fulltext3 - Substrate Arrays for Fluorescence-Based Enzyme...

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Substrate Arrays for Fluorescence-Based Enzyme Fingerprinting and High-Throughput Screening J EAN -L OUIS R EYMOND Department of Chemistry and Biochemistry, University of Berne, Berne, Switzerland Indirect release of fluorogenic phenols such as umbelliferone was used as a chemical principle to prepare fluorogenic substrates for a variety of enzymes in which the enzyme-reactive group is separated from the fluorescent reporter group, allowing structural and functional diversifica- tion. Fluorescent probes were obtained for enzymes useful for asymmetric synthesis including aldolase catalytic antibodies, transaldolases, alcohol dehydrogenases, lipases, epoxide hydrolases, proteases, and Bayer-Villiger monoxygenases. Arrays of structurally related substrates were pre- pared and used to record the activity of enzymes on multiple substrates simultaneously in parallel formats such as microtiter plates, microarrays, and substrate cocktails, leading to enzyme-specific activity patterns. Arrays of nonlabeled substrates were assayed using indirect product sensors such as the adrenaline-periodate back-titration assay for 1,2-diols or the copper–calcein assay for amino acids. Many of the fluorogenic substrates and sensors described here are either commercially available or accessible in one or two simple synthetic steps and can be used for high-throughput screening of enzyme activities. Key words: catalytic antibodies; enzyme assays; fluorogenic substrates; chemosensors; activity fingerprinting; profiling; proteases; lipases; epoxide hydrolases; alcohol deydrogenases; Bayer- Villiger monoxygenases; microarrays; fluorescence; high-throughput screening; acridone; umbel- liferone; adrenaline; fluorescein. Introduction One of the main activities in biocatalysis consists in developing enzymes for industrial applications, par- ticularly in the areas of fine chemical synthesis, food processing, and polymers. Enzymes are discovered and improved by screening various microbial collections and mutant libraries for the desired activity. Enzyme assays, which make up analytical methods for the mea- surement of enzyme activity, play a critical role in this process, especially assays suitable for high-throughput screening in microtiter plate or in agar plates. 1 The facilitating role of fluorescence assays for high- throughput screening during enzyme discovery can be illustrated by two examples in the area of catalytic antibodies. Antibodies with catalytic activities can be discovered by screening series of monoclonal antibod- ies binding specifically to small molecules mimick- ing the reaction’s transition state. 2 Antibody 14D9, Address for correspondence: Jean-Louis Reymond, Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, 3012 Berne, Switzerland. Voice: + 41 31 631 43 25; fax: + 41 31 631 80 57. jean-louis.reymond@ioc.unibe.ch
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fulltext3 - Substrate Arrays for Fluorescence-Based Enzyme...

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