Isolation and Biochemical Characterization of Two Novel Metagenome-Derived Esterases

Abstract

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...

An unusual feruloyl esterase belonging to family VIII esterases and displaying a broad substrate range

“…This clearly differentiates them from bona fide β-lactamases. EstU1, on the other hand, cleaves the amide bond of first generation β-lactam antibiotics, although it is more efficient at hydrolysing short chain esters [48]. Other family VIII esterases (EstM-N1, EstM-N2, EstC) have demonstrated activity on nitrocefin, although it is not clear from the data presented whether this constitutes deacetylation or amide bond hydrolysis [18,37].…”

“…As a result, family VIII esterases are typically tested for their ability to hydrolyse a variety of β-lactam substrates ( Table 2). The majority either lack the activity or show negligible activity, despite the high sequence identity to the β-lactamases, while others have been described as exhibiting "promiscuous β-lactamase activity" [18,21,41,47,48,49]. Consequently, it has been suggested that these esterases have evolved from the class C β-lactamases, where some have maintained this remnant activity, while others have lost the capability due to steric interference resulting from structural evolution [18,41,50].…”

Relationships between Substrate Promiscuity and Chiral Selectivity of Esterases from Phylogenetically and Environmentally Diverse Microorganisms

“…Recent examples include those preferably hydrolyzing one of the chiral esters in racemic mixtures of ibuprofen esters [9,10]; ketoprofen esters [11][12][13][14]; solketal esters [15]; esters of phenylalkyl carboxylic acids, 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate and 3,7-dimethyl-1,6-octadien-3-yl acetate [16,17]; methyl 3-phenylglycidate [18]; 1-phenylethyl acetate [19,20]; ofloxacin butyl ester [21]; 1-octin-3-ol, 3-chlor-1-phenyl-1-propanol, trimethylsilylbutinol, cis/trans-1,2-cyclohexanediol, and isopropylidenglycerol acetate [22]; glycidyl butyrate [23]; methyl-mandelate, glycidyl-4-nitrobenzoate, methyl-3-bromo-2-methyl propionate, methyl lactate, menthyl acetate, neomenthyl acetate, pantolactone, and methyl 3-hydroxybutyrate [22,24,25]; 1-octin-3-ol, 3-chlor-1-phenyl-1-propanol, and trimethylsilylbutinol [22]; methyl-3-hydroxy-2-methylpropionate [26]; and esters of secondary alcohols [27,28], to cite some. The advances in metagenomics techniques and screening methods have allowed the discovery of these and other selective esterases [29].…”

Gene Encoding Nitrilase from Soil Sample of Lombok Gold Mine Industry using Metagenomics Approach

“…Demand for novel biocatalysts is continuously increased and 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 metagenomics approach to investigate the catalytic potential of uncultured microorganisms [2].…”