scholarly journals Hydrogel Microvalves as Control Elements for Parallelized Enzymatic Cascade Reactions in Microfluidics

Micromachines ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 167
Author(s):  
Franziska Obst ◽  
Anthony Beck ◽  
Chayan Bishayee ◽  
Philipp J. Mehner ◽  
Andreas Richter ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Microfluidic Devices ◽  
Cascade Reactions ◽  
Stimuli Responsive ◽  
Enzyme Cascade ◽  
Enzyme Substrate ◽  
Vis Spectroscopy ◽  
Substrate Conversion ◽  
Path Dependent ◽  
Fluid Pathway

Compartmentalized microfluidic devices with immobilized catalysts are a valuable tool for overcoming the incompatibility challenge in (bio) catalytic cascade reactions and high-throughput screening of multiple reaction parameters. To achieve flow control in microfluidics, stimuli-responsive hydrogel microvalves were previously introduced. However, an application of this valve concept for the control of multistep reactions was not yet shown. To fill this gap, we show the integration of thermoresponsive poly(N-isopropylacrylamide) (PNiPAAm) microvalves (diameter: 500 and 600 µm) into PDMS-on-glass microfluidic devices for the control of parallelized enzyme-catalyzed cascade reactions. As a proof-of-principle, the biocatalysts glucose oxidase (GOx), horseradish peroxidase (HRP) and myoglobin (Myo) were immobilized in photopatterned hydrogel dot arrays (diameter of the dots: 350 µm, amount of enzymes: 0.13–2.3 µg) within three compartments of the device. Switching of the microvalves was achieved within 4 to 6 s and thereby the fluid pathway of the enzyme substrate solution (5 mmol/L) in the device was determined. Consequently, either the enzyme cascade reaction GOx-HRP or GOx-Myo was performed and continuously quantified by ultraviolet-visible (UV-Vis) spectroscopy. The functionality of the microvalves was shown in four hourly switching cycles and visualized by the path-dependent substrate conversion.

scholarly journals Microfluidics Featuring Multilayered Hydrogel Assemblies Enable Real-Time NMR-Monitoring of Enzyme Cascade Reactions

2020 ◽  
Author(s):  
Nurdiana Nordin ◽  
lorenzo bordonali ◽  
Hossein Davoodi ◽  
Novindi Dwi Ratnawati ◽  
Gudrun Gygli ◽  
...  
Keyword(s):  
Real Time ◽  
Functional Performance ◽  
Cascade Reactions ◽  
Inhibitory Effect ◽  
Resonance Spectroscopy ◽  
Reaction Cell ◽  
Enzyme Cascade ◽  
Sensing Applications ◽  
Progress Curve ◽  
Michaelis Constants

Compartmentalized chemical reactions at the microscale are interesting from many perspectives including (multi)functional surfaces and biotechnology. Monitoring the molecular content as a measure of functional performance at these small scales is challenging. As a means to address this challenge, we leverage microtechnology and biocompatible materials to integrate a compact, reconfigurable reaction cell featuring electrochemical functionality with high-resolution nuclear magnetic resonance spectroscopy (NMR). We demonstrate the operation of this system by monitoring the activity of enzymes immobilized in chemically distinct layers within a multi-layered chitosan hydrogel assembly. As a benchmark, we observed the parallel activities of urease (Urs), catalase (Cat), and glucose oxidase (GOx) by recording NMR spectra to extract reagent and product concentrations in real-time. As a result, simultaneous monitoring of a cooperative enzymatic process (GOx + Cat) together with an independent process (Urs) is achieved. Using Michaelis-Menten progress curve analysis of the NMR data, kinetic data is extracted: in the case of GOx, the Michaelis constants (K<sub>M</sub>) are consistent with previous reports, while for Urs, deviations are observed, attributed to an inhibitory effect under our reaction conditions. The system therefore enables the construction of complex reaction cascades with spatial control, as would be interesting in, for example, metabolic engineering and multiplexed sensing applications.

Multi‐enzyme Cascade Reactions in Metal‐organic Frameworks

2020 ◽  
Vol 20 (10) ◽  
pp. 1100-1116 ◽  
Author(s):  
Jieying Liang ◽  
Kang Liang
Keyword(s):  
Metal Organic Frameworks ◽  
Cascade Reactions ◽  
Enzyme Cascade ◽  
Metal Organic

scholarly journals Recent Advances in Enzymatic and Chemoenzymatic Cascade Processes

Catalysts ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1258
Author(s):  
Noelia Losada-Garcia ◽  
Zaida Cabrera ◽  
Paulina Urrutia ◽  
Carla Garcia-Sanz ◽  
Alicia Andreu ◽  
...  
Keyword(s):  
Organic Chemistry ◽  
Solid Phase ◽  
Cascade Reactions ◽  
Review Article ◽  
The Novel ◽  
Enzyme Cascade ◽  
Cascade Processes ◽  
Novel Strategy ◽  
Substantial Interest

