scholarly journals Rapid Methods for High-Throughput Detection of Sulfoxides

2009 ◽  
Vol 75 (14) ◽  
pp. 4711-4719 ◽  
Author(s):  
Janna Shainsky ◽  
Netta-Lee Derry ◽  
Yael Leichtmann-Bardoogo ◽  
Thomas K. Wood ◽  
Ayelet Fishman
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Selective Inhibition ◽  
Saturation Mutagenesis ◽  
Screening Methods ◽  
Acid Activation ◽  
Special Equipment ◽  
Whole Cell ◽  
Microtiter Plates

ABSTRACT Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

From Classical to High Throughput Screening Methods for Feruloyl Esterases: A Review

2016 ◽  
Vol 19 (8) ◽  
pp. 616-626 ◽  
Author(s):  
Lorena Ramírez-Velasco ◽  
Mariana Armendáriz-Ruiz ◽  
Jorge Alberto Rodríguez-González ◽  
Marcelo Müller-Santos ◽  
Ali Asaff-Torres ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Methods ◽  
Feruloyl Esterases

scholarly journals High-throughput Screening and Sensitized Bacteria Identify an M. tuberculosis Dihydrofolate Reductase Inhibitor with Whole Cell Activity

PLoS ONE ◽  
2012 ◽  
Vol 7 (6) ◽  
pp. e39961 ◽  
Author(s):  
Anuradha Kumar ◽  
Meng Zhang ◽  
Linyun Zhu ◽  
Reiling P. Liao ◽  
Charles Mutai ◽  
...  
Keyword(s):  
High Throughput ◽  
Dihydrofolate Reductase ◽  
High Throughput Screening ◽  
Reductase Inhibitor ◽  
Cell Activity ◽  
Whole Cell

scholarly journals High throughput method development and optimised production of leaf protein concentrates with potential to support the agri-industry

2021 ◽  
Author(s):  
Ajay Iyer ◽  
Lisa Guerrier ◽  
Salomé Leveque ◽  
Charles S. Bestwick ◽  
Sylvia H. Duncan ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Extraction ◽  
Leaf Protein ◽  
Protein Recovery ◽  
Screening Methods ◽  
Extraction Technology ◽  
Protein Concentrates ◽  
Ethanol Precipitation

AbstractInvasive plants offer an interesting and unconventional source of protein and the considerable investment made towards their eradication can potentially be salvaged through their revalorisation. To identify viable sources, effective and high-throughput screening methods are required, as well as efficient procedures to isolate these components. Rigorous assessment of low-cost, high-throughput screening assays for total sugar, phenolics and protein was performed, and ninhydrin, Lever and Fast Blue assays were found to be most suitable owing to high reliability scores and false positive errors less than 1%. These assays were used to characterise invasive Scottish plants such as Gorse (Ulex europeans), Broom (Cystisus scoparius) and Fireweed (Chamaenerion angustifolium). Protein extraction (alkali-, heat- and enzyme assisted) were tested on these plants, and further purification (acid and ethanol precipitation, as well as ultrafiltration) procedures were tested on Gorse, based on protein recovery values. Cellulase treatment and ethanol precipitation gave the highest protein recovery (64.0 ± 0.5%) and purity (96.8 ± 0.1%) with Gorse. The amino acid profile of the purified protein revealed high levels of essential amino acids (34.8 ± 0.0%). Comparison of results with preceding literature revealed a strong association between amino acid profiles and overall protein recovery with the extraction method employed. The final purity of the protein concentrates was closely associated to the protein content of the initial plant mass. Leaf protein extraction technology can effectively raise crop harvest indices, revalorise underutilised plants and waste streams.

Evaluation of Methods To Assess the Biofilm-Forming Ability of Listeria monocytogenes

2012 ◽  
Vol 75 (8) ◽  
pp. 1411-1417 ◽  
Author(s):  
ANTÓNIO LOURENÇO ◽  
FRANCISCO REGO ◽  
LUISA BRITO ◽  
JOSEPH F. FRANK
Keyword(s):  
Stainless Steel ◽  
Listeria Monocytogenes ◽  
High Throughput ◽  
Biofilm Formation ◽  
High Throughput Screening ◽  
Screening Methods ◽  
Genetic Lineage ◽  
Production Lines ◽  
Genetic Characteristics

The contamination of ready-to-eat products with Listeria monocytogenes has been related to the presence of biofilms in production lines, as biofilms protect cells from chemical sanitizers. The ability of L. monocytogenes to produce biofilms is often evaluated using in vitro methodologies. This work aims to compare the most frequently used methodologies, including high-throughput screening methods based on microplates (crystal violet and the Calgary Biofilm Device) and methods based on CFU enumeration and microscopy after growth on stainless steel. Thirty isolates with diverse origins and genetic characteristics were evaluated. No (or low) correlations between methods were observed. The only significant correlation was found between the methods using stainless steel. No statistically significant correlation (P &gt; 0.05) was detected among genetic lineage, serovar, and biofilm-forming ability. Because results indicate that biofilm formation is influenced by the surface material, the extrapolation of results from high-throughput methods using microplates to more industrially relevant surfaces should be undertaken with caution.

High-throughput Screening Methods Developed for Oxidoreductases

Enzyme Assays ◽  
2006 ◽  
pp. 77-93 ◽  
Author(s):  
Tyler W. Johannes ◽  
Ryan D. Woodyer ◽  
Huimin Zhao
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Methods

scholarly journals High-Throughput Screening for Drugs That Inhibit Papain-Like Protease in SARS-CoV-2

2020 ◽  
Vol 25 (10) ◽  
pp. 1152-1161
Author(s):  
Emery Smith ◽  
Meredith E. Davis-Gardner ◽  
Ruben D. Garcia-Ordonez ◽  
Tu-Trinh Nguyen ◽  
Mitchell Hull ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Clinical Stage ◽  
Severe Pneumonia ◽  
Selective Inhibition ◽  
Peptide Cleavage ◽  
Global Pandemic ◽  
Drug Registration ◽  
Medical Countermeasures

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019 has triggered an ongoing global pandemic whereby infection may result in a lethal severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, millions of confirmed cases and hundreds of thousands of deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. The purported development of a vaccine could require at least 1–4 years, while the typical timeline from hit finding to drug registration of an antiviral is >10 years. Thus, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we developed and initiated a high-throughput cell-based screen that incorporates the essential viral papain-like protease (PLpro) and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or US Food and Drug Administration (FDA)-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Here, we report the identification of four clinically relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral PLpro.

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