Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge Haliclona simulans
Identifieur interne : 000319 ( Ncbi/Merge ); précédent : 000318; suivant : 000320Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge Haliclona simulans
Auteurs : Joseph Selvin [Inde] ; Jonathan Kennedy [Irlande (pays)] ; David Ph Lejon [Irlande (pays), France] ; G Seghal Kiran [Inde] ; Alan Dw Dobson [Irlande (pays)]Source :
- Microbial Cell Factories [ 1475-2859 ] ; 2012.
Abstract
Lipases (EC 3.1.1.3) catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome.
A metagenomic library was constructed from the marine sponge
We have isolated a novel halo tolerant lipase following a functional screen of a marine sponge fosmid metagenomic library. The activity and stability profile of the recombinant enzyme over a wide range of salinity, pH and temperature; and in the presence of organic solvent and metal ions suggests a utility for this enzyme in a variety of industrial applications.
Url:
DOI: 10.1186/1475-2859-11-72
PubMed: 22657530
PubMed Central: 3544137
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge <italic>Haliclona simulans</italic>
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<author><name sortKey="Kennedy, Jonathan" sort="Kennedy, Jonathan" uniqKey="Kennedy J" first="Jonathan" last="Kennedy">Jonathan Kennedy</name>
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<country xml:lang="fr">France</country>
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<author><name sortKey="Kiran, G Seghal" sort="Kiran, G Seghal" uniqKey="Kiran G" first="G Seghal" last="Kiran">G Seghal Kiran</name>
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<author><name sortKey="Dobson, Alan Dw" sort="Dobson, Alan Dw" uniqKey="Dobson A" first="Alan Dw" last="Dobson">Alan Dw Dobson</name>
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<series><title level="j">Microbial Cell Factories</title>
<idno type="eISSN">1475-2859</idno>
<imprint><date when="2012">2012</date>
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<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>Lipases (EC 3.1.1.3) catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome.</p>
</sec>
<sec><title>Results</title>
<p>A metagenomic library was constructed from the marine sponge <italic>Haliclona simulans</italic>
in the pCC1fos vector, containing approximately 48,000 fosmid clones. High throughput plate screening on 1% tributyrin agar resulted in the identification of 58 positive lipase clones. Following sequence analysis of the 10 most highly active fosmid clones the pCC1fos53E1 clone was found to contain a putative lipase gene <italic>lpc</italic>
53E1, encoded by 387 amino acids and with a predicted molecular mass of 41.87 kDa. Sequence analysis of the predicted amino acid sequence of Lpc53E1 revealed that it is a member of the group VIII family of lipases possessing the SXTK motif, related to type C β-lactamases. Heterologous expression of <italic>lpc</italic>
53E1 in <italic>E. coli</italic>
and the subsequent biochemical characterization of the recombinant protein, showed an enzyme with the highest substrate specificity for long chain fatty acyl esters. Optimal activity was observed with <italic>p</italic>
- nitrophenyl palmitate (C<sub>16</sub>
) at 40°C, in the presence of 5 M NaCl at pH 7; while in addition the recombinant enzyme displayed activity across broad pH (3–12) and temperature (4 -60°C) ranges and high levels of stability in the presence of various solvents at NaCl concentrations as high as 5 M and at temperatures ranging from 10 to 80°C. A maximum lipase activity of 2,700 U/mg was observed with 10 mM <italic>p</italic>
-nitrophenyl palmitate as substrate, in the presence of 5 mM Ca<sup>2+</sup>
and 5 M NaCl, and a reaction time of 15 min at pH 7 and 40°C; while K<sub>M</sub>
and <italic>Vmax</italic>
values were calculated to be 1.093 mM<sup>-1</sup>
and 50 μmol/min, respectively.</p>
</sec>
