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...

Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90°C and between pH 6.5 and 10; the half-life of the enzymes at 75°C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53-2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds.

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