Cascade reactions have been described as efficient and universal tools, and are of substantial interest in synthetic organic chemistry. This review article provides an overview of the novel and recent achievements in enzyme cascade processes catalyzed by multi-enzymatic or chemoenzymatic systems. The examples here selected collect the advances related to the application of the sequential use of enzymes in natural or genetically modified combination; second, the important combination of enzymes and metal complex systems, and finally we described the application of biocatalytic biohybrid systems on in situ catalytic solid-phase as a novel strategy. Examples of efficient and interesting enzymatic catalytic cascade processes in organic chemistry, in the production of important industrial products, such as the designing of novel biosensors or bio-chemocatalytic systems for medicinal chemistry application, are discussed

scholarly journals Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

2004 ◽  
Vol 383 (3) ◽  
pp. 551-559 ◽  
Author(s):  
Sheraz GUL ◽  
Richard BROWN ◽  
Earl MAY ◽  
Marie MAZZULLA ◽  
Martin G. SMYTH ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dna Ligase ◽  
Protection Assay ◽  
Dna Ligases ◽  
Enzyme Substrate ◽  
Ic50 Values ◽  
Acridinium Ester

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the Km values for NAD+ (2.75±0.1 μM) and the acridinium-ester-labelled DNA substrate (2.5±0.2 nM). A study of the pH-dependencies of kcat, Km and kcat/Km has revealed values of kinetically influential ionizations within the enzyme–substrate complexes (kcat) and free enzyme (kcat/Km). In each case, the curves were shown to be composed of one kinetically influential ionization, for kcat, pKa=6.6±0.1 and kcat/Km, pKa=7.1±0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30±0.86 μM for doxorubicin and 1.40±0.07 μM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 μl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

Enzyme cascade reactions: synthesis of furandicarboxylic acid (FDCA) and carboxylic acids using oxidases in tandem

2015 ◽  
Vol 17 (6) ◽  
pp. 3271-3275 ◽  
Author(s):  
Shane M. McKenna ◽  
Silke Leimkühler ◽  
Susanne Herter ◽  
Nicholas J. Turner ◽  
Andrew J. Carnell
Keyword(s):  
Carboxylic Acids ◽  
Cascade Reactions ◽  
Preparative Scale ◽  
Mild Conditions ◽  
Enzyme Cascade ◽  
Furandicarboxylic Acid

Three enzymes are combined under mild conditions for the preparative scale oxidation of HMF to FDCA and a range of 10 alcohols.

scholarly journals Multi-enzyme cascade reactions using protein–polymer surfactant self-standing films

2017 ◽  
Vol 53 (13) ◽  
pp. 2094-2097 ◽  
Author(s):  
Thomas Farrugia ◽  
Adam W. Perriman ◽  
Kamendra P. Sharma ◽  
Stephen Mann
Keyword(s):  
Cascade Reactions ◽  
Hierarchical Assembly ◽  
Protein Polymer ◽  
Enzyme Cascade ◽  
Catalytically Active ◽  
Polymer Surfactant

Self-supporting bio-catalytically active multi-enzyme films fabricated via hierarchical assembly of enzyme–polymer surfactant nanoconjugates are capable of sustaining cascade reactions.

Fusion proteins of an enoate reductase and a Baeyer-Villiger monooxygenase facilitate the synthesis of chiral lactones

2017 ◽  
Vol 398 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Christin Peters ◽  
Florian Rudroff ◽  
Marko D. Mihovilovic ◽  
Uwe T. Bornscheuer
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Reaction Rates ◽  
Cascade Reactions ◽  
Product Formation ◽  
Similar Reaction ◽  
Enzyme Cascade ◽  
Enoate Reductase ◽  
Chiral Lactones

Abstract Nature uses the advantages of fusion proteins for multi-step reactions to facilitate the metabolism in cells as the conversion of substrates through intermediates to the final product can take place more rapidly and with less side-product formation. In a similar fashion, also for enzyme cascade reactions, the fusion of biocatalysts involved can be advantageous. In the present study, we investigated fusion of an alcohol dehydrogenase (ADH), an enoate reductase (ERED) and a Baeyer-Villiger monooxygenase (BVMO) to enable the synthesis of (chiral) lactones starting from unsaturated alcohols as substrates. The domain order and various linkers were studied to find optimal conditions with respect to expression levels and enzymatic activities. Best results were achieved for the ERED xenobiotic reductase B (XenB) from Pseudomonas putida and the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp., whereas none of the ADHs studied could be fused successfully. This fusion protein together with separately supplied ADH resulted in similar reaction rates in in vivo biocatalysis reactions. After 1.5 h we could detect 40% more dihydrocarvone lactone in in vivo reactions with the fusion protein and ADH then with the single enzymes.

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