Cloning and characterization of a carboxylesterase from Bacillus coagulans 81-11
A genomic library of Bacillus coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4 kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723 bp open readin...
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| Veröffentlicht in: | Current microbiology 2005-04, Vol.50 (4), p.196-201 |
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| creator | Mnisi, Stephens M Louw, Maureen E Theron, Jacques |
| description | A genomic library of Bacillus coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4 kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723 bp open reading frame (ORF), designated estC1, encoding a protein of 240 amino acids with an estimated molecular mass of 27,528 Da and a pI of 9.15. The deduced amino acid sequence of the estC1 gene exhibited significant amino acid sequence identity with carboxylesterases from thermophilic Geobacillus spp. and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl (p-NP) esters with different acyl chain lengths as the substrate confirmed the esterase activity. EstC1 exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50 degrees C, although the enzyme displayed stability at temperatures up to 60 degrees C. |
| doi_str_mv | 10.1007/s00284-004-4423-3 |
| format | Article |
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A 2.4 kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723 bp open reading frame (ORF), designated estC1, encoding a protein of 240 amino acids with an estimated molecular mass of 27,528 Da and a pI of 9.15. The deduced amino acid sequence of the estC1 gene exhibited significant amino acid sequence identity with carboxylesterases from thermophilic Geobacillus spp. and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl (p-NP) esters with different acyl chain lengths as the substrate confirmed the esterase activity. EstC1 exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. 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A 2.4 kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723 bp open reading frame (ORF), designated estC1, encoding a protein of 240 amino acids with an estimated molecular mass of 27,528 Da and a pI of 9.15. The deduced amino acid sequence of the estC1 gene exhibited significant amino acid sequence identity with carboxylesterases from thermophilic Geobacillus spp. and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl (p-NP) esters with different acyl chain lengths as the substrate confirmed the esterase activity. EstC1 exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50 degrees C, although the enzyme displayed stability at temperatures up to 60 degrees C.</description><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Bacillus - enzymology</subject><subject>Bacillus - genetics</subject><subject>Bacillus coagulans</subject><subject>Bacteria</subject><subject>Bacteriology</subject><subject>Carboxylesterase - chemistry</subject><subject>Carboxylesterase - genetics</subject><subject>Carboxylesterase - metabolism</subject><subject>Cloning</subject><subject>Cloning, Molecular</subject><subject>DNA, Bacterial - analysis</subject><subject>DNA, Bacterial - genetics</subject><subject>E coli</subject><subject>Enzyme Stability</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Esters</subject><subject>Genome, Bacterial</subject><subject>Geobacillus</subject><subject>Microbiology</subject><subject>Molecular Sequence Data</subject><issn>0343-8651</issn><issn>1432-0991</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNqFkU2LFDEQhoMo7rj6A7xI8OAtWpXK51EHv2BBD3oO6XSy9tLTWZNpcP319jIDghdPdajnfaniYew5wmsEsG86gHRKACihlCRBD9gOFUkB3uNDtgNSJJzReMGe9H4DgNIDPmYXqD1IZcyOfd3PdZmWax6XkacfscV0zG36HY9TXXgtPPIU21B_3c25b5vYMy-tHvi7mKZ5XjtPNV6vc1w6dygQn7JHJc49PzvPS_b9w_tv-0_i6svHz_u3VyKRtkchlbW2gHdj1BYGXQbEsRRtrSpKkbfRGByMd0BmINIqyyi1NSNR8pBHumSvTr23rf5ct9vCYeopz9slua49GOvIG9D_BdEqIx3gBr78B7ypa1u2J4L12jnSxm0QnqDUau8tl3DbpkNsdwEh3EsJJylhkxLupQTaMi_OxetwyOPfxNkC_QGvToUS</recordid><startdate>200504</startdate><enddate>200504</enddate><creator>Mnisi, Stephens M</creator><creator>Louw, Maureen E</creator><creator>Theron, Jacques</creator><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>M7N</scope><scope>M7P</scope><scope>MBDVC</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>200504</creationdate><title>Cloning and characterization of a carboxylesterase from Bacillus coagulans 81-11</title><author>Mnisi, Stephens M ; Louw, Maureen E ; Theron, Jacques</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c357t-24777f098da570b5fb11dff5774f44397a661b698036b3354e2a2576d33c90ed3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Amino Acid Sequence</topic><topic>Amino acids</topic><topic>Bacillus - enzymology</topic><topic>Bacillus - genetics</topic><topic>Bacillus coagulans</topic><topic>Bacteria</topic><topic>Bacteriology</topic><topic>Carboxylesterase - chemistry</topic><topic>Carboxylesterase - genetics</topic><topic>Carboxylesterase - metabolism</topic><topic>Cloning</topic><topic>Cloning, Molecular</topic><topic>DNA, Bacterial - analysis</topic><topic>DNA, Bacterial - genetics</topic><topic>E coli</topic><topic>Enzyme Stability</topic><topic>Enzymes</topic><topic>Escherichia coli</topic><topic>Esters</topic><topic>Genome, Bacterial</topic><topic>Geobacillus</topic><topic>Microbiology</topic><topic>Molecular Sequence Data</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Mnisi, Stephens M</creatorcontrib><creatorcontrib>Louw, Maureen E</creatorcontrib><creatorcontrib>Theron, Jacques</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Research Library (Alumni Edition)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>Research Library Prep</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Research Library</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Research Library (Corporate)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Current microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Mnisi, Stephens M</au><au>Louw, Maureen E</au><au>Theron, Jacques</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cloning and characterization of a carboxylesterase from Bacillus coagulans 81-11</atitle><jtitle>Current microbiology</jtitle><addtitle>Curr Microbiol</addtitle><date>2005-04</date><risdate>2005</risdate><volume>50</volume><issue>4</issue><spage>196</spage><epage>201</epage><pages>196-201</pages><issn>0343-8651</issn><eissn>1432-0991</eissn><abstract>A genomic library of Bacillus coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4 kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723 bp open reading frame (ORF), designated estC1, encoding a protein of 240 amino acids with an estimated molecular mass of 27,528 Da and a pI of 9.15. The deduced amino acid sequence of the estC1 gene exhibited significant amino acid sequence identity with carboxylesterases from thermophilic Geobacillus spp. and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl (p-NP) esters with different acyl chain lengths as the substrate confirmed the esterase activity. EstC1 exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50 degrees C, although the enzyme displayed stability at temperatures up to 60 degrees C.</abstract><cop>United States</cop><pub>Springer Nature B.V</pub><pmid>15902466</pmid><doi>10.1007/s00284-004-4423-3</doi><tpages>6</tpages></addata></record> |
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| subjects | Amino Acid Sequence Amino acids Bacillus - enzymology Bacillus - genetics Bacillus coagulans Bacteria Bacteriology Carboxylesterase - chemistry Carboxylesterase - genetics Carboxylesterase - metabolism Cloning Cloning, Molecular DNA, Bacterial - analysis DNA, Bacterial - genetics E coli Enzyme Stability Enzymes Escherichia coli Esters Genome, Bacterial Geobacillus Microbiology Molecular Sequence Data |
| title | Cloning and characterization of a carboxylesterase from Bacillus coagulans 81-11 |
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