A Sensitive and Selective High‐Throughput Screening Fluorescence Assay for Lipases and Esterases

Abstract

Long-chain fatty acid esters of 7-(3,4-dihydroxybutyloxy)-2H-1-benzopyran-2-one (6) such as octanoate 2a are shown to be exceptionally sensitive and selective fluorogenic substrates for lipases and esterases. Umbelliferone (8) is released upon hydrolysis of the ester function in 2a in the presence of bovine serum albumin and sodium periodate. These substrates are at least by one order of magnitude more sensitive to lipases than the commercial fluorogenic substrate 4-methylumbelliferyl heptanoate. Furthermore, they are stable to a broad range of pH-induced-and thermal-hydrolysis conditions and do not react with non-catalytic proteins such as bovine serum albumin (BSA).Introduction. ± Enzyme assays are indispensable tools in enzymology, where they are used to identify enzymes and to evaluate their purity and activity [1]. When an enzyme is discovered, one important question is always the identity of its natural substrate, which is presumably also its best substrate in terms of kinetic behavior. In the context of enzyme assays, the related problem is to find the optimal method for any given enzyme, taking into consideration that assays giving spectroscopic signals are preferred for high-throughput applications, for example, in the context of biodiversity mining and directed evolution [2]. Herein, we report that C 8 ÀC 14 aliphatic esters of the fluorogenic diol 6 are exceptionally selective and sensitive probes for lipases (Scheme 1). These substrates are stable under a variety of conditions, but are rapidly hydrolyzed whenever only traces of an active lipase are present. The assay is applicable for high-throughput screening.

Enzyme Substrates Protective Encapsulation within Polymeric Microspheres

“…Moreover depending on conditions the magnitude of the effect is different, and in some cases even accidental contact of 4-MOU with alkaline solution can significantly deteriorate analytical usefulness of substrate. It should be stressed that degradation of coumarin derivatives has been previously demonstrated by Nyfeler, et al [3]. In this work it was observed that commercially available 4-methylumbelliferone acyl esters spontaneously hydrolyze at pH 8.8 and higher.…”

“…This effect was observed, e.g. for coumarines derivatives used as substrates [1][2][3]. Occurrence of spontaneous decomposition, especially priori analysis, can result in false elevated results of determination; affecting also the sensitivity of determination.…”

Fluorescence Assays for Biotransformations

“…In addition, the functional group reacting with the enzyme is non -activated and therefore much less prone to uncatalyzed spontaneous hydrolysis in the assay medium. The Clips -O substrate 19 is particularly reactive with lipases because the enzyme reactive ester is part of a 1,2 -diol monoester function resembling the natural glyceride ester substrates of these enzymes [33] .…”

Mixed Carbonates as Useful Substrates for a Fluorogenic Assay for Lipases and Esterases

“…They used a chiral secondary alcohol linked to an umbelliferone with an ether bond; this was oxidized to the corresponding ketone by alcohol dehydrogenase, then treatment with bovine serum albumin (BSA) released the fluorescent product by b-elimination. [8] This technique was later adapted for variety of different enzymes, such as lipases, esterases, [9] aldolases, [10] amidases, [11] phosphatases, [12] epoxide hydrolases, [13] and Bayer-Villiger monooxygenases. [14] Various enzymes were examined, and activity was detected as a timedependent increase in fluorescence intensity.…”