (trifluoromethyl) sulfonyl amide anions, were used as reaction media to synthesize N-acetyl-L-tyrosine propyl ester by transesterification with alpha-chymotrypsin at 2% (v/v) water content at 50 degrees C. The synthetic activity was reduced by the increase in alkyl chains length of cations and by increases in anion size, which was related to the decrease in polarity. Incubation of the enzyme (with and without substrate) in ionic liquids exhibited first-order deactivation kinetics at 50 degrees C, allowing determination of deactivation rate constants and half-life times (1-3 h). Ionic liquids showed a clear relative stabilization effect on the enzyme, which was improved by increased chain length of the alkyl substituents on the imidazolium ring cations and the anion size. This effect was 10-times enhanced by the presence of substrate. For example, 1-butyl-3-methylimidazolium hexafluorophosphate increased the alpha-chymotrypsin half-life by 200 times in the presence of substrate with respect to the 1-propanol medium. These results show that ionic liquids are excellent enzyme-stabilizing agents and reaction media for clean biocatalysis in non-conventional conditions.

Enzymatic hydrolysis of proteins from yellowfin tuna (Thunnus albacares) wastes using Alcalase

Abstract: Enzymatic hydrolysis of tuna stomach proteins by Alcalase was investigated in a batch reactor. The influence of the process variables (enzyme/substrate ratio; effect of intermediate substrate and enzyme addition) was studied with regards to the extent of proteolytic degradation and to the molecular weight distribution of the peptides. A linear correlation was found between the degree of hydrolysis (DH) and the enzyme concentration. After addition of extra substrate during the course of hydrolysis, the final DH obtained was proportional to the substrate added, suggesting that the concentration of hydrolysable bonds was one of the main factors controlling the hydrolysis rate. Preliminary results showed that tuna protein hydrolysates performed effectively as nitrogenous source in microbial growth media.

p-Hydroxyphenylacetate decarboxylase from Clostridium difficile. A novel glycyl radical enzyme catalysing the formation of p-cresol.

Abstract: The human pathogenic bacterium Clostridium difficile is a versatile organism concerning its ability to ferment amino acids. The formation of p-cresol as the main fermentation product of tyrosine by C. difficile is unique among clostridial species. The enzyme responsible for p-cresol formation is p-hydroxyphenylacetate decarboxylase. The enzyme was purified from C. difficile strain DMSZ 1296T and initially characterized. The N-terminal amino-acid sequence was 100% identical to an open reading frame in the unfinished genome of C. difficile strain 630. The ORF encoded a protein of the same size as the purified decarboxylase and was very similar to pyruvate formate-lyase-like proteins from Escherichia coli and Archaeoglobus fulgidus. The enzyme decarboxylated p-hydroxyphenylacetate (Km = 2.8 mm) and 3,4-dihydroxyphenylacetate (Km = 0.5 mm). It was competitively inhibited by the substrate analogues p-hydroxyphenylacetylamide and p-hydroxymandelate with Ki values of 0.7 mm and 0.48 mm, respectively. The protein was readily and irreversibly inactivated by molecular oxygen. Although the purified enzyme was active in the presence of sodium sulfide, there are some indications for an as yet unidentified low molecular mass cofactor that is required for catalytic activity in vivo. Based on the identification of p-hydroxyphenylacetate decarboxylase as a novel glycyl radical enzyme and the substrate specificity of the enzyme, a catalytic mechanism involving ketyl radicals as intermediates is proposed.

Variants with altered properties

Abstract: The present invention relates to variants (mutants) of polypeptides, in particular Termamyl-like alpha-amylases, which variant has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent alpha-amylase: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, Ca2+ dependency, specific activity, and solubility, in particular under production conditions.

Catalase-like activity of horseradish peroxidase: relationship to enzyme inactivation by H2O2.

Abstract: H2O2 is the usual oxidizing substrate of horseradish peroxidase C (HRP-C). In the absence in the reaction medium of a one-electron donor substrate, H2O2 is able to act as both oxidizing and reducing substrate. However, under these conditions the enzyme also undergoes a progressive loss of activity. There are several pathways that maintain the activity of the enzyme by recovering the ferric form, one of which is the decomposition of H2O2 to molecular oxygen in a similar way to the action of catalase. This production of oxygen has been kinetically characterized with a Clark-type electrode coupled to an oxygraph. HRP-C exhibits a weak catalase-like activity, the initial reaction rate of which is hyperbolically dependent on the H2O2 concentration, with values for K(2) (affinity of the first intermediate, compound I, for H2O2) and k(3) (apparent rate constant controlling catalase activity) of 4.0 +/- 0.6 mM and 1.78 +/- 0.12 s(-1) respectively. Oxygen production by HRP-C is favoured at pH values greater than approx. 6.5; under similar conditions HRP-C is also much less sensitive to inactivation during incubations with H2O2. We therefore suggest that this pathway is a major protective mechanism of HRP-C against such inactivation.

High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis.

Abstract: Chitinase A (ChiA) from the bacterium Serratia marcescens is a hydrolytic enzyme, which cleaves ‚-1,4-glycosidic bonds of the natural biopolymer chitin to generate di- N-acetyl-chitobiose. The refined structure of ChiA at 1.55 A shows that residue Asp313, which is located near the catalytic proton donor residue Glu315, is found in two alternative conformations of equal occupancy. In addition, the structures of the cocrystallized mutant proteins D313A, E315Q, Y390F, and D391A with octa- or hexa- N-acetyl-glucosamine have been refined at high resolution and the interactions with the substrate have been characterized. The obtained results clearly show that the active site is a semiclosed tunnel. Upon binding, the enzyme bends and rotates the substrate in the vicinity of the scissile bond. Furthermore, the enzyme imposes a critical "chair" to "boat" conformational change on the sugar residue bound to the -1 subsite. According to our results, we suggest that residues Asp313 and Tyr390 along with Glu315 play a central role in the catalysis. We propose that after the protonation of the substrate glycosidic bond, Asp313 that interacts with Asp311 flips to its alternative position where it interacts with Glu315 thus forcing the substrate acetamido group of -1 sugar to rotate around the C2-N2 bond. As a result of these structural changes, the water molecule that is hydrogen-bonded to Tyr390 and the NH of the acetamido group is displaced to a position that allows the completion of hydrolysis. The presented results suggest a mechanism for ChiA that modifies the earlier proposed "substrate assisted" catalysis.

Spectroscopic Studies of Substrate Interactions with Clavaminate Synthase 2, a Multifunctional α-KG-Dependent Non-Heme Iron Enzyme: Correlation with Mechanisms and Reactivities

Abstract: Using a single ferrous active site, clavaminate synthase 2 (CS2) activates O2 and catalyzes the hydroxylation of deoxyguanidinoproclavaminic acid (DGPC), the oxidative ring closure of proclavaminic acid (PC), and the desaturation of dihydroclavaminic acid (and a substrate analogue, deoxyproclavaminic acid (DPC)), each coupled to the oxidative decarboxylation of cosubstrate, α-ketoglutarate (α-KG). CS2 can also catalyze an uncoupled decarboxylation of α-KG both in the absence and in the presence of substrate, which results in enzyme deactivation. Resting CS2/FeII has a six-coordinate FeII site, and α-KG binds to the iron in a bidentate mode. The active site becomes five-coordinate only when both substrate and α-KG are bound, the latter still in a bidentate mode. Absorption, CD, MCD, and VTVH MCD studies of the interaction of CS2 with DGPC, PC, and DPC provide significant molecular level insight into the structure/function correlations of this multifunctional enzyme. There are varying amounts of six-coordin...

The Reaction Mechanism of the Enzyme-Catalyzed Central Cleavage of β-Carotene to Retinal

Abstract: Seeing things as they really are: The enzyme catalyzing the central cleavage of β-carotene (1) to retinal (2) is not, as previously thought, a dioxygenase. Incubation of the substrate analogue α-carotene in the presence of highly enriched 17 O2 and H218 O revealed a monooxygenase mechanism.

Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals.

Abstract: An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein The purified wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize N6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2,6-dichlorophenol-indophenol 2,3-Dimethoxy-5-methyl-1,4-benzoquinone, a precursor of the naturally occurring electron acceptor ubiquinone, readily interacts with the enzyme in micromolar concentrations Typical flavoenzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity Presence of the flavin cofactor in the enzyme was confirmed by differential pulse polarography and by measuring the fluorescence emission spectrum Possible existence of a second redox centre is discussed

Method for vapour deposition of a film onto a substrate

Abstract: Method for chemical vapor deposition of a film onto a substrate. Before bulk chemical vapor deposition the substrate is subjected to a nucleation treatment. The nucleation treatment comprises atomic layer deposition wherein the substrate is alternatingly and sequentially exposed to pulses of at least two mutually reactive gaseous reactants wherein the nucleation temperature is chosen to prevent condensation of either of the used reactants and to prevent substantial thermal decomposition of each of the reactants individually.

Crystal structure of a monocotyledon (maize ZMGlu1) beta-glucosidase and a model of its complex with p-nitrophenyl beta-D-thioglucoside.

Abstract: The maize beta-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli, purified, crystallized and its structure solved at 2.5 Angstrom resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the non-hydrolysable inhibitor p-nitrophenyl beta-D-thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.

ALD reactor and method with controlled wall temperature

Abstract: The present invention relates to improved methods and apparatus for atomic layer deposition (ALD) of thin films on substrates such as wafers and flat panel displays. The invention provides an ALD reactor comprising a first temperature regulating system to control the temperature of the substrate and a second temperature regulating system to independently control the temperature of the reaction chamber walls. The invention also provides a method for ALD of a film onto a substrate in a reaction chamber, in which the temperature of the substrate is maintained to maximize ALD on the substrate while the temperature of the reaction chamber walls is set to minimize film growth thereon, whether by ALD, condensation, physisorption or thermal decomposition. The temperature of the walls may be maintained at the same temperature as the substrate, or higher or lower than the substrate temperature, depending upon the particular reaction being used.

Purification and Characterization of Bifunctional Alginate Lyase from Alteromonas sp. Strain No. 272 and Its Action on Saturated Oligomeric Substrates

Abstract: A marine bacterium (strain No. 272) isolated from sea mud in Omura Bay produced an alginate lyase and was classified as an Alteromonas species. The enzyme was purified from the culture medium of the bacterium by DEAE-Cellulofine, Sephadex G-100 gel chromatography to an electrophoretically homogeneous state in the presence and absence of SDS. The molecular mass of the enzyme was 23 and 33.9 kDa on Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis, respectively, with an isoelectric point of 3.8. The predominant secondary structure of the enzyme was found to be most likely beta-structure by circular dichroism. The enzyme was most active at pH 7.5-8.0 and stable around pH 5-11. The enzyme was more labile in Tris-HCI buffer (pH 7.0) to heat treatment, than in phosphate buffer (pH 7.0). No of metal ions significantly affected the enzyme activity. The enzyme acted on sodium alginate in an endo-type manner and on two components of alginate, poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate, as judged by routine ultraviolet assay (235 nm) and circular dichroic spectral changes of the substrates. However, the coexisting poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate apparently interacted with the enzyme in a competitive manner. Although the enzyme depolymerized alginate in an endo-type, it did not act on trimeric guluronate and mannuronate, but on the tetramers or more. The kinetic analyses showed that kcat/Km for each oligomer was larger for the guluronate oligomers than for the mannuronate ones, and that the subsite structure of the enzyme most likely consisted of six binding sites from the intrinsic reaction rate constant (kint) and intrinsic substrate binding constant (Kint).

Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11.

Abstract: The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myo-inositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+ In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme Ca2+ turned out to be also required for the substrate because the phytase is only able to hydrolyze the calcium-phytate complex In fact, both an excess amount of free Ca2+ and an excess of free phytate, which is not complexed with each other, can act as competitive inhibitors The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate The Ca2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate