Fingerprints of lipases and esterases have been recorded by using an array of chiral fluorogenic aliphatic esters of increasing chain length (C(4)-C(16)). Classification of the enzyme series was carried out with selectivity data by clustering and principal component analysis (PCA). Enzymes were classified on the basis of selectivity for chain length (C(4)-C(6) vs. C(10)-C(16)) and of middle-chain-length (C(8)-C(10)) reactivity. A minimum set of nine substrates was defined by cluster analysis of relative reactivities of each substrate for the different enzymes. This selectivity-based analysis is general. It does not require a common reference substrate to react with all enzymes or vice versa, and is independent of knowing the exact concentration of active protein in the enzyme samples.

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.

The branched glycerol analogs 1 and 2 were prepared. Mono-ester derivatives of these triols undergo a chromogenic or fluorogenic reaction in the presence of NaIO 4 . In contrast, both the diesters and the triols are themselves not chromogenic or fluorogenic. Diester derivatives of these triols can be used as probes for lipases. The tris-phosphate derivative of 1 is a fluorogenic substrate for various phosphatases.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 kinetics behavior. In the context of enzyme assays, the related problem is to find the optimal assay method for any given enzyme, in particular the most-sensitive method. Simple assays giving readily recordable spectroscopic signals are preferred in the context of high-throughput screening for new enzymes by biodiversity mining or directed evolution. Herein, we report the synthesis and evaluation of the branched triols 1 and 2 as building blocks for enzyme substrates. Triols 1 and 2 are close analogs of glycerol, and their ester derivatives can be used to prepare chromogenic and fluorogenic analogs of glycerides, the natural substrates of lipases. The synthesis of these triols and their derivatives and their applications in enzyme assays are presented.

Reações de biocatálise foram realizadas em microplacas (200 µL) visando a utilização de substratos fluorogênicos (100 µmol L -1 ) para prospecção rápida de epóxido hidrolases (EHs) e Baeyer-Villiger monoxigenases (BVMOs) em microrganismos (células inteiras). Um protocolo final foi alcançado para EHs, com a detecção de 3 novas fontes enzimáticas (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). O ensaio fluorogênico para BVMO não ocorreu como esperado. A abordagem de algumas variáveis envolvidas (aeração; pH) proporcionou a detecção inédita da atividade enzimática de BVM em T. cutaneum.Biocatalysis reactions were performed on microtiter plates (200 µL) aiming at the utilization of fluorogenic substrates (100 µmol L -1 ) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monoxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum.

No abstract