Optimization of Spectrophotometric and Fluorometric Assays Using Alternative Substrates for the High-Throughput Screening of Lipase Activity

The effects of reaction conditions on the spectrophotometric and fluorometric assays using alternative substrates (p-nitrophenyl palmitate and 4-methylumbelliferyl oleate) were investigated to optimize them for the high-throughput screening of lipase activity from agricultural products. Four model lipases from Chromobacterium viscosum, Pseudomonas fluorescens, Sus scrofa pancreas, and wheat germ (Triticum aestivum) were allowed to hydrolyze the alternative substrates at different substrate concentrations (1–5 mM), operating pH (5.0–8.0), and operating temperatures (25–55°C). The results show that both the spectrophotometric and fluorometric assays worked well at the standard reaction conditions (pH 7.0 and 30°C) for finding a typical lipase, although pH conditions should be considered to detect the catalytic activity of lipases, which are applicable to more acidic or alkaline pH circumstances. To validate the optimized conditions, the high-throughput screening of lipase activity was conducted using 17 domestic agricultural products. A pileus of Pleurotus eryngii showed the highest activity in both the spectrophotometric (633.42 μU/mg) and fluorometric (101.77 μU/mg) assays. The results of this research provide practical information for the high-throughput screening of lipases using alternative substrates on microplates.

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Analysis of Complexation Interactions between Metal Ions and Drugs under Pseudo-physiological pH Conditions by a High-throughput Screening Method Using a Solid-phase Extraction Cartridge

Analytical Sciences ◽

10.2116/analsci.19p413 ◽

2020 ◽

Vol 36 (6) ◽

pp. 709-715

Author(s):

Yukiko MORIIWA ◽

Naoko SUZUKI ◽

Atsushi SHOJI ◽

Akio YANAGIDA

Keyword(s):

Solid Phase Extraction ◽

Metal Ions ◽

High Throughput ◽

High Throughput Screening ◽

Solid Phase ◽

Screening Method ◽

Phase Extraction ◽

Solid Phase Extraction Cartridge ◽

Ph Conditions

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Preparation of Metal Oxides Containing ppm Levels of Pd as Catalysts for the Reduction of Nitroarene and Evaluation of Their Catalytic Activity by the Fluorescence-Based High-Throughput Screening Method

Catalysts ◽

10.3390/catal10050542 ◽

2020 ◽

Vol 10 (5) ◽

pp. 542

Author(s):

Taeho Lim ◽

Min Su Han

Keyword(s):

Catalytic Activity ◽

Metal Oxides ◽

High Throughput ◽

High Throughput Screening ◽

Screening Method ◽

Reduction Reaction ◽

Precipitation Method ◽

Reaction Conditions ◽

One Pot ◽

Co Precipitation

Herein, an easily accessible and efficient green method for the reduction of nitroarene compounds was developed using metal oxide catalysts. Heterogeneous metal oxides with or without Pd were prepared by a simple and scalable co-precipitation method and used for the reduction of nitroarenes. A fluorescence-based high-throughput screening (HTS) method was also developed for the rapid analysis of the reaction conditions. The catalytic activity of the metal oxides and reaction conditions were rapidly screened by the fluorescence-based HTS method, and Pd/CuO showed the highest catalytic activity under mild reaction conditions. After identifying the optimal reaction conditions, various nitroarenes were reduced to the corresponding aniline derivatives by Pd/CuO (0.005 mol% of Pd) under these conditions. Furthermore, the Pd/CuO catalyst was used for the one-pot Suzuki–Miyaura cross-coupling/reduction reaction. A gram-scale reaction (20 mmol) was successfully performed using the present method, and Pd/CuO showed high reusability without a loss of catalytic activity for five cycles.

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A High-Throughput Screen of the GTPase Activity of Escherichia coli EngA to Find an Inhibitor of Bacterial Ribosome Biogenesis

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057113486001 ◽

2013 ◽

Vol 18 (7) ◽

pp. 830-836 ◽

Cited By ~ 15

Author(s):

Amrita Bharat ◽

Jan E. Blanchard ◽

Eric D. Brown

Keyword(s):

Escherichia Coli ◽

High Throughput ◽

High Throughput Screening ◽

Ribosome Biogenesis ◽

Signal Amplitude ◽

Low Noise ◽

High Throughput Screen ◽

Reaction Conditions ◽

Screening Process ◽

Bacterial Ribosome

The synthesis of ribosomes is an essential process, which is aided by a variety of trans-acting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in a 384-well density. Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Furthermore, we describe the elimination of nonspecific inhibitors that were detergent sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counterscreens for nonspecificity, but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The high-throughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis.

