Development of Fluorescent Assay for Monitoring of Dehalogenase Activity

Nevolova, Sarka; Manaskova, Elisabet; Mazurenko, Stanislav; Damborský, Jiřı́; Prokop, Zbyněk

doi:10.1002/biot.201800144

Cited by 9 publications

(8 citation statements)

References 20 publications

(16 reference statements)

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“…8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). 27 The reaction mixture acidification results in a decrease of the fluorescence signal that is monitored with a planar laser-induced fluorescence by a single optical fibre. 28 A reaction progress as an end-point measurement is recorded after passing through the incubation chamber inside a detection cube bringing the tubing and the optical fiber to a close proximity at a perpendicular orientation (Figure 2A).…”

Section: Resultsmentioning

confidence: 99%

Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics

et al. 2019

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A number of novel protein sequences dramatically increases in public databases, however, there are only very few functional annotations. This significantly limits the exploration of the available protein diversity obtained by next-generation sequencing. Conventional methods cannot keep up with current challenges and a development of a new generation of biochemical techniques is essential. The great potential of microfluidic technology with reduced sample requirements and powerful throughput can bring an adequate capacity for functional annotation of the rising protein diversity. However, even the most promising dropletbased systems still need to address a number of limitations. The leakage of hydrophobic compounds from water compartments to the carrier oil represents one of the major problems which limits the utilization of these systems to operate with hydrophilic reagents. Herein, we present an approach using a novel way of substrate delivery in droplet microfluidics applied to high-throughput functional characterization of enzymes converting hydrophobic substrates. The substrate delivery is based on the partitioning of hydrophobic chemicals between the oil and water phases. We applied a controlled distribution of hydrophobic haloalkanes from oil to reaction water droplets to perform activity screening of eight model enzymes from the haloalkane dehalogenase family. The droplet-on-demand microfluidic system reduces the reaction volume 65 000 times and increases the speed of the analysis almost 100 times, compared to the conventional method. Additionally, the microfluidic setup enables a convenient determination of temperature optima for a set of mesophilic and engineered hyper stable enzyme variants at the working range 5 to 90 °C. A quantitative comparison of the microfluidic data and the results from the the conventional method showed a high consistency with R 2 = 0.89 and 0.95 for the substrate specificity and temperature optima analysis, respectively. The microfluidic method demonstrated a high precision and an advanced analytical throughput >20,000 reactions per day. The presented substrate delivery approach extends the scope of microfluidics applications for high-throughput analysis of reactions including compounds with limited water solubility.

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Section: Resultsmentioning

confidence: 99%

Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics

et al. 2019

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“…For this reason, the Iwasaki assay for halide ions has remained popular in dehalogenase assays, despite the involvement of extremely toxic mercury derivatives . In microtiter plate format, variations of the Iwasaki assay were reported to have detection limits for halide ions between 20 and 270 μM …”

Section: Introductionmentioning

confidence: 99%

An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation

et al. 2020

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Halide assays are important for the study of enzymatic dehalogenation, a topic of great industrial and scientific importance. Here we describe the development of a very sensitive halide assay that can detect less than a picomole of bromide ions, making it very useful for quantifying enzymatic dehalogenation products. Halides are oxidised under mild conditions using the vanadium‐dependent chloroperoxidase from Curvularia inaequalis, forming hypohalous acids that are detected using aminophenyl fluorescein. The assay is up to three orders of magnitude more sensitive than currently available alternatives, with detection limits of 20 nM for bromide and 1 μM for chloride and iodide. We demonstrate that the assay can be used to determine specific activities of dehalogenases and validate this by comparison to a well‐established GC‐MS method. This new assay will facilitate the identification and characterisation of novel dehalogenases and may also be of interest to those studying other halide‐producing enzymes.

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“…We evaluated their photophysical properties and compared their sensitivity with the commercially available fluorescence pH-sensor 8-hydroxy-pyrene-1,3,6-trisulfonic acid (HPTS), previously applied in monitoring the enzymatic activity at near-alkaline conditions. 30,31 We demonstrate the applicability of the selected derivatives to monitor the activity of a model enzyme, HLDs. The application of the new pH sensors compatible with the enzymes operating at alkaline conditions will enable the development of advanced sensitive biosensors, 30,31 high throughput screening for novel biocatalysts, 9 and sensitive microfluidic devices for advanced enzymology research and engineering.…”

Section: ■ Introductionmentioning

confidence: 99%

Fluorescent pH Indicators for Neutral to Near-Alkaline Conditions Based on 9-Iminopyronin Derivatives

et al. 2019

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Monitoring enzymatic activities at pH ranges compatible with their physiological optimum using fluorescent assays is important for high-throughput screening and engineering of novel biocatalysts as well as applications in biosensing and diagnostics. However, the number of fluorescent dyes exhibiting necessary optical properties at alkaline pHs is limited. Here, we report the design, synthesis, and biochemical application of fluorescent 9-iminopyronin derivatives possessing unique and distinct emission properties to provide optimal sensitivity at neutral to near-alkaline conditions. They exist in two acid−base forms, allowing for a dual-emission ratiometric sensing with a linear response of pH between 6.0 and 8.5 and detection sensitivity of a 0.06 pH unit. Selected fluorophores were used as the sensors for monitoring the enzymatic hydrolytic activity of a model enzyme haloalkane dehalogenase.

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Development of Fluorescent Assay for Monitoring of Dehalogenase Activity

Cited by 9 publications

References 20 publications

Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics

Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics

An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation

Fluorescent pH Indicators for Neutral to Near-Alkaline Conditions Based on 9-Iminopyronin Derivatives

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