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1.
This is the continuation of our studies to use very basic information on enzyme to predict optimal reaction parameters in enzymatic reactions because the gap between available enzyme sequences and their available reaction parameters is widening. In this study, 23 features selected from 540 plus features of individual amino acid as well as a feature combined whole protein information were screened as independents in a 20-1 feedforward backpropagation neural network for predicting optimal pH in beta-glucosidase’s hydrolytic reaction because this enzyme drew attention recently due to its role in biofuel industry. The results show that 11 features can be used as independents for the prediction, while the feature of amino acid distribution probability works better than the rest independents for the prediction. Our study paves a way to predict the optimal reaction parameters of enzymes based on the amino acid features of enzyme sequences. 相似文献
2.
Beatriz M. Brena Jonas Lidholm Francisco Batista-Viera Jan Carlsson 《Applied biochemistry and biotechnology》1998,75(2-3):323-341
A new approach for the control and interruption of enzymatic reactions via selective enzyme immobilization has been developed. The technique was exemplified by the use of three model enzymes with the corresponding macromolecular substrates: α-amylase/starch, trypsin/ insoluble collagen, and alkaline phosphatase/plasmid DNA. Prior to incubation with its substrate, each enzyme was provided withde novo thiol-groups by a two-step reaction involvingN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and DTT. The chemical modification was achieved such that at least 80% of the native enzyme activity was preserved in all cases. In order to interrupt rapidly the reactions in which the enzymes were used, the modified enzyme was immobilized by reaction via its thiol groups on a thiolsulfinate-agarose derivative. The gel-bound enzyme could then be easily removed from unreacted substrate and product by filtration or centrifugation. Comparative studies showed that the immobilized enzymes had much lower activities in the reactions studied than the corresponding soluble ones. The potential for enzyme reuse was also demonstrated with the a-amylase derivatives, which were quantitatively released and eluted in fully active form from the agarose. We have shown that it is possible to achieve practically complete enzyme immobilization in short times and thus to control the progress of the reactions. Because of its simplicity and high efficiency, this approach may represent an interesting alternative for biotechnological processes involving macromolecular or solid substrates. 相似文献
3.
Norbert Strter William N. Lipscomb Thomas Klabunde Bernt Krebs 《Angewandte Chemie (International ed. in English)》1996,35(18):2024-2055
Numerous studies, both in enzymatic and nonenzymatic catalysis, have been undertaken to understand the way by which metal ions, especially zinc ions, promote the hydrolysis of phosphate ester and amide bonds. Hydrolases containing one metal ion in the active site, termed mononuclear metallohydrolases, such as carboxypeptidase. A and thermolysin were among the first enzymes to have their structures unraveled by X-ray crystallography. In recent years an increasing number of metalloenzymes have been identified that use two or more adjacent metal ions in the catalysis of phosphoryl-transfer reactions (R-OPO3 + R′-OH → R′-OPO3 + R-OH; in the case of the phosphatase reaction R′-OH is a water molecule) and carbonyl-transfer reactions, for example, in peptidases or other amidases. These dinuclear metalloenzymes catalyze a great variety of these reactions, including hydrolytic cleavage of phosphomono-, -di- and -triester bonds, phosphoanhydride bonds as well as of peptide bonds or urea. In addition, the formation of the phosphodiester bond of RNA and DNA by polymerases is catalyzed by a two-metal ion mechanism. A remarkable diversity is also seen in the structures of the active sites of these di- and trinuclear metalloenzymes, even for enzymes that catalyze very similar reactions. The determination of the structure of a substrate, product, stable intermediate, or a reaction coordinate analogue compound bound to an active or inactivated enzyme is a powerful approach to investigate mechanistic details of enzyme action. Such studies have been applied to several of the metalloenzymes reviewed in this article; together with many other biochemical studies they provide a growing body of information on how the two (or more) metal ions cooperate to achieve efficient catalysis. 相似文献
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We present a simple model to rationalize the effects of environment on the reaction barrier frequencies derived from free energy profiles. These frequencies are relevant in deviations of a rate constant from its transition state theory value and in determining which environmental dynamics participate in the reaction. In particular, this simple model can be used to understand the changes in the reaction barrier frequencies of an enzymatic catalyzed reaction and the corresponding uncatalyzed process in aqueous solution, a change which has implications for dynamical environmental effects on the enzymatic reaction. Two possible cases are analyzed, in which the polarity (charge separation/localization) of the reacting system increases or decreases as the reaction advances. A simple modeling of the environment′s effects allows the explanation of an unusual “inverse” effect on the reaction frequencies, that is, a free energy barrier lowering accompanied by an increase of the reaction frequency, a behavior observed in some enzymes. The model predictions are successfully compared with results from full simulations for four different enzyme reactions. 相似文献
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In the present paper, the reaction of chemical immobilization of catalase on a crosslinked macromolecular carrier of a polysaccharide structure (gellan) is studied. The influence of some reaction parameters (enzyme/carrier, activator/carrier ratios, duration) on the activity of enzymatic products is analyzed. The kinetics of the biocatalytic process, stability under different pH and temperature conditions, and the inhibitors effect were studied for the immobilized enzymes. 相似文献
8.
Su‐Juan Li Chen Wang Zeng‐Qiang Wu Jing‐Juan Xu Prof. Xing‐Hua Xia Prof. Hong‐Yuan Chen Prof. 《Chemistry (Weinheim an der Bergstrasse, Germany)》2010,16(33):10186-10194
To understand the fundamentals of enzymatic reactions confined in micro‐/nanosystems, the construction of a small enzyme reactor coupled with an integrated real‐time detection system for monitoring the kinetic information is a significant challenge. Nano‐enzyme array reactors were fabricated by covalently linking enzymes to the inner channels of a porous anodic alumina (PAA) membrane. The mechanical stability of this nanodevice enables us to integrate an electrochemical detector for the real‐time monitoring of the formation of the enzyme reaction product by sputtering a thin Pt film on one side of the PAA membrane. Because the enzymatic reaction is confined in a limited nanospace, the mass transport of the substrate would influence the reaction kinetics considerably. Therefore, the oxidation of glucose by dissolved oxygen catalyzed by immobilized glucose oxidase was used as a model to investigate the mass‐transport‐related enzymatic reaction kinetics in confined nanospaces. The activity and stability of the enzyme immobilized in the nanochannels was enhanced. In this nano‐enzyme reactor, the enzymatic reaction was controlled by mass transport if the flux was low. With an increase in the flux (e.g., >50 μL min?1), the enzymatic reaction kinetics became the rate‐determining step. This change resulted in the decrease in the conversion efficiency of the nano‐enzyme reactor and the apparent Michaelis–Menten constant with an increase in substrate flux. This nanodevice integrated with an electrochemical detector could help to understand the fundamentals of enzymatic reactions confined in nanospaces and provide a platform for the design of highly efficient enzyme reactors. In addition, we believe that such nanodevices will find widespread applications in biosensing, drug screening, and biochemical synthesis. 相似文献
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Apostolakis J Sacher O Körner R Gasteiger J 《Journal of chemical information and modeling》2008,48(6):1190-1198
The correct identification of the reacting bonds and atoms is a prerequisite for the analysis of the reaction mechanism. We have recently developed a method based on the Imaginary Transition State Energy Minimization approach for automatically determining the reaction center information and the atom-atom mapping numbers. We test here the accuracy of this ITSE approach by comparing the predictions of the method against more than 1500 manually annotated reactions from BioPath, a comprehensive database of biochemical reactions. The results show high agreement between manually annotated mappings and computational predictions (98.4%), with significant discrepancies in only 24 cases out of 1542 (1.6%). This result validates both the computational prediction and the database, at the same time, as the results of the former agree with expert knowledge and the latter appears largely self-consistent, and consistent with a simple principle. In 10 of the discrepant cases, simple chemical arguments or independent literature studies support the predicted reaction center. In five reaction instances the differences in the automatically and manually annotated mappings are described in detail. Finally, in approximately 200 cases the algorithm finds alternate reaction centers, which need to be studied on a case by case basis, as the exact choice of the alternative may depend on the enzyme catalyzing the reaction. 相似文献
11.
V. Podzemná M. Slováková L. Kourková L. Svoboda 《Journal of Thermal Analysis and Calorimetry》2010,101(2):715-719
In this article, the monitoring of an enzymatic reaction by means of a miniaturized batch type IC-calorimeter was performed.
The aim of this work was focused on an investigation of enthalpy and rate of enzymatic reaction of trypsin with Nα-benzoyl-l-arginine-p-nitroanilide hydrochloride (BApNA). Both the parameters were determined for reactions in different buffers and for varying
concentration of enzyme at 37 °C. The rate of reaction decreased with the increasing concentration of enzyme caused by trypsin
autolysis. 相似文献
12.
Reitz M von Homeyer A Gasteiger J 《Journal of chemical information and modeling》2006,46(6):2333-2341
A method for the generation of intermediates of enzyme-catalyzed reactions is presented. These intermediates can be used as three-dimensional structural queries for searching for inhibitors of enzymatic reactions. The intermediates can be considered as being structurally quite close to transition-state analogues. For this application, a database containing detailed chemical information on metabolic reactions is used. The likely three-dimensional structure of the intermediates of enzyme-catalyzed reactions can be generated from the information in the database. For three reactions catalyzed by the enzymes AMP deaminase (EC code 3.5.4.6), triose phosphate isomerase (EC code 5.3.1.1), and arginase II (EC code 3.5.3.1), we show how a 3D model of these intermediates can be superimposed onto known inhibitors of these enzymes by a program that uses a genetic algorithm. For this, we test different methods for the superimposition using information on the enzymatic binding site, using information on physicochemical properties calculated from the molecular structure, or without having any information in the superimposition process. We show that these inhibitors are most similar to the corresponding intermediates regarding the 3D structure. 相似文献
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Priti Kumar Roy Sumit Nandi Mithun Kumar Ghosh 《Journal of mathematical chemistry》2013,51(10):2704-2717
Double substrate enzyme kinetics has a leading role for product quantification and optimization in different chemical and biochemical sectors. Mathematical approach for controlling these reactions in different stages by suitable parameters adds a new dimension in this interdisciplinary field of research. Applying control theoretic approach in the reversible backward stages of double substrate enzymatic model, time economization with regard to product formation is significant. In this article, we formulate a double substrate mathematical model of enzymatic dynamical reaction system with control measures with a view to observe the effect of changes of these measures with respect to the concentration of substrates, enzyme, complexes and finally product. Here, Pontryagin Minimum Principle is used for observing the effect of control measures in the system dynamics with the help of Hamiltonian. We compare the relevant numerical solutions for the substrates, enzyme, complexes and product concentration profile for a specified time interval with respect to control factors. 相似文献
15.