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Use of a Novel Homogeneous Fluorescent Technology in High Throughput Screening

CrossRef Listing of Deleted DOIs ◽

10.1177/108705719600100407 ◽

1996 ◽

Vol 1 (4) ◽

pp. 203-210 ◽

Cited By ~ 50

Author(s):

Janet M. Kolb ◽

Gregory Yamanaka ◽

Susan P. Manly

Keyword(s):

Time Delay ◽

High Throughput ◽

High Throughput Screening ◽

Time Resolved Fluorescence ◽

Reaction Conditions ◽

Time Resolved ◽

Attractive Candidate ◽

Endogenous Fluorescence ◽

High Throughput Screens ◽

Fluorescent Compounds

A new fluorescent technology called homogeneous time-resolved fluorescence (HTRF) is sensitive, homogeneous, and quite tolerant to extremes in reaction conditions. These characteristics make this technique an attractive candidate for use in high throughput screens. The assay system uses a pair of fluorescent compounds to label biomolecules. The long-lived nature of the fluorescence of one of them, europium cryptate, facilitates the homogeneous nature of the assay. Furthermore, the introduction of a time delay in reading the signal eliminates the principal difficulty in applying fluorescence to screening formats, that of endogenous fluorescence of samples tested (especially natural products). This technique is robust and sensitive, and we report here its utility in a high throughput screening format.

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Comparative Analysis of Universal Protein Extraction Methodologies for Screening of Lipase Activity from Agricultural Products

Catalysts ◽

10.3390/catal11070816 ◽

2021 ◽

Vol 11 (7) ◽

pp. 816

Author(s):

Jisu Ha ◽

Jun-Young Park ◽

Yoonseok Choi ◽

Pahn-Shick Chang ◽

Kyung-Min Park

Keyword(s):

Lipase Activity ◽

High Throughput Screening ◽

Protein Extraction ◽

Extraction Yield ◽

Agricultural Products ◽

Acetone Precipitation ◽

Commercial Kits ◽

Protein Assays ◽

Plant Sources ◽

Universal Protein

Protein extraction techniques are absolutely required for the research of biological catalysts. The present study compared four universal protein extraction methodologies (ammonium sulfate precipitation, TCA/acetone precipitation, and two commercial kits) to provide practical information on protein extraction in order to discover a novel lipase in agricultural products. Yields of protein extraction from 24 domestic agricultural products and their specific activities were evaluated and compared with each other. TCA/acetone precipitation showed a relatively higher extraction yield (on average, 3.41 ± 1.08 mg protein/0.1 g sample) in crude protein extraction, whereas the Pierce™ Plant Total Protein Extraction Kit showed the highest specific lipase activity on average in both spectrophotometric (266.61 ± 235.78 μU/mg protein) and fluorometric (41.52 ± 32.63 μU/mg protein) assays. Our results suggest that commercial kits for the rapid extraction of soluble functional proteins would be a better choice than conventional precipitation techniques to perform the high-throughput screening of enzyme activity from plant sources. Finally, several agricultural products such as cordyceps, pepper, bracken, and hemp, all of which exhibited an excellent specific lipase activity, were proposed as promising candidates for a source of novel lipases.

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High-Throughput Microplate Phosphorylation Assays Based on DevR-DevS/Rv2027c 2-Component Signal Transduction Pathway to Screen for Novel Antitubercular Compounds

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104272090 ◽

2005 ◽

Vol 10 (3) ◽

pp. 215-224 ◽

Cited By ~ 16

Author(s):

Deepak Kumar Saini ◽

Jaya Sivaswami Tyagi

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Response Regulator ◽

Signal Transduction Pathway ◽

Cellulose Ester ◽

Reaction Conditions ◽

Component Systems ◽

Novel Target ◽

Radioactivity Retention ◽

Transfer Properties

DevR-DevS (Rv3133c-Rv3132c) and DevR-Rv2027c have been established through their autophosphorylation and phospho-transfer properties to constitute bonafide regulatory 2-component systems of Mycobacterium tuberculosis. DevR has also been shown by others to play a key regulatory role in the expression of M. tuberculosis genes comprising the dormancy regulon. The authors describe high-throughput phosphorylation assays in a microplate format using DevS and Rv2027c histidine kinases and DevR response regulator proteins from M. tuberculosis. The assays were designed to measure [γ-32P]ATP-dependent autophosphorylation of DevS/Rv2027c and also the phosphotransfer reaction to DevR. First, the optimal reaction conditions were established using the conventional method of radiolabeling the 2-component proteins by [γ-32P]ATP and followed by gel electrophoresis-based analysis. Next, the assays were converted to a high-throughput format in which the radiolabeled protein retained on a filter using mixed cellulose ester-based 96-well filter plates was analyzed for radioactivity retention by scintillation counting. The utility of these assays to screen for inhibitors is illustrated using 2-mercaptobenzimidazole, ethidium bromide, and EDTA. The high quality and flexibility of these assays will enable their use in high-throughput screening for new antitubercular compounds directed against 2-component systems that comprise a novel target in dormant mycobacteria.

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