scholarly journals Simple Colorimetric Inhibition Assay of Heme Crystallization for High-Throughput Screening of Antimalarial Compounds

2006 ◽  
Vol 51 (1) ◽  
pp. 350-353 ◽  
Author(s):  
Nguyen Tien Huy ◽  
Dinh Thanh Uyen ◽  
Atsushi Maeda ◽  
Dai Thi Xuan Trang ◽  
Tatsuo Oida ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Assay ◽  
Tween 20 ◽  
Inhibition Assay ◽  
Microplate Reader ◽  
Antimalarial Compounds

ABSTRACT Current assays for screening new antimalarials need initiators of β-hematin formation that require laborious preparation, special devices, and substrates. In this study, based on reduction of heme absorption in β-hematin formation, we developed a simple colorimetric assay using Tween 20 as an initiator and a microplate reader for high-throughput screening of inhibitors of β-hematin formation.

scholarly journals Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX

2018 ◽  
Vol 84 (16) ◽  
Author(s):  
Wei Zhou ◽  
Rui Huang ◽  
Zhiguang Zhu ◽  
Yi-Heng P. Job Zhang
Keyword(s):  
High Activity ◽  
Double Layer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Specific Activity ◽  
Colorimetric Assay ◽  
Petri Dish ◽  
Rapid Identification ◽  
Thermobifida Fusca ◽  
Content Type

ABSTRACT Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.

Phospholipid/Polydiacetylene Vesicle-Based Colorimetric Assay for High-Throughput Screening of Bacteriocins and Halocins

2016 ◽  
Vol 182 (1) ◽  
pp. 142-154 ◽  
Author(s):  
Manoj Kumar Yadav ◽  
Vijay Kumar ◽  
Bijender Singh ◽  
Santosh Kumar Tiwari
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Assay

scholarly journals Simple Colorimetric Trypanothione Reductase-Based Assay for High-Throughput Screening of Drugs against Leishmania Intracellular Amastigotes

2013 ◽  
Vol 58 (1) ◽  
pp. 527-535 ◽  
Author(s):  
Erika van den Bogaart ◽  
Gerard J. Schoone ◽  
Paul England ◽  
Dorien Faber ◽  
Kristina M. Orrling ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Genetic Recombination ◽  
Colorimetric Assay ◽  
Reference Method ◽  
Published Data ◽  
Content Type ◽  
Intracellular Amastigotes ◽  
Efficient Test ◽  
Enzymatic Reduction

ABSTRACTCritical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity againstLeishmaniaclinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of aLeishmanianative enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth ofLeishmaniaparasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays.

ChemInform Abstract: A Rapid, Sensitive Colorimetric Assay for the High-Throughput Screening of Transaminases in Liquid or Solid-Phase.

ChemInform ◽  
2016 ◽  
Vol 47 (15) ◽  
pp. no-no
Author(s):  
D. Baud ◽  
N. Ladkau ◽  
T. S. Moody ◽  
J. M. Ward ◽  
H. C. Hailes
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Solid Phase ◽  
Colorimetric Assay

scholarly journals New High-Throughput Fluorimetric Assay for Discovering Inhibitors of UDP-N-Acetylmuramyl-l-Alanine: d-Glutamate (MurD) Ligase

2009 ◽  
Vol 14 (4) ◽  
pp. 412-418 ◽  
Author(s):  
Katja Kristan ◽  
Miha Kotnik ◽  
Marko Oblak ◽  
Uroš Urleb
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Antibacterial Agents ◽  
Enzyme System ◽  
The Novel ◽  
Inhibition Assay ◽  
Structural Class ◽  
Bacterial Enzyme ◽  
Bacterial Peptidoglycan ◽  
Murd Ligase

A novel assay for monitoring the activity of the bacterial enzyme UDP- N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD ligase) is presented. MurD, which belongs to an enzyme family of Mur ligases, is essential for the synthesis of bacterial peptidoglycan and therefore represents an attractive target for the discovery of novel antibacterial agents. The inhibition assay described in this article is amenable to high-throughput screening. It is based on the detection of the accumulation of adenosine 5′-diphosphate (ADP), a product of the reaction catalyzed by MurD ligase, by conversion to a fluorescent signal via a coupled enzyme system, using the ADP Quest™ assay kit from DiscoveRx. The novel assay has been validated by obtaining KM,app values for substrates D-Glu, UDP- N-acetylmuramyl-L-alanine (UMA) and ATP that are in agreement with the data reported in the literature. A counterscreen assay was introduced to eliminate false positives, and some of the known MurD inhibitors have been retested to compare the data measured with different methods. Moreover, a focused library of around 1000 compounds was screened for the inhibition of MurD to assess the performance and robustness of the assay. Finally, a novel MurD inhibitor belonging to a new structural class, with an IC50 value of 105 µM, was discovered. ( Journal of Biomolecular Screening 2009:412-418)

scholarly journals A rapid, sensitive colorimetric assay for the high-throughput screening of transaminases in liquid or solid-phase

2015 ◽  
Vol 51 (97) ◽  
pp. 17225-17228 ◽  
Author(s):  
D. Baud ◽  
N. Ladkau ◽  
T. S. Moody ◽  
J. M. Ward ◽  
H. C. Hailes
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Solid Phase ◽  
Colorimetric Assay ◽  
Red Light ◽  
Highly Sensitive

Red light for transaminases. A highly sensitive colorimetric assay using an inexpensive amine donor has been established for use in high-throughput transaminase screens.

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