Characterization of two soil metagenome-derived lipases with high specificity for p-nitrophenyl palmitate
Two novel genes (pwtsB and pwtsC) encoding lipases were isolated by screening the soil metagenomic library. Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecula...
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| Veröffentlicht in: | Archives of microbiology 2009-03, Vol.191 (3), p.233-240 |
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| creator | Wei, Ping Bai, Liping Song, Wengang Hao, Gangping |
| description | Two novel genes (pwtsB and pwtsC) encoding lipases were isolated by screening the soil metagenomic library. Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecular weight of 35 kDa. Furthermore, both genes were cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzymes were purified by Ni-nitrilotriacetic acid affinity chromatography and characterized by spectrophotometric with different p-nitrophenyl esters. The results showed that PWTSB displayed a high degree of activity and stability at 20°C with an optimal pH of around 8.0, and PWTSC at 40°C with an optimal pH of around 7.0. P-nitrophenyl palmitate (p-NPP) was identified as the best substrate of PWTSB and PWTSC. The specific activities of PWTSB and PWTSC were 150 and 166 U/mg, respectively toward p-NPP at 30°C, about 20-fold higher than that toward p-nitrophenyl butyrate (C4) and caprylate (C8). In conclusion, our results suggest that PWTSB is a cold adapt lipase and PWTSC is a thermostable lipase to long-chain p-nitrophenyl esters. |
| doi_str_mv | 10.1007/s00203-008-0448-5 |
| format | Article |
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Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecular weight of 35 kDa. Furthermore, both genes were cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzymes were purified by Ni-nitrilotriacetic acid affinity chromatography and characterized by spectrophotometric with different p-nitrophenyl esters. The results showed that PWTSB displayed a high degree of activity and stability at 20°C with an optimal pH of around 8.0, and PWTSC at 40°C with an optimal pH of around 7.0. P-nitrophenyl palmitate (p-NPP) was identified as the best substrate of PWTSB and PWTSC. The specific activities of PWTSB and PWTSC were 150 and 166 U/mg, respectively toward p-NPP at 30°C, about 20-fold higher than that toward p-nitrophenyl butyrate (C4) and caprylate (C8). In conclusion, our results suggest that PWTSB is a cold adapt lipase and PWTSC is a thermostable lipase to long-chain p-nitrophenyl esters.</description><identifier>ISSN: 0302-8933</identifier><identifier>EISSN: 1432-072X</identifier><identifier>DOI: 10.1007/s00203-008-0448-5</identifier><identifier>PMID: 19043691</identifier><identifier>CODEN: AMICCW</identifier><language>eng</language><publisher>Berlin/Heidelberg: Berlin/Heidelberg : Springer-Verlag</publisher><subject>Amino Acid Sequence ; Amino acids ; Bacteriology ; Biochemistry ; Biological and medical sciences ; Biomedical and Life Sciences ; Biotechnology ; Cell Biology ; Cloning ; DNA, Bacterial - isolation & purification ; E coli ; Ecology ; Enzyme Stability ; Enzymes ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Esters ; Fundamental and applied biological sciences. Psychology ; Gene Library ; Genes ; Genetics ; Genome, Bacterial ; Hydrogen-Ion Concentration ; Life Sciences ; Lipase - genetics ; Lipase - metabolism ; Microbial Ecology ; Microbiology ; Microorganisms ; Molecular Sequence Data ; Molecular weight ; Nitrilotriacetic acid ; Original Paper ; Palmitates - metabolism ; Plasmids ; Proteins ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Sequence Alignment ; Soil Microbiology ; Substrate Specificity ; Temperature</subject><ispartof>Archives of microbiology, 2009-03, Vol.191 (3), p.233-240</ispartof><rights>Springer-Verlag 2008</rights><rights>2009 INIST-CNRS</rights><rights>Springer-Verlag 2009</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c423t-b61b321940a0580eff28062dd69260c1242a87f868cebf7bdb5185a7af9453053</citedby><cites>FETCH-LOGICAL-c423t-b61b321940a0580eff28062dd69260c1242a87f868cebf7bdb5185a7af9453053</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00203-008-0448-5$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00203-008-0448-5$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=21182537$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19043691$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wei, Ping</creatorcontrib><creatorcontrib>Bai, Liping</creatorcontrib><creatorcontrib>Song, Wengang</creatorcontrib><creatorcontrib>Hao, Gangping</creatorcontrib><title>Characterization of two soil metagenome-derived lipases with high specificity for p-nitrophenyl palmitate</title><title>Archives of microbiology</title><addtitle>Arch Microbiol</addtitle><addtitle>Arch Microbiol</addtitle><description>Two novel genes (pwtsB and pwtsC) encoding lipases were isolated by screening the soil metagenomic library. Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecular weight of 35 kDa. Furthermore, both genes were cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzymes were purified by Ni-nitrilotriacetic acid affinity chromatography and characterized by spectrophotometric with different p-nitrophenyl esters. The results showed that PWTSB displayed a high degree of activity and stability at 20°C with an optimal pH of around 8.0, and PWTSC at 40°C with an optimal pH of around 7.0. P-nitrophenyl palmitate (p-NPP) was identified as the best substrate of PWTSB and PWTSC. The specific activities of PWTSB and PWTSC were 150 and 166 U/mg, respectively toward p-NPP at 30°C, about 20-fold higher than that toward p-nitrophenyl butyrate (C4) and caprylate (C8). In conclusion, our results suggest that PWTSB is a cold adapt lipase and PWTSC is a thermostable lipase to long-chain p-nitrophenyl esters.</description><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Bacteriology</subject><subject>Biochemistry</subject><subject>Biological and medical sciences</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnology</subject><subject>Cell Biology</subject><subject>Cloning</subject><subject>DNA, Bacterial - isolation & purification</subject><subject>E coli</subject><subject>Ecology</subject><subject>Enzyme Stability</subject><subject>Enzymes</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Esters</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Library</subject><subject>Genes</subject><subject>Genetics</subject><subject>Genome, Bacterial</subject><subject>Hydrogen-Ion Concentration</subject><subject>Life Sciences</subject><subject>Lipase - genetics</subject><subject>Lipase - metabolism</subject><subject>Microbial Ecology</subject><subject>Microbiology</subject><subject>Microorganisms</subject><subject>Molecular Sequence Data</subject><subject>Molecular weight</subject><subject>Nitrilotriacetic acid</subject><subject>Original Paper</subject><subject>Palmitates - metabolism</subject><subject>Plasmids</subject><subject>Proteins</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sequence Alignment</subject><subject>Soil Microbiology</subject><subject>Substrate Specificity</subject><subject>Temperature</subject><issn>0302-8933</issn><issn>1432-072X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp9kU-LFDEQxYMo7uzqB_CiQdBbtPKn0-mjDLoKCx50wVtIp5PpLN2dNsm4jJ_ebnpQ8eChqEP96tWjHkLPKLyhAPXbDMCAEwBFQAhFqgdoRwVnBGr27SHaAQdGVMP5BbrM-Q6AMqXUY3RBGxBcNnSHwr43ydjiUvhpSogTjh6X-4hzDAMeXTEHN8XRkW4hfrgOD2E22WV8H0qP-3DocZ6dDT7YUE7Yx4RnMoWS4ty76TTg2QxjKKa4J-iRN0N2T8_9Ct1-eP91_5HcfL7-tH93Q6xgvJBW0pYz2ggwUClw3jMFknWdbJgES5lgRtVeSWVd6-u2ayuqKlMb34iKQ8Wv0OtNd07x-9HloseQrRsGM7l4zFrKhguQcgFf_gPexWOaFm-aNg1fa4XoBtkUc07O6zmF0aSTpqDXEPQWgl5C0GsIenXw_Cx8bEfX_dk4f30BXp0Bk60ZfDKTDfk3xyhVrOL1wrGNy8toOrj0l8P_XH-xLXkTtTmkRfj2CwPKgVaN5ELwX6cFqJo</recordid><startdate>20090301</startdate><enddate>20090301</enddate><creator>Wei, Ping</creator><creator>Bai, Liping</creator><creator>Song, Wengang</creator><creator>Hao, Gangping</creator><general>Berlin/Heidelberg : Springer-Verlag</general><general>Springer-Verlag</general><general>Springer</general><general>Springer Nature B.V</general><scope>FBQ</scope><scope>IQODW</scope><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>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</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>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20090301</creationdate><title>Characterization of two soil metagenome-derived lipases with high specificity for p-nitrophenyl palmitate</title><author>Wei, Ping ; Bai, Liping ; Song, Wengang ; Hao, Gangping</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c423t-b61b321940a0580eff28062dd69260c1242a87f868cebf7bdb5185a7af9453053</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>Amino Acid Sequence</topic><topic>Amino acids</topic><topic>Bacteriology</topic><topic>Biochemistry</topic><topic>Biological and medical sciences</topic><topic>Biomedical and Life Sciences</topic><topic>Biotechnology</topic><topic>Cell