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.

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 High-Throughput Analysis of Selected Urinary Hydroxy Polycyclic Aromatic Hydrocarbons by an Innovative Automated Solid-Phase Microextraction

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 1869 ◽  
Author(s):  
Stefano Dugheri ◽  
Alessandro Bonari ◽  
Matteo Gentili ◽  
Giovanni Cappelli ◽  
Ilenia Pompilio ◽  
...  
Keyword(s):  
Polycyclic Aromatic Hydrocarbons ◽  
High Throughput ◽  
Aromatic Hydrocarbons ◽  
High Throughput Screening ◽  
Solid Phase Microextraction ◽  
Solid Phase ◽  
User Friendliness ◽  
High Throughput Analysis ◽  
Polycyclic Aromatic ◽  
User Friendly

High-throughput screening of samples is the strategy of choice to detect occupational exposure biomarkers, yet it requires a user-friendly apparatus that gives relatively prompt results while ensuring high degrees of selectivity, precision, accuracy and automation, particularly in the preparation process. Miniaturization has attracted much attention in analytical chemistry and has driven solvent and sample savings as easier automation, the latter thanks to the introduction on the market of the three axis autosampler. In light of the above, this contribution describes a novel user-friendly solid-phase microextraction (SPME) off- and on-line platform coupled with gas chromatography and triple quadrupole-mass spectrometry to determine urinary metabolites of polycyclic aromatic hydrocarbons 1- and 2-hydroxy-naphthalene, 9-hydroxy-phenanthrene, 1-hydroxy-pyrene, 3- and 9-hydroxy-benzoantracene, and 3-hydroxy-benzo[a]pyrene. In this new procedure, chromatography’s sensitivity is combined with the user-friendliness of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide on-fiber SPME derivatization using direct immersion sampling; moreover, specific isotope-labelled internal standards provide quantitative accuracy. The detection limits for the seven OH-PAHs ranged from 0.25 to 4.52 ng/L. Intra-(from 2.5 to 3.0%) and inter-session (from 2.4 to 3.9%) repeatability was also evaluated. This method serves to identify suitable risk-control strategies for occupational hygiene conservation programs.

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.

scholarly journals Drug discovery from Nature: automated high-quality sample preparation

2000 ◽  
Vol 22 (5) ◽  
pp. 149-157 ◽  
Author(s):  
Ralf Thiericke
Keyword(s):  
Drug Discovery ◽  
Sample Preparation ◽  
High Throughput ◽  
High Throughput Screening ◽  
Solid Phase ◽  
Structural Diversity ◽  
Synthetic Compound ◽  
Natural Origin ◽  
Screening Programs ◽  
Compound Libraries

Secondary metabolites from plants, animals and microorganisms have been proven to be an outstanding source for new and innovative drugs and show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds:: often generated in time consuming and for the most part manual processes. As quality and quantity of the provided samples play a pivotal role in the success of high-throughput screening programs this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of a modified Zymark RapidTrace®SPE workstation an easy-to-handle and effective fractionation method has been developed which allows the generation of highquality samples from natural origin, fulfilling the requirements of an integration into high-throughput screening programs.

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 A Rapid Transglutaminase Assay for High-Throughput Screening Applications

2006 ◽  
Vol 11 (7) ◽  
pp. 836-843 ◽  
Author(s):  
Yu-Wei Wu ◽  
Yu-Hui Tsai
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Enzyme Inhibitors ◽  
Competitive Inhibition ◽  
Fluorescent Substrate ◽  
Highly Sensitive ◽  
The Family ◽  
Fluorescent Molecules ◽  
Inhibition Pattern ◽  
Guinea Pig Liver

Transglutaminases (TGs) are widely distributed enzymes that catalyze posttranslational modification of proteins by Ca2+-dependent cross-linking reactions. The family members of TGs participate in many significant processes of biological functions such as tissue regeneration, cell differentiation, apoptosis, and certain pathologies. A novel technique for TG activity assay was developed in this study. It was based on the rapid capturing, fluorescence quenching, and fast separation of the unreacted fluorescent molecules from the macromolecular product with magnetic dextran-coated charcoal. As few as 3 ng of guinea pig liver transglutaminase (gpTG) could be detected by the method; activities of 96 TG samples could be measured within an hour. The Km of gpTG determined by this method for monodansylcadaverine (dansyl-CAD) and N, N-dimethylcasein was 14 and 5 μM, respectively. A typical competitive inhibition pattern of cystamine on dansyl-CAD for gpTG activity was also demonstrated. The application of this technique is not limited to the use of dansyl-CAD as the fluorescent substrate of TG; other small fluor-labeled TG substrates may substitute dansyl-CAD. Finally, this method is rapid, highly sensitive, and inexpensive. It is suitable not only for high-throughput screening of enzymes or enzyme inhibitors but also for enzyme kinetic analysis.

scholarly journals Microbiological High Throughput Screening: An Opportunity for the Lead Discovery Process

2000 ◽  
Vol 5 (1) ◽  
pp. 13-21 ◽  
Author(s):  
Marie-Helene Beydon ◽  
Alain Fournier ◽  
Lionel Drugeault ◽  
Jerome Becquart
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Solid Phase ◽  
Discovery Process ◽  
Robotic Platform ◽  
Lead Discovery ◽  
Innovative Solutions ◽  
Different Types ◽  
Microbial Screening ◽  
Low Costs

Microbial HTS has been implemented at Rhone-Poulenc Rorer through the development of a dedicated robotic platform. This robot (Turbo) has been designed with the aim of fully integrating microbial HTS into the lead discovery processes. Innovative solutions have been found to reach high throughput as well as flexibility. This opens up new prospects for solid-phase microbial screening, taking advantage of the easy implementation and the very low costs of such screens. The different types of microbial screens done in our laboratory, as well as the throughputs and outputs obtained, are described. Some of the specific aspects of microbial HTS, as compared to biochemical and cell-based assays, are also discussed.

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