Browse
Submit Data
Databases
API
Help

Dataset Information

18 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Automated Determination of Oxygen-Dependent Enzyme Kinetics in a Tube-in-Tube Flow Reactor.

ABSTRACT: Enzyme-mediated oxidation is of particular interest to synthetic organic chemists. However, the implementation of such systems demands knowledge of enzyme kinetics. Conventionally collecting kinetic data for biocatalytic oxidations is fraught with difficulties such as low oxygen solubility in water and limited oxygen supply. Here, we present a novel method for the collection of such kinetic data using a pressurized tube-in-tube reactor, operated in the low-dispersed flow regime to generate time-series data, with minimal material consumption. Experimental development and validation of the instrument revealed not only the high degree of accuracy of the kinetic data obtained, but also the necessity of making measurements in this way to enable the accurate evaluation of high K_MO enzyme systems. For the first time, this paves the way to integrate kinetic data into the protein engineering cycle.

SUBMITTER: Ringborg RH

PROVIDER: S-EPMC5768025 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Automated Determination of Oxygen-Dependent Enzyme Kinetics in a Tube-in-Tube Flow Reactor.

Ringborg Rolf H RH Toftgaard Pedersen Asbjørn A Woodley John M JM

ChemCatChem 20170810 17

Enzyme-mediated oxidation is of particular interest to synthetic organic chemists. However, the implementation of such systems demands knowledge of enzyme kinetics. Conventionally collecting kinetic data for biocatalytic oxidations is fraught with difficulties such as low oxygen solubility in water and limited oxygen supply. Here, we present a novel method for the collection of such kinetic data using a pressurized tube-in-tube reactor, operated in the low-dispersed flow regime to generate time- ...[more]

PMID: 29399209

Similar Datasets

Kinetics Studies on a Multicomponent Knoevenagel-Michael Domino Reaction by an Automated Flow Reactor.

Project description:The optimization of complex chemical reaction systems is often a troublesome and time-consuming process. The application of modern technologies, including automated reactors and analytics, opens the avenue for generating large data sets on chemical reaction processes in a short period of time. In this work, an automated flow reactor is used to present detailed kinetics and mechanistic studies about an amine-catalyzed Knoevenagel-Michael domino reaction to yield tetrahydrochromene derivatives. High-performance monoliths as catalyst supports and online coupled HPLC analysis allow for time-efficient data generation. We show that the two-step multicomponent domino reaction does not follow the kinetics of consecutive reaction steps proceeding independently from each other. Instead, the starting materials of both individual reactions compete for the active sites on the heterogeneous catalyst, which lowers the rate constants of both steps. This knowledge was used to implement a more efficient experimental setup which increased the turnover numbers of the catalyst, without adjusting common reaction parameters like temperature, reaction time, and concentrations.

| S-EPMC6511915 | biostudies-literature

Automated flow injection method for the high precision determination of drift tube ion mobility collision cross sections.

Project description:The field of ion mobility-based omics studies requires high-quality collision cross section (CCS) libraries to effectively utilize CCS as a molecular descriptor. Absolute CCS values with the highest precision are obtained on drift tube instruments by measuring the drift time of ions at multiple drift voltages, commonly referred to as a 'stepped field' experiment. However, generating large scale absolute CCS libraries from drift tube instruments is time consuming due to the current lack of high-throughput methods. This communication reports a fully automated stepped-field method to acquire absolute CCS on commercially available equipment. Using a drift tube ion mobility-mass spectrometer (DTIM-MS) coupled to a minimally modified liquid chromatography (LC) system, CCS values can be measured online with a carefully timed flow injection analysis (FIA) experiment. Results demonstrate that the FIA stepped-field method yields CCS values which are of high analytical precision (<0.4% relative standard deviation, RSD) and accuracy (?0.4% difference) comparable to CCS values obtained using traditional direct-infusion stepped-field experiments. This high-throughput CCS method consumes very little sample volume (20 ?L) and will expedite the generation of large-scale CCS libraries to support molecular identification within global untargeted studies.

| S-EPMC5869168 | biostudies-literature

Catalytic Chan-Lam coupling using a 'tube-in-tube' reactor to deliver molecular oxygen as an oxidant.

Project description:A flow system to perform Chan-Lam coupling reactions of various amines and arylboronic acids has been realised employing molecular oxygen as an oxidant for the re-oxidation of the copper catalyst enabling a catalytic process. A tube-in-tube gas reactor has been used to simplify the delivery of the oxygen accelerating the optimisation phase and allowing easy access to elevated pressures. A small exemplification library of heteroaromatic products has been prepared and the process has been shown to be robust over extended reaction times.

| S-EPMC4979635 | biostudies-literature

An automated method to evaluate the enzyme kinetics of ?-glucosidases.

Project description:Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize ?-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time-consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of ?-glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.

| S-EPMC5275727 | biostudies-literature

Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels.

Project description:The availability of oxygen (O(2)) is a critical parameter affecting vascular tube formation. In this study, we hypothesize that dissolved oxygen (DO) levels in collagen gels change during the three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs) in atmospheric conditions and that such changes affect the kinetics of tube formation through the production of reactive oxygen species (ROS). We demonstrate a decrease in O(2) tension during 3D cultures of HUVECs. Noninvasive measurements of DO levels during culture under atmospheric conditions revealed a profound decrease that reached as low as 2% O(2) at the end of 24 h. After media replacement, DO levels rose rapidly and equilibrated at ?15% O(2), creating a reoxygenated environment. To accurately estimate DO gradients in 3D collagen gels, we developed a 3D mathematical model and determined the Michaelis-Menten parameters, V(max) and K(m), of HUVECs in collagen gels. We detected an increase in ROS levels throughout the culture period. Using diphenyliodonium to inhibit ROS production resulted in the complete inhibition of tube formation. Interference RNA studies further showed that hypoxia-inducible factors (HIFs)-1? and -2? are not involved in the formation of 3D tubes in collagen gels. We conclude that ROS affect the tubulogenesis process through HIF?-independent pathways, where the levels of ROS are influenced by the uncontrolled variations in DO levels. This study is the first demonstration of the critical and unexpected role of O(2) during 3D in vitro culture models of tubulogenesis in atmospheric conditions.

| S-EPMC3154554 | biostudies-literature

Automated Glycan Assembly in a Variable-Bed Flow Reactor Provides Insights into Oligosaccharide-Resin Interactions.

Project description:A pressure-based variable-bed flow reactor built for peptide synthesis and capable of real-time monitoring of resin swelling was adapted for automated glycan assembly. In the context of the solid-phase synthesis of several oligosaccharides, the coupling efficiencies, resin growth patterns, and saccharide solvation during the synthesis were determined. The presented work provides the first estimation of on-resin oligosaccharide solvation and an alternative technique to UV-vis monitoring.

| S-EPMC7281783 | biostudies-literature

Automated in vitro evolution of a translation-coupled RNA replication system in a droplet flow reactor.

Project description:Automation is a useful strategy to make laborious evolutionary experiments faster and easier. To date, several types of continuous flow reactors have been developed for the automated evolutionary experiments of viruses and bacteria. However, the development of a flow reactor applicable to compartmentalized in vitro self-replication systems is still a challenge. In this study, we demonstrate automated in vitro evolution of a translation-coupled RNA system in a droplet flow reactor for the first time. This reactor contains approximately 1010 micro-scale droplets (average diameter is approximately 0.8 ?m), which continuously fuse and divide among each other at a controllable rate. In the droplets, an RNA (artificial genomic RNA) replicate through the translation of self-encoded RNA replicase with spontaneously appearing parasitic RNA. We performed two automated replication experiments for more than 400?hours with different mixing intensities. We found that several mutations displayed increased frequencies in the genomic RNA populations and the dominant RNA mutants acquired the ability to replicate faster or acquired resistance to the parasitic RNA, demonstrating that Darwinian evolution occurred during the long-term replication. The droplet flow reactor we developed can be a useful tool to perform in vitro evolutionary experiments of translation-coupled systems.

| S-EPMC6082869 | biostudies-other

Rapid determination of enzyme kinetics from fluorescence: overcoming the inner filter effect.

Project description:Fluorescence change is convenient for monitoring enzyme kinetics. Unfortunately, it loses linearity as the absorbance of the fluorescent substrate increases with concentration. When the sum of absorbance at excitation and emission wavelengths exceeds 0.08, this inner filtering effect (IFE) alters apparent initial velocities, K(m), and k(cat). The IFE distortion of apparent initial velocities can be corrected without doing fluorophore dilution assays. Using the substrate's extinction coefficients at excitation and emission wavelengths, the inner filter effect can be modeled during curve fitting for more accurate Michaelis-Menten parameters. A faster and simpler approach is to derive k(cat) and K(m) from progress curves. Strategies to obtain reliable and reproducible estimates of k(cat) and K(m) from only two or three progress curves are illustrated using matrix metalloproteinase 12 and alkaline phosphatase. Accurate estimates of concentration of enzyme-active sites and specificity constant k(cat)/K(m) (from one progress curve with [S]<<K(m)) confer accuracy, freedom of choices of [S], and robustness to k(cat) and K(m) globally fitted to a few progress curves. The economies of the progress curve approach make accurate k(cat) and K(m) more accessible from fluorescence measurements.

| S-EPMC2211277 | biostudies-literature

The kinetics of TEM1 antibiotic degrading enzymes that are displayed on Ure2 protein nanofibrils in a flow reactor.

Project description:Enzymatic functionalization of cross-? structured protein nanofibrils has hitherto resulted in a severe reduction of the catalytic efficiency of high turnover biocatalysts. It has been speculated that steric restrictions and mass transport pose limits on the attached enzymes, but detailed kinetics analyzing this have not yet been reported. For a more comprehensive understanding, we studied protein nanofibrils endowed with TEM1, a ?-lactamase from Escherichia coli. The packing density of TEM1 along the fibrils was controlled by co-fibrillation; in other words, the N-terminal ureidosuccinate transporter Ure2(1-80) from Saccharomyces cerevisiae was simultaneously aggregated with the chimeric proteins TEM1-Ure2(1-80). The mature fibrils were trapped in a column, and the rate of ampicillin hydrolysis was recorded using a continuous substrate flow. The turnover rate was plotted as a function of substrate molecules available per enzyme per second, which demonstrated that an elevated substrate availability counteracts mass transport limitations. To analyze this data set, we derived a kinetic model, which makes it possible to easily characterize and compare enzymes packed in columns. The functional TEM1 nanofibrils possess 80% of the catalytic turnover rate compared to free TEM1 in solution. Altogether, we have created protein nanofibrils that can effectively hydrolyze ?-lactam antibiotic contaminations and provided a groundwork strategy for other highly functional enzymatic nanofibrils.

| S-EPMC5912753 | biostudies-literature

Stopped-flow kinetics of oxygen uptake and release by embryonic red blood cells.

Project description:The processes of O2 uptake and release by the three embryonic haemoglobins contained within early mouse embryonic red blood cells have been studied using dual-wavelength stopped-flow kinetic spectroscopy. The rate of O2 uptake in the pseudo-spherical, nucleated, embryonic red blood cells exhibits a greater than first-order dependence on O2 concentration. The time courses for the release from the red blood cells into dithionite-containing solutions tends towards a limiting rate at high dithionite concentrations. The rates of both the uptake and release processes observed in the embryonic cells are compared with those previously seen for adult mouse red blood cells. A new mathematical model is described which accurately simulates both uptake and release experimental data for the nucleated embryonic red blood cells.

| S-EPMC1138929 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data