Fluorescence lipase assay

Scheme 1.5 Fluorescence lipase assay on a solid support.

Figure 2. Principle of the fluorescence lipase assay. The fluorogenic substrate contains a fluorophore and a fluorescence quencher. Release of the quencher fatty acid by a lipolytic enzyme leads to a continuous increase in fluorescence intensity from which enzyme activity can be determined.

H. S. Hendrickson, Fluorescence-based assays of lipases, phospholipases, and... 

Hendrickson, H. S. 1994. Fluorescence-Based Assays of Lipases, Phospholipases, and Other Lipolytic Enzymes. Analytical Biochemistry 219 8. 

One of the first fluorescence-based ee assays uses umbelliferone (14) as the built-in fluorophore and works for several different types of enzymatic reactions 70,86). In an initial investigation, the system was used to monitor the hydrolytic kinetic resolution of chiral acetates (e.g., rac-11) (Fig. 8). It is based on a sequence of two coupled enzymatic steps that converts a pair of enantiomeric alcohols formed by the asymmetric hydrolysis under study (e.g., R - and (5)-12) into a fluorescent product (e.g., 14). In the first step, (R)- and (5)-ll are subjected separately to hydrolysis in reactions catalyzed by a mutant enzyme (lipase or esterase). The goal of the assay is to measure the enantioselectivity of this kinetic resolution. The relative amount of R)- and ( S)-12 produced after a given reaction time is a measure of the enantioselectivity and can be ascertained rapidly, but not directly. 

Thirty different esterases and lipases were tested. The rate of release of 14 in the wells of standard microtiter plates was monitored by fluorescence (70,86). Control experiments ensured that the apparent rate of release of 14 is directly proportional to the rate of acetate hydrolysis. The predicted and observed E and ee values (as checked by standard HPLC assay on a chiral phase) were found to match within + 20%. Only in one case was a larger discrepancy observed, a result that was believed to be caused by the occurrence of an unusually low value of Km for one of the enantiomers. 

Another fluorescence-based method for assaying activity and enantioselectivity of synthetic catalysts, specifically in the acylation of chiral alcohols, was recently reported [27]. The idea is to use a molecular sensor that fluoresces upon formation of an acidic product (acetic acid). Adaptation to high-throughput evaluation of enantioselective lipases or esterases needs to be demonstrated. 

This tripeptide provided moderate levels of selectivity (s < 12.6) for the KR of certain amide-containing sec-alcohols (see Section 8.2.1.4), but displayed essentially no selectivity for KR of aryl alkyl sec-alcohols. However, Miller subsequently devised an elegant fluorescent chemosensor-based screening protocol to assay for acylation [111, 115, 117, 119], and this facilitated the identification of octapeptide 28 as a much more efficient catalyst for KR of sec-alcohols via acylation [115]. The substrate scope includes not only aryl alkyl sec-alcohols but also alkyl alkyl sec-alcohols for which lipases and other organocatalysts invariably perform poorly (Scheme 8.7). 

Continuous assays are rarely described for lipases, but are frequently described for esterases. Similar to lipases, esterases hydrolyze ester bonds. However, in contrast to lipases, their substrates are water-soluble and thus water-soluble fluorescent substrates can be used to measure their enzymatic activity. Some of these water-soluble substrates have been proposed for the measurement of lipolytic ac-... 

We synthesized fluorogenic alkyldiacylglycerols and carboxylic acid esters that are useful substrates for the fast and accurate determination of activity and stereopreference of lipolytic enzymes in aqueous systems and organic solvents. The respective lipids can also be used for the discrimination of lipase and esterase activities. The continuous fluorescence assay can be performed using a cheap fluorometer or, for the analysis of many different samples at a time, a fluorescence plate reader... 

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