Functional-Based Screening Methods for Lipases, Esterases, and Phospholipases in Metagenomic Libraries

Reyes‐Duarte, Dolores; Ferrer, Manuel; Garcı́a-Arellano, Humberto

doi:10.1007/978-1-61779-600-5_6

Cited by 36 publications

(29 citation statements)

References 14 publications

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“…Approximately 11,520 fosmid clones from the metagenomic library of Lake Arreo sediment (nearly 342 Mbp of metagenome DNA) were screened using plate-based screens for hydrolytic activity against ␣-naphthyl acetate, a model esterase substrate (29). Ten positive clones (incidence of positive clones, 1/1,152) were identified.…”

Section: Resultsmentioning

confidence: 99%

“…Approximately 11,520 clones, plated onto small (12.5-by 12.5-cm) petri plates (each containing 96 clones) with LB agar containing chloramphenicol (12.5 g/ml) and the induction solution (Epicentre Biotechnologies, WI), as recommended by the supplier to induce a high fosmid copy number, were screened with ␣-naphthyl acetate under previously described conditions (29 Cloning, expression, and purification of selected proteins. The cloning, expression, and purification of selected His 6 -tagged proteins in the Ek/LIC 46 vector and E. coli BL21 were performed as described elsewhere (32), except that protein expression was performed using 1.0 mM isopropyl-␤-D-galactopyranoside for 16 h at 16°C.…”

Section: Methodsmentioning

confidence: 99%

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Biochemical Diversity of Carboxyl Esterases and Lipases from Lake Arreo (Spain): a Metagenomic Approach

Martínez-Martínez

Alcaide

Tchigvintsev

et al. 2013

Appl Environ Microbiol

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The esterases and lipases from the ␣/␤ hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity, and activities. Here, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46=N, 2°59=W; altitude, 655 m). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from Lake Arreo suggests that its sediment contains moderately halophilic and cold-adapted proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origins. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics, as they exhibit maximal activity at alkaline pH (8.0 to 8.5) and temperature of 16 to 40°C, and they are stimulated (1.5 to 2.2 times) by chloride ions (0.1 to 1.2 M), reflecting an adaptation to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e., production of enantiomers and sugar esters), because these enzymes are salt tolerant and are active at low temperatures and against a broad range of substrates. As an example, the ability of a single protein to hydrolyze triacylglycerols, (non)halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones, and chiral epoxides to a similar extent was demonstrated. E sterases and lipases from the ␣/␤ hydrolase family have received considerable attention, because they are widely distributed within the microbial communities operating in most of environments where they have important physiological functions (1) and because they are one of the most important groups of biocatalysts for biotechnological applications (2-4). Upon searching the list of genes using Pfam (the protein family database [5]) from the approximately 140 metagenomic projects in various stages of sequencing on the GOLD website (Genomes OnLine Database; http://www.genomesonline.org/) and the available sequences of esterases and lipases, more than 72,000 predicted esterases/lipases of the ␣/␤ hydrolase superfamily were retrieved, which revealed the richness of uncultured biodiversity (6), to provide wide collections of such biocatalysts. This is one of the largest protein families with available sequences. In relation to the cultivation-independent methods used to identify them, it should be highlighted that sequence-based metagenomics only provide the presumptive compositional and functional blueprint represented in the community genome (7,8), but at the same time, this method causes serious problems regarding both sequencing errors (9) and the erroneous assignment of substrate specificity (10). In contrast, the activity-directed techniques have been shown to provide a direct view of known or new protein families and functionalities (for examples, see referen...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Biochemical Diversity of Carboxyl Esterases and Lipases from Lake Arreo (Spain): a Metagenomic Approach

Martínez-Martínez

Alcaide

Tchigvintsev

et al. 2013

Appl Environ Microbiol

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“…1) (19). Total DNA was extracted from the gill chambers of the collected specimens as previously described (19); from this DNA, a large-insert pCCFOS1 fosmid library was generated using the Escherichia coli EPI300-T1 R strain (Epicentre Biotechnologies; Madison, WI, USA), and the library was scored for the ability to hydrolyze ␣-naphthyl acetate and tributyrin (27,30). Positive clones were selected, and their DNA inserts were sequenced using a Roche 454 GS FLX Ti sequencer (454 Life Sciences, Branford, CT, USA) at Life Sequencing SL (Valencia, Spain) or were completely Sanger sequenced using universal primers and subsequent primer walking.…”

Section: Methodsmentioning

confidence: 99%

“…A subset of the 27,200 clones from the R. exoculata gill chamber microbiome library generated in this study, which included nearly 816 Mbp of community genomes, was scored for the ability of individual clones to hydrolyze ␣-naphthyl acetate, as reported previously (30). The subset of clones also was screened for carboxyl esterase activity using tributyrin plates (30).…”

Section: Metagenome Library Construction and Screening For Carboxyl Ementioning

confidence: 99%

Identification and Characterization of Carboxyl Esterases of Gill Chamber-Associated Microbiota in the Deep-Sea Shrimp Rimicaris exoculata by Using Functional Metagenomics

Alcaide

Tchigvintsev

Martínez-Martínez

et al. 2015

Appl Environ Microbiol

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iThe shrimp Rimicaris exoculata dominates the fauna in deep-sea hydrothermal vent sites along the Mid-Atlantic Ridge (depth, 2,320 m). Here, we identified and biochemically characterized three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library. These proteins exhibit low to moderate identity to known esterase sequences (<52%) and to each other (11.9 to 63.7%) and appear to have originated from unknown species or from genera of Proteobacteria related to Thiothrix/Leucothrix (MGS-RG1/RG2) and to the Rhodobacteraceae group (MGS-RG3). A library of 131 esters and 31 additional esterase/lipase preparations was used to evaluate the activity profiles of these enzymes. All 3 of these enzymes had greater esterase than lipase activity and exhibited specific activities with ester substrates (<356 U mg ؊1 ) in the range of similar enzymes. MGS-RG3 was inhibited by salts and pressure and had a low optimal temperature (30°C), and its substrate profile clustered within a group of low-activity and substrate-restricted marine enzymes. In contrast, MGS-RG1 and MGS-RG2 were most active at 45 to 50°C and were salt activated and barotolerant. They also exhibited wider substrate profiles that were close to those of highly active promiscuous enzymes from a marine hydrothermal vent (MGS-RG2) and from a cold brackish lake (MGS-RG1). The data presented are discussed in the context of promoting the examination of enzyme activities of taxa found in habitats that have been neglected for enzyme prospecting; the enzymes found in these taxa may reflect distinct habitat-specific adaptations and may constitute new sources of rare reaction specificities. Metagenomics provides a means for the discovery of entirely new enzymes in microorganisms and their communities without the need to culture these microorganisms as individual species, which is technically very difficult (1-5). Although the discovery of new enzyme activities has progressed considerably (6), it has not managed to go beyond the effective identification of enzymatic activities at a rather limited number of environmental sites. While an extensive search in the specialized literature and public databases indicated that microbial communities from approximately 1,800 different sites worldwide have been examined for their genomic content, only in approximately 200 of those locations (11% of the total) have new active clones or enzymes been identified and/or partially characterized. Thus, only a tiny fraction of the Earth's biosphere has been explored for the purpose of enzyme discovery, despite the fact that natural microbial diversity has been recognized to be the major source of new microbes (7). Such diversity also contains novel genes and functions (8) whose activities remain mostly unknown but whose potential to contribute to an innovation-based economy is recognized (9, 10).One of the habitats less explored to date in terms of examining enzyme repertoires is the deep-sea realm, where...

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“…Crude-cell-lysate-based methods usually have low throughput and are less reproducible (Felczykowska, Dydecka et al 2014). Lipases/esterases constitute a major fraction of enzymes derived from metagenomic screening simply due to the availability of easy high-throughput screening methods (Taupp, Mewis et al 2011, Reyes-Duarte, Ferrer et al 2012. Apart from these direct screening methods, more sophisticated highthroughput screening technologies have recently been developed.…”

Section: Function-based Screening For Detection Of Novel Enzymesmentioning

confidence: 99%

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

Rashid

Stingl

2015

Biotechnology Advances

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Functional-Based Screening Methods for Lipases, Esterases, and Phospholipases in Metagenomic Libraries

Cited by 36 publications

References 14 publications

Biochemical Diversity of Carboxyl Esterases and Lipases from Lake Arreo (Spain): a Metagenomic Approach

Biochemical Diversity of Carboxyl Esterases and Lipases from Lake Arreo (Spain): a Metagenomic Approach

Identification and Characterization of Carboxyl Esterases of Gill Chamber-Associated Microbiota in the Deep-Sea Shrimp Rimicaris exoculata by Using Functional Metagenomics

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

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