Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase
Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product...
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| Veröffentlicht in: | Applied microbiology and biotechnology 2009-03, Vol.82 (3), p.479-490 |
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| creator | Doukyu, Noriyuki Shibata, Kanpei Ogino, Hiroyasu Sagermann, Martin |
| description | Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53-62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. K m and V max values of the cholesterol oxidase were estimated from Lineweaver-Burk plots. The V max/K m ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures. |
| doi_str_mv | 10.1007/s00253-008-1775-9 |
| format | Article |
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DS-1 cholesterol oxidase</title><source>MEDLINE</source><source>SpringerLink Journals - AutoHoldings</source><creator>Doukyu, Noriyuki ; Shibata, Kanpei ; Ogino, Hiroyasu ; Sagermann, Martin</creator><creatorcontrib>Doukyu, Noriyuki ; Shibata, Kanpei ; Ogino, Hiroyasu ; Sagermann, Martin</creatorcontrib><description>Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53-62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. K m and V max values of the cholesterol oxidase were estimated from Lineweaver-Burk plots. The V max/K m ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures.</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-008-1775-9</identifier><identifier>PMID: 19015844</identifier><identifier>CODEN: AMBIDG</identifier><language>eng</language><publisher>Berlin/Heidelberg: Berlin/Heidelberg : Springer-Verlag</publisher><subject>Amino Acid Sequence ; Amino acids ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - isolation & purification ; Bacterial Proteins - metabolism ; Base Sequence ; Biological and medical sciences ; Biotechnologically Relevant Enzymes and Proteins ; Biotechnology ; Burkholderia cepacia ; Chemicals ; Cholesterol ; Cholesterol oxidase ; Cholesterol Oxidase - chemistry ; Cholesterol Oxidase - genetics ; Cholesterol Oxidase - isolation & purification ; Cholesterol Oxidase - metabolism ; Chromobacterium ; Chromobacterium - chemistry ; Chromobacterium - enzymology ; Chromobacterium - genetics ; Cloning ; Cloning, Molecular ; Detergent ; Detergents ; E coli ; Enzyme Stability ; Enzymes ; Escherichia coli ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Fundamental and applied biological sciences. Psychology ; Gene Expression ; Genetic research ; High temperature ; Kinetics ; Laboratories ; Life Sciences ; Microbial Genetics and Genomics ; Microbiology ; Molecular Sequence Data ; Molecular Weight ; Organic solvent ; Organic solvents ; Peptides ; Physicochemical properties ; Pseudomonas aeruginosa ; Sequence Analysis ; Sodium ; Solvents ; Spectral analysis ; Studies ; thermal stability</subject><ispartof>Applied microbiology and biotechnology, 2009-03, Vol.82 (3), p.479-490</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-c520t-ab0be24df2514f63220c490f1dd1a231d4e5640239fcd6d1cfe79dc1acd016163</citedby><cites>FETCH-LOGICAL-c520t-ab0be24df2514f63220c490f1dd1a231d4e5640239fcd6d1cfe79dc1acd016163</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/s00253-008-1775-9$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00253-008-1775-9$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,777,781,27905,27906,41469,42538,51300</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=21148399$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19015844$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Doukyu, Noriyuki</creatorcontrib><creatorcontrib>Shibata, Kanpei</creatorcontrib><creatorcontrib>Ogino, Hiroyasu</creatorcontrib><creatorcontrib>Sagermann, Martin</creatorcontrib><title>Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase</title><title>Applied microbiology and biotechnology</title><addtitle>Appl Microbiol Biotechnol</addtitle><addtitle>Appl Microbiol Biotechnol</addtitle><description>Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53-62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. K m and V max values of the cholesterol oxidase were estimated from Lineweaver-Burk plots. The V max/K m ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures.</description><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - isolation & purification</subject><subject>Bacterial Proteins - metabolism</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Biotechnologically Relevant Enzymes and Proteins</subject><subject>Biotechnology</subject><subject>Burkholderia cepacia</subject><subject>Chemicals</subject><subject>Cholesterol</subject><subject>Cholesterol oxidase</subject><subject>Cholesterol Oxidase - chemistry</subject><subject>Cholesterol Oxidase - genetics</subject><subject>Cholesterol Oxidase - isolation & purification</subject><subject>Cholesterol Oxidase - metabolism</subject><subject>Chromobacterium</subject><subject>Chromobacterium - chemistry</subject><subject>Chromobacterium - enzymology</subject><subject>Chromobacterium - genetics</subject><subject>Cloning</subject><subject>Cloning, Molecular</subject><subject>Detergent</subject><subject>Detergents</subject><subject>E coli</subject><subject>Enzyme Stability</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression</subject><subject>Genetic research</subject><subject>High temperature</subject><subject>Kinetics</subject><subject>Laboratories</subject><subject>Life Sciences</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Molecular Sequence Data</subject><subject>Molecular Weight</subject><subject>Organic solvent</subject><subject>Organic solvents</subject><subject>Peptides</subject><subject>Physicochemical properties</subject><subject>Pseudomonas aeruginosa</subject><subject>Sequence Analysis</subject><subject>Sodium</subject><subject>Solvents</subject><subject>Spectral analysis</subject><subject>Studies</subject><subject>thermal stability</subject><issn>0175-7598</issn><issn>1432-0614</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNqFkU9v1DAQxS0EokvhA3ABCwlOTZmxHSc-VstfqRKH0rPltZ1tVkm8eDZS--3xKisqcYCTrfHvvfHMY-w1wiUCNB8JQNSyAmgrbJq6Mk_YCpUUFWhUT9kKsBSb2rRn7AXRDgBFq_VzdoYGsG6VWrHdekhTP20vOMVfc5x85G5ywwP1dFFugcf7fY5EfZp46rjj2zhFXrgUioqv73Ia08b5Q8z9PHLaX_JPNxVyf5eGSKWaBp7u--AovmTPOjdQfHU6z9ntl88_19-q6x9fv6-vritfCzhUbgObKFToRI2q01II8MpAhyGgExKDirVWIKTpfNABfRcbEzw6HwA1annOPiy--5zKSHSