5,5-Dialkylluciferins are thermal stable substrates for bioluminescence-based detection systems

Autor:	Ethan Edward Strauss, Paul Otto, Monika G. Wood, Mary P. Hall, Michael P Killoran, Lance P. Encell, Ce Shi, Thomas Machleidt, Keith V. Wood, Thomas A. Kirkland
Rok vydání:	2020
Předmět:	0301 basic medicine Luminescence Alkylation Firefly Luciferin Protein Engineering 01 natural sciences Biochemistry Substrate Specificity Adenosine Triphosphate Luciferases Firefly Macromolecular Engineering Thermostability chemistry.chemical_classification Multidisciplinary Physics Electromagnetic Radiation Luciferase Assay Temperature Luciferin Enzymes Bioassays and Physiological Analysis Physical Sciences Medicine Engineering and Technology Bioluminescence Oxidoreductases Luciferase Research Article Directed Evolution Evolutionary Processes Science Bioengineering Research and Analysis Methods 03 medical and health sciences Genetics Point Mutation Enzyme Assays Evolutionary Biology Luminescent Agents 010405 organic chemistry Substrate (chemistry) Biology and Life Sciences Proteins Protein engineering Combinatorial chemistry 0104 chemical sciences Luminescent Proteins 030104 developmental biology Enzyme chemistry Mutagenesis Reagent Luminescent Measurements Mutation Enzymology Indicators and Reagents Biochemical Analysis
Zdroj:	PLoS ONE PLoS ONE, Vol 15, Iss 12, p e0243747 (2020)
ISSN:	1932-6203
Popis:	Firefly luciferase-based ATP detection assays are frequently used as a sensitive, cost-efficient method for monitoring hygiene in many industrial settings. Solutions of detection reagent, containing a mixture of a substrate and luciferase enzyme that produces photons in the presence of ATP, are relatively unstable and maintain only a limited shelf life even under refrigerated conditions. It is therefore common for the individual performing a hygiene test to manually prepare fresh reagent at the time of monitoring. To simplify sample processing, a liquid detection reagent with improved thermal stability is needed. The engineered firefly luciferase, Ultra-Glo™, fulfills one aspect of this need and has been valuable for hygiene monitoring because of its high resistance to chemical and thermal inactivation. However, solutions containing both Ultra-Glo™ luciferase and its substrate luciferin gradually lose the ability to effectively detect ATP over time. We demonstrate here that dehydroluciferin, a prevalent oxidative breakdown product of luciferin, is a potent inhibitor of Ultra-Glo™ luciferase and that its formation in the detection reagent is responsible for the decreased ability to detect ATP. We subsequently found that dialkylation at the 5-position of luciferin (e.g., 5,5-dimethylluciferin) prevents degradation to dehydroluciferin and improves substrate thermostability in solution. However, since 5,5-dialkylluciferins are poorly utilized by Ultra-Glo™ luciferase as substrates, we used structural optimization of the luciferin dialkyl modification and protein engineering of Ultra-Glo™ to develop a luciferase/luciferin pair that shows improved total reagent stability in solution at ambient temperature. The results of our studies outline a novel luciferase/luciferin system that could serve as foundations for the next generation of bioluminescence ATP detection assays with desirable reagent stability.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0ef2bf1e48d69be03ac6ff5852c2614a https://pubmed.ncbi.nlm.nih.gov/33315907 Zobrazit plný text záznamu Plný text ve formátu PDF Plný text ve formátu HTML
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