<sec><title>Conclusion</title>
<p>We have isolated a novel halo tolerant lipase following a functional screen of a marine sponge fosmid metagenomic library. The activity and stability profile of the recombinant enzyme over a wide range of salinity, pH and temperature; and in the presence of organic solvent and metal ions suggests a utility for this enzyme in a variety of industrial applications.</p>
</sec>
</div>
</front>
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<pmc article-type="research-article" xml:lang="en"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Microb Cell Fact</journal-id>
<journal-id journal-id-type="iso-abbrev">Microb. Cell Fact</journal-id>
<journal-title-group><journal-title>Microbial Cell Factories</journal-title>
</journal-title-group>
<issn pub-type="epub">1475-2859</issn>
<publisher><publisher-name>BioMed Central</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">22657530</article-id>
<article-id pub-id-type="pmc">3544137</article-id>
<article-id pub-id-type="publisher-id">1475-2859-11-72</article-id>
<article-id pub-id-type="doi">10.1186/1475-2859-11-72</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research</subject>
</subj-group>
</article-categories>
<title-group><article-title>Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge <italic>Haliclona simulans</italic>
</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" id="A1"><name><surname>Selvin</surname>
<given-names>Joseph</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>jselvin@bdu.ac.in</email>
</contrib>
<contrib contrib-type="author" id="A2"><name><surname>Kennedy</surname>
<given-names>Jonathan</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<email>jonathan.kennedy@ucc.ie</email>
</contrib>
<contrib contrib-type="author" id="A3"><name><surname>Lejon</surname>
<given-names>David PH</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<xref ref-type="aff" rid="I4">4</xref>
<email>david.lejon@ujf-grenoble.fr</email>
</contrib>
<contrib contrib-type="author" id="A4"><name><surname>Kiran</surname>
<given-names>G Seghal</given-names>
</name>
<xref ref-type="aff" rid="I5">5</xref>
<email>seghalkiran@gmail.com</email>
</contrib>
<contrib contrib-type="author" corresp="yes" id="A5"><name><surname>Dobson</surname>
<given-names>Alan DW</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<xref ref-type="aff" rid="I3">3</xref>
<email>a.dobson@ucc.ie</email>
</contrib>
</contrib-group>
<aff id="I1"><label>1</label>
Microbial Genomics Research Unit, Department of Bioinformatics, Bharathidasan University, Tiruchirapalli, India</aff>
<aff id="I2"><label>2</label>
Marine Biotechnology Centre, Environmental Research Institute, University College Cork, Cork, Ireland</aff>
<aff id="I3"><label>3</label>
Department of Microbiology, University College Cork, Cork, Ireland</aff>
<aff id="I4"><label>4</label>
Laboratoire d’étude des Transferts en Hydrologie et Environnement, UMR 5564 (CNRS/INPG/IRD/UJF), Université de Grenoble, Grenoble, France</aff>
<aff id="I5"><label>5</label>
Department of Microbiology, Pondicherry University, Puducherry, India</aff>
<pub-date pub-type="collection"><year>2012</year>
</pub-date>
<pub-date pub-type="epub"><day>1</day>
<month>6</month>
<year>2012</year>
</pub-date>
<volume>11</volume>
<fpage>72</fpage>
<lpage>72</lpage>
<history><date date-type="received"><day>20</day>
<month>2</month>
<year>2012</year>
</date>
<date date-type="accepted"><day>4</day>
<month>5</month>
<year>2012</year>
</date>
</history>
<permissions><copyright-statement>Copyright ©2012 Selvin et al.; licensee BioMed Central Ltd.</copyright-statement>
<copyright-year>2012</copyright-year>
<copyright-holder>Selvin et al.; licensee BioMed Central Ltd.</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0">http://creativecommons.org/licenses/by/2.0</ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
</license>
</permissions>
<self-uri xlink:href="http://www.microbialcellfactories.com/content/11/1/72"></self-uri>
<abstract><sec><title>Background</title>
<p>Lipases (EC 3.1.1.3) catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome.</p>
</sec>
<sec><title>Results</title>
<p>A metagenomic library was constructed from the marine sponge <italic>Haliclona simulans</italic>
in the pCC1fos vector, containing approximately 48,000 fosmid clones. High throughput plate screening on 1% tributyrin agar resulted in the identification of 58 positive lipase clones. Following sequence analysis of the 10 most highly active fosmid clones the pCC1fos53E1 clone was found to contain a putative lipase gene <italic>lpc</italic>
53E1, encoded by 387 amino acids and with a predicted molecular mass of 41.87 kDa. Sequence analysis of the predicted amino acid sequence of Lpc53E1 revealed that it is a member of the group VIII family of lipases possessing the SXTK motif, related to type C β-lactamases. Heterologous expression of <italic>lpc</italic>
53E1 in <italic>E. coli</italic>
and the subsequent biochemical characterization of the recombinant protein, showed an enzyme with the highest substrate specificity for long chain fatty acyl esters. Optimal activity was observed with <italic>p</italic>
- nitrophenyl palmitate (C<sub>16</sub>
) at 40°C, in the presence of 5 M NaCl at pH 7; while in addition the recombinant enzyme displayed activity across broad pH (3–12) and temperature (4 -60°C) ranges and high levels of stability in the presence of various solvents at NaCl concentrations as high as 5 M and at temperatures ranging from 10 to 80°C. A maximum lipase activity of 2,700 U/mg was observed with 10 mM <italic>p</italic>
-nitrophenyl palmitate as substrate, in the presence of 5 mM Ca<sup>2+</sup>
and 5 M NaCl, and a reaction time of 15 min at pH 7 and 40°C; while K<sub>M</sub>
and <italic>Vmax</italic>
values were calculated to be 1.093 mM<sup>-1</sup>
and 50 μmol/min, respectively.</p>
</sec>
<sec><title>Conclusion</title>
<p>We have isolated a novel halo tolerant lipase following a functional screen of a marine sponge fosmid metagenomic library. The activity and stability profile of the recombinant enzyme over a wide range of salinity, pH and temperature; and in the presence of organic solvent and metal ions suggests a utility for this enzyme in a variety of industrial applications.</p>
</sec>
</abstract>
<kwd-group><kwd>Metagenomic library</kwd>
<kwd>Functional screening</kwd>
<kwd>Lipase</kwd>
<kwd>Marine sponge</kwd>
</kwd-group>
</article-meta>
</front>
</pmc>
<affiliations><list><country><li>France</li>
<li>Inde</li>
<li>Irlande (pays)</li>
</country>
<settlement><li>Grenoble</li>
</settlement>
</list>
<tree><country name="Inde"><noRegion><name sortKey="Selvin, Joseph" sort="Selvin, Joseph" uniqKey="Selvin J" first="Joseph" last="Selvin">Joseph Selvin</name>
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<name sortKey="Kiran, G Seghal" sort="Kiran, G Seghal" uniqKey="Kiran G" first="G Seghal" last="Kiran">G Seghal Kiran</name>
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<country name="Irlande (pays)"><noRegion><name sortKey="Kennedy, Jonathan" sort="Kennedy, Jonathan" uniqKey="Kennedy J" first="Jonathan" last="Kennedy">Jonathan Kennedy</name>
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<name sortKey="Dobson, Alan Dw" sort="Dobson, Alan Dw" uniqKey="Dobson A" first="Alan Dw" last="Dobson">Alan Dw Dobson</name>
<name sortKey="Dobson, Alan Dw" sort="Dobson, Alan Dw" uniqKey="Dobson A" first="Alan Dw" last="Dobson">Alan Dw Dobson</name>
<name sortKey="Lejon, David Ph" sort="Lejon, David Ph" uniqKey="Lejon D" first="David Ph" last="Lejon">David Ph Lejon</name>
</country>
<country name="France"><noRegion><name sortKey="Lejon, David Ph" sort="Lejon, David Ph" uniqKey="Lejon D" first="David Ph" last="Lejon">David Ph Lejon</name>
</noRegion>
</country>
</tree>
</affiliations>
</record>
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