Biology</topic><topic>Cloning</topic><topic>DNA, Bacterial - isolation & purification</topic><topic>E coli</topic><topic>Ecology</topic><topic>Enzyme Stability</topic><topic>Enzymes</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - metabolism</topic><topic>Esters</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Library</topic><topic>Genes</topic><topic>Genetics</topic><topic>Genome, Bacterial</topic><topic>Hydrogen-Ion Concentration</topic><topic>Life Sciences</topic><topic>Lipase - genetics</topic><topic>Lipase - metabolism</topic><topic>Microbial Ecology</topic><topic>Microbiology</topic><topic>Microorganisms</topic><topic>Molecular Sequence Data</topic><topic>Molecular weight</topic><topic>Nitrilotriacetic acid</topic><topic>Original Paper</topic><topic>Palmitates - metabolism</topic><topic>Plasmids</topic><topic>Proteins</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sequence Alignment</topic><topic>Soil Microbiology</topic><topic>Substrate Specificity</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wei, Ping</creatorcontrib><creatorcontrib>Bai, Liping</creatorcontrib><creatorcontrib>Song, Wengang</creatorcontrib><creatorcontrib>Hao, Gangping</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><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>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>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>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</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>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>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</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>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Archives of microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wei, Ping</au><au>Bai, Liping</au><au>Song, Wengang</au><au>Hao, Gangping</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization of two soil metagenome-derived lipases with high specificity for p-nitrophenyl palmitate</atitle><jtitle>Archives of microbiology</jtitle><stitle>Arch Microbiol</stitle><addtitle>Arch Microbiol</addtitle><date>2009-03-01</date><risdate>2009</risdate><volume>191</volume><issue>3</issue><spage>233</spage><epage>240</epage><pages>233-240</pages><issn>0302-8933</issn><eissn>1432-072X</eissn><coden>AMICCW</coden><abstract>Two novel genes (pwtsB and pwtsC) encoding lipases were isolated by screening the soil metagenomic library. Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecular weight of 35 kDa. Furthermore, both genes were cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzymes were purified by Ni-nitrilotriacetic acid affinity chromatography and characterized by spectrophotometric with different p-nitrophenyl esters. The results showed that PWTSB displayed a high degree of activity and stability at 20°C with an optimal pH of around 8.0, and PWTSC at 40°C with an optimal pH of around 7.0. P-nitrophenyl palmitate (p-NPP) was identified as the best substrate of PWTSB and PWTSC. The specific activities of PWTSB and PWTSC were 150 and 166 U/mg, respectively toward p-NPP at 30°C, about 20-fold higher than that toward p-nitrophenyl butyrate (C4) and caprylate (C8). In conclusion, our results suggest that PWTSB is a cold adapt lipase and PWTSC is a thermostable lipase to long-chain p-nitrophenyl esters.</abstract><cop>Berlin/Heidelberg</cop><pub>Berlin/Heidelberg : Springer-Verlag</pub><pmid>19043691</pmid><doi>10.1007/s00203-008-0448-5</doi><tpages>8</tpages></addata></record> |
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| subjects | Amino Acid Sequence Amino acids Bacteriology Biochemistry Biological and medical sciences Biomedical and Life Sciences Biotechnology Cell Biology Cloning DNA, Bacterial - isolation & purification E coli Ecology Enzyme Stability Enzymes Escherichia coli - genetics Escherichia coli - metabolism Esters Fundamental and applied biological sciences. Psychology Gene Library Genes Genetics Genome, Bacterial Hydrogen-Ion Concentration Life Sciences Lipase - genetics Lipase - metabolism Microbial Ecology Microbiology Microorganisms Molecular Sequence Data Molecular weight Nitrilotriacetic acid Original Paper Palmitates - metabolism Plasmids Proteins Recombinant Proteins - genetics Recombinant Proteins - metabolism Sequence Alignment Soil Microbiology Substrate Specificity Temperature |
| title | Characterization of two soil metagenome-derived lipases with high specificity for p-nitrophenyl palmitate |
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