This was the continuation of our previous study along the same line with more focus on technical details because the data
are usually divided into two datasets, one for model development and the other for model validation during the development
of predictive model. The widely used validation method is the delete-1 jackknife validation. However, no systematical studies
were conducted to determine whether the jackknife validation with different deletions works better because the number of validations
with different deletions increases in a factorial fashion. Therefore it is only small dataset that can be used for such an
exhausted study. Cellulase is an enzyme playing an important role in modern industry, and many parameters related to cellulase
in enzymatic reactions were poorly documented. With increased interests in cellulases in bio-fuel industry, the prediction
of parameters in enzymatic reactions is listed on agenda. In this study, two aims were defined (a) which amino acid property
works better to predict the temperature optimum and (b) with which deletion the jackknife validation works. The results showed
that the amino acid distribution probability works better in predicting the optimum temperature of catalytic reaction by cellulase,
and the delete-4, more precisely one-fifth deletion, jackknife validation works better. 相似文献
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Gel points, predicted using Ahmed‐Rolfes‐Stepto (ARS) theory and a Monte‐Carlo (MC) simulation method accounting for intramolecular reaction, are compared with experimental data for polyester (PES)‐, polyurethane (PU)‐ and poly(dimethyhl siloxane) (PDMS)‐forming polymerisations. The PES and PU polymerisations were from stoichiometric reactions at different initial dilutions and the PDMS ones were from critical‐ratio experiments at different dilutions of one reactant. The predictions use realistic chain statistics to define intramolecular reaction probabilities and employ no arbitrary parameters. Universal plots of excess reaction at gelation versus ring‐forming parameter are devised to enable the experimental data and theoretical predictions to be compared critically. It is shown that various gel points can be predicted by MC simulations, depending on the criterion for gelation used. Due to the lengthy computations needed and the uncertainties in the predictions, MC simulation is not a viable approach. Although inconsistencies are noted in the measured gel points, so that a unified interpretation of the data cannot be achieved, ARS theory is shown to be the preferred basis for gel‐point prediction. It is also concluded that, before one can be certain of agreement between experiment and predictions, more experimental systems at different initial dilutions and ratios of reactants need to be studied and the various methods used for detecting gel points need to be compared. 相似文献
18.
Rosgaard L Andric P Dam-Johansen K Pedersen S Meyer AS 《Applied biochemistry and biotechnology》2007,143(1):27-40
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. 相似文献
19.
We firstly transformed the traditional Michaelis-Menten equation into an off-line form which can be used for evaluating the Michaelis-Menten constant after the enzymatic reaction. For experimental estimation of the kinetics of enzymatic reactions, we have developed a facile and effective method by integrating an enzyme microreactor into direct-printing polymer microchips. Strong nonspecific adsorption of proteins was utilized to effectively immobilize enzymes onto the microchannel wall, forming the integrated on-column enzyme microreactor in a microchip. The properties of the integrated enzyme microreactor were evaluated by using the enzymatic reaction of glucose oxidase (GOx) with its substrate glucose as a model system. The reaction product, hydrogen peroxide, was electrochemically (EC) analyzed using a Pt microelectrode. The data for enzyme kinetics using our off-line form of the Michaelis-Menten equation was obtained (K(m) = 2.64 mM), which is much smaller than that reported in solution (K(m) = 6.0 mM). Due to the hydrophobic property and the native mesoscopic structure of the poly(ethylene terephthalate) film, the immobilized enzyme in the microreactor shows good stability and bioactivity under the flowing conditions. 相似文献
20.
Greving M Cheng X Reindl W Bowen B Deng K Louie K Nyman M Cohen J Singh A Simmons B Adams P Siuzdak G Northen T 《Analytical and bioanalytical chemistry》2012,403(3):707-711
Mass spectrometry (MS)-based enzyme assay has been shown to be a useful tool for screening enzymatic activities from environmental samples. Recently, reported approaches for high-specificity multiplexed characterization of enzymatic activities allow for providing detailed information on the range of enzymatic products and monitoring multiple enzymatic reactions. However, the throughput has been limited by the slow liquid-liquid handling and manual analysis. This rapid communication demonstrates the integration of acoustic sample deposition with nanostructure initiator mass spectrometry (NIMS) imaging to provide reproducible measurements of multiple enzymatic reactions at a throughput that is tenfold to 100-fold faster than conventional MS-based enzyme assay. It also provides a simple means for the visualization of multiple reactions and reaction pathways. 相似文献