Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries
Abstract Background The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields. Results Here we present a no...
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ftbiomed:oai:biomedcentral.com:s12858-016-0057-x 2023-05-15T15:03:52+02:00 Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries De Santi, Concetta Altermark, Bjørn Pierechod, Marcin Ambrosino, Luca de Pascale, Donatella Willassen, Nils-Peder 2016-01-19 http://www.biomedcentral.com/1471-2091/17/1 en eng BioMed Central Ltd. http://www.biomedcentral.com/1471-2091/17/1 Copyright 2016 De Santi et al. Metagenomics libraries Cold-active esterase Salt Homology modeling Biotechnological applications Research article 2016 ftbiomed 2016-01-24T01:08:33Z Abstract Background The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields. Results Here we present a novel esterase gene ( lip3 ) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme. The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified from Escherichia coli , preferentially hydrolyzed short and medium length p -nitrophenyl esters with the best substrate being p -nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 °C and a pH optimum of 8.0. Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, β-mercaptoethanol and high concentrations of salt. The enzyme was also highly activated by NaCl. Conclusions The biochemical characterization and homology model reveals a novel esterase originating from the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes. Article in Journal/Newspaper Arctic BioMed Central Arctic |
institution |
Open Polar |
collection |
BioMed Central |
op_collection_id |
ftbiomed |
language |
English |
topic |
Metagenomics libraries Cold-active esterase Salt Homology modeling Biotechnological applications |
spellingShingle |
Metagenomics libraries Cold-active esterase Salt Homology modeling Biotechnological applications De Santi, Concetta Altermark, Bjørn Pierechod, Marcin Ambrosino, Luca de Pascale, Donatella Willassen, Nils-Peder Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries |
topic_facet |
Metagenomics libraries Cold-active esterase Salt Homology modeling Biotechnological applications |
description |
Abstract Background The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields. Results Here we present a novel esterase gene ( lip3 ) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme. The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified from Escherichia coli , preferentially hydrolyzed short and medium length p -nitrophenyl esters with the best substrate being p -nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 °C and a pH optimum of 8.0. Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, β-mercaptoethanol and high concentrations of salt. The enzyme was also highly activated by NaCl. Conclusions The biochemical characterization and homology model reveals a novel esterase originating from the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes. |
format |
Article in Journal/Newspaper |
author |
De Santi, Concetta Altermark, Bjørn Pierechod, Marcin Ambrosino, Luca de Pascale, Donatella Willassen, Nils-Peder |
author_facet |
De Santi, Concetta Altermark, Bjørn Pierechod, Marcin Ambrosino, Luca de Pascale, Donatella Willassen, Nils-Peder |
author_sort |
De Santi, Concetta |
title |
Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries |
title_short |
Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries |
title_full |
Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries |
title_fullStr |
Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries |
title_full_unstemmed |
Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries |
title_sort |
characterization of a cold-active and salt tolerant esterase identified by functional screening of arctic metagenomic libraries |
publisher |
BioMed Central Ltd. |
publishDate |
2016 |
url |
http://www.biomedcentral.com/1471-2091/17/1 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_relation |
http://www.biomedcentral.com/1471-2091/17/1 |
op_rights |
Copyright 2016 De Santi et al. |
_version_ |
1766335719432781824 |