scholarly journals High-Throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2 Mac1 Macrodomain

2021 ◽  
Author(s):  
Morgan Dasovich ◽  
Junlin Zhuo ◽  
Jack A. Goodman ◽  
Ajit Thomas ◽  
Robert Lyle McPherson ◽  
...  
Keyword(s):  
High Throughput ◽  
Activity Assay

A quantitative, high-throughput urease activity assay for comparison and rapid screening of ureolytic bacteria

2022 ◽  
pp. 112738
Author(s):  
Ming-Juan Cui ◽  
Aloysius Teng ◽  
Jian Chu ◽  
Bin Cao
Keyword(s):  
High Throughput ◽  
Urease Activity ◽  
Rapid Screening ◽  
Activity Assay ◽  
Ureolytic Bacteria

scholarly journals A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase based HRP colorimetric method for lytic polysaccharide monooxygenase activity assay

2021 ◽  
Author(s):  
Gang Liu
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Direct Method ◽  
Total Concentration ◽  
Colorimetric Method ◽  
Industrial Applications ◽  
Assay Method ◽  
Activity Assay ◽  
Lytic Polysaccharide Monooxygenase ◽  
Monooxygenase Activity

Abstract Background The AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) are ubiquitous and diverse group of enzymes amongst the fungal kingdom. They catalyze the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for secondary biorefinery applications. Screening of AA9 LPMOs for desirable properties is crucial for biorefinery industrial applications. However, robust, high-throughput and direct method for AA9 LPMO activity assay, which is prerequisite for screening of LPMOs with excellent properties, is still lacking. Here, we have described a gluco-oligosaccharide oxidase (GOOX) based horseradish peroxidase (HRP) colorimetric method for AA9 LPMO activity assay. Results We cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and set up a SsGOOX based HRP colorimetric method for cellobiose concentration assay. Then we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G in T. reesei, purified the recombinant proteins, and analyzed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1 type (class 1) LPMO, while TtAA9G was characterized as a C4 type (class 2) LPMO. Finally, the SsGOOX based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from LPMO reaction, and consequently, the activities of both C1 and C4 types of LPMOs were analyzed. These LPMOs could be effectively analyzed with limits of detection (LoDs) lower than 30 nmol/L, and standard curves between A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained. Conclusions A novel, sensitive and accurate assay method that directly targets the main activity of both C1 and C4 type of AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate ready for high-throughput screening of AA9 LPMOs with high properties.

scholarly journals A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O ‐GlcNAc Transferase

2020 ◽  
Vol 59 (24) ◽  
pp. 9601-9609 ◽  
Author(s):  
Matthew G. Alteen ◽  
Christina Gros ◽  
Richard W. Meek ◽  
David A. Cardoso ◽  
Jil A. Busmann ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Activity Assay ◽  
Glcnac Transferase

scholarly journals A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

2019 ◽  
Vol 58 (30) ◽  
pp. 10114-10119 ◽  
Author(s):  
Tristan de Rond ◽  
Jian Gao ◽  
Amin Zargar ◽  
Markus de Raad ◽  
Jack Cunha ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cytochrome P450 ◽  
High Throughput ◽  
Mass Spectrometric ◽  
Activity Assay ◽  
Enzyme Activity Assay

scholarly journals Discovery of Hepatitis C Virus NS3 Helicase Inhibitors by a Multiplexed, High-Throughput Helicase Activity Assay Based on Graphene Oxide

2013 ◽  
Vol 125 (8) ◽  
pp. 2396-2400 ◽  
Author(s):  
Hongje Jang ◽  
Soo-Ryoon Ryoo ◽  
Young-Kwan Kim ◽  
Soojin Yoon ◽  
Henna Kim ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
Helicase Activity ◽  
Activity Assay

Human cytochrome P450 expression in bacteria: Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4

2018 ◽  
Vol 158 ◽  
pp. 134-140 ◽  
Author(s):  
Francisco Esteves ◽  
Diana Campelo ◽  
Philippe Urban ◽  
Sophie Bozonnet ◽  
Thomas Lautier ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
High Throughput ◽  
Activity Assay ◽  
Whole Cell ◽  
Human Cytochrome ◽  
P450 Expression

A robust and quantitative assay platform for multiplexed, high throughput screening of protein kinase inhibitors

2016 ◽  
Vol 52 (81) ◽  
pp. 12112-12115 ◽  
Author(s):  
Jieon Lee ◽  
Il-Soo Park ◽  
Ginam Park ◽  
Kyukwang Cho ◽  
Hee-Sung Park ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Protein Kinase Activity ◽  
Activity Assay ◽  
Kinase Activity Assay

We present a new platform for multiplexed protein kinase activity assay using TiO2decorated graphene oxide (GO), which is applicable to high throughput inhibitor screening.

Multiplex isotope dimethyl labeling of substrate peptides for high throughput kinase activity assay via quantitative MALDI MS

2014 ◽  
Vol 50 (90) ◽  
pp. 13960-13962 ◽  
Author(s):  
Zhenzhen Deng ◽  
Mingliang Ye ◽  
Yangyang Bian ◽  
Zheyi Liu ◽  
Fangjie Liu ◽  
...  
Keyword(s):  
High Throughput ◽  
Kinase Activity ◽  
Maldi Ms ◽  
Time Dependent ◽  
Activity Assay ◽  
Dimethyl Labeling ◽  
Labeling Approach ◽  
Kinase Activity Assay

A multiplex isotope dimethyl labeling approach allowed MALDI MS to monitor the time dependent consumption of substrates and generation of products in one spot.

scholarly journals Novel LC/MS/MS and High-Throughput Mass Spectrometric Assays for Monoacylglycerol Acyltransferase Inhibitors

2017 ◽  
Vol 22 (4) ◽  
pp. 433-439
Author(s):  
Jenson Qi ◽  
John A. Masucci ◽  
Wensheng Lang ◽  
Margery A. Connelly ◽  
Gary W. Caldwell ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Metabolic Diseases ◽  
Host Cells ◽  
Activity Assay ◽  
Highly Sensitive ◽  
Liquid Chromatography Tandem Mass ◽  
Acyltransferase Inhibitors ◽  
Tlc Analysis ◽  
Monoacylglycerol Acyltransferase

Monoacylglycerol acyltransferase enzymes (MGAT1, MGAT2, and MGAT3) convert monoacylglycerol to diacylglycerol (DAG). MGAT1 and MGAT2 are both implicated in obesity-related metabolic diseases. Conventional MGAT enzyme assays use radioactive substrates, wherein the product of the MGAT-catalyzed reaction is usually resolved by time-consuming thin layer chromatography (TLC) analysis. Furthermore, microsomal membrane preparations typically contain endogenous diacylglycerol acyltransferase (DGAT) from the host cells, and these DGAT activities can further acylate DAG to form triglyceride (TG). Our mass spectrometry (liquid chromatography–tandem mass spectrometry, or LC/MS/MS) MGAT2 assay measures human recombinant MGAT2-catalyzed formation of didecanoyl-glycerol from 1-decanoyl-rac-glycerol and decanoyl-CoA, to produce predominantly 1,3-didecanoyl-glycerol. Unlike 1,2-DAG, 1,3-didecanoyl-glycerol is proved to be not susceptible to further acylation to TG. 1,3-Didecanoyl-glycerol product can be readily solubilized and directly subjected to high-throughput mass spectrometry (HTMS) without further extraction in a 384-well format. We also have established the LC/MS/MS MGAT activity assay in the intestinal microsomes from various species. Our assay is proved to be highly sensitive, and thus it allows measurement of endogenous MGAT activity in cell lysates and tissue preparations. The implementation of the HTMS MGAT activity assay has facilitated the robust screening and evaluation of MGAT inhibitors for the treatment of metabolic diseases.

Development of high-throughput spermidine synthase activity assay using homogeneous time-resolved fluorescence

2006 ◽  
Vol 351 (2) ◽  
pp. 229-240 ◽  
Author(s):  
Koji Enomoto ◽  
Tohru Nagasaki ◽  
Akira Yamauchi ◽  
Junji Onoda ◽  
Katsunori Sakai ◽  
...  
Keyword(s):  
High Throughput ◽  
Spermidine Synthase ◽  
Activity Assay ◽  
Time Resolved Fluorescence ◽  
Time Resolved
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