The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to ␤-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50°C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47°C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (؉)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications.Modern biotechnology has a steadily increasing demand for novel biocatalysts, thereby prompting the development of novel experimental approaches to find and identify novel biocatalyst-encoding genes. Recently, there has been an increase in the number of studies using a metagenomic approach to investigate the catalytic potential of uncultured microorganisms (8, 28). The term metagenome was introduced to describe the genomes of complex microbial communities found in natural habitats, only a small fraction of which can be cultured (1, 17). Investigation of metagenomes became possible after the development of strategies for the isolation and cloning of environmental DNA (41, 47). Modern metagenomic developments and cloning strategies have recently been reviewed in detail (8,9,16,46). Once constructed, metagenomic libraries can be screened for a wide range of ecologically and biotechnologically interesting phenotypes (44).In the search for novel biocatalysts, there are various metagenomic strategies that are used for targeting specific catalyst characteristics such as substrate range or temperature and pH optima. One approach is to generate the metagenomic library from soils or sediments that are known to harbor a high level of microbial diversity and thus, potentially, a wide diversity of biocatalysts (7). This approach has been used successfully to find a wide variety of novel catalysts and secondary metabolites (9,19,20,27,30,39). A further development of this approach is to create the metagenomic library from an environment that has been subjected to extreme conditions in the likelihood that enzymes from such an environment will be able to f...

A high-throughput enzyme assay is described that uses 1 microL or less of enzyme solution for each test. Enzyme solutions are deposited by robotic handling in a throughput of over 1000 tests/h on the surface of silica gel plates that have been preimpregnated with fluorogenic substrates. The reaction is quantitated by fluorescence. The method is compatible with water-insoluble substrates (lipases), water-soluble substrates (glycosidases), whole-protein substrates (proteases), and enzyme inhibition measurements. Hydrolytically labile umbelliferyl esters can be used to assay lipases in this format without background hydrolysis. High throughput and reproducibility were tested by fingerprint analysis of lipases and esterases against 37 different fluorogenic ester substrates. A set of eight fluorogenic unbelliferyl esters was selected for optimal activity screening of lipases and esterases on silica gel plates.

Monoalkylation or acylation of fluorescein (1) with various acyloxymethyl or acyl halides afforded, respectively, a series of ether-(2) and ester-functionalized (3) fluorogenic probes. The highly reactive and water-soluble substrates release fluorescein (1) upon reaction with lipases and esterases within seconds or minutes, both under fully aqueous conditions or in the presence of DMSO (20%) as a co-solvent. The most-reactive substrates in the two series were the octanoic acid derivatives 2f (= 2-{6-[(octanoyloxy)methoxy]-3-oxo-3H-xanthen-9-yl}benzoic acid) and 3a (= 2-[6-(octanoyloxy)-3-oxo-3H-xanthen-9-yl]benzoic acid). Esterases were found to generally react faster under aqueous conditions, while lipases were more reactive in the presence of DMSO as a co-solvent.Introduction. -High-throughput-screening (HTS) enzyme assays are essential tools for enzyme discovery and engineering [1]. In particular, assays for lipases and esterases are in great demand due to the importance of this enzyme class in industrial biotechnology [2], and for their role as disease markers [3].Lipases can be assayed by following the hydrolysis of triglycerides, which are the presumed natural substrates of these enzymes, or with the aid of titration Unfortunately all of these known substrates are far from ideal because 1) they are insoluble in aqueous media, requiring co-solvents for reaction, and 2) they show a high level of non-specific hydrolysis with non-catalytic proteins [4]. HTS Assays with improved performances on both points would, thus, be highly desirable. The problem of non-specific hydrolysis can be solved with FRET-type substrates [9], or esters of aliphatic alcohols releasing a colored or fluorescent phenol via a secondary reaction sequence such as the cleavage of a 1,2-diol [10] or a cyanohydrin [11] followed by belimination [12]. Simple esters of umbelliferone can also be used in a solid-supported assay by adsorption on silica gel, which suppresses background hydrolysis [13]. However, these modified substrates still show very limited aqueous solubility, and require addition of co-solvents for a reliable reaction.Herein we show that monoalkylation and monoacylation of fluorescein (1) yields highly reactive fluorogenic substrates of type 2 and 3 suitable for lipase and esterase

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Enzyme screening technology has undergone massive developments in recent years, particularly in the area of high-throughput screening and microarray methods. Screening consists of testing each sample of a sample library individually for the targeted reaction. This requires enzyme assays that accurately test relevant parameters of the reaction, such as catalytic turnover with a given substrate and selectivity parameters such as enantio- and regioselectivity. Enzyme assays also play an important role outside of enzyme screening, in particular for drug screening, medical diagnostics, and in the area of cellular and tissue imaging. In the 1990s, methods for high-throughput screening of enzyme activities were perceived as a critical bottleneck. As illustrated partly in this chapter, a large repertoire of efficient screening strategies are available today that allow testing of almost any reaction with high-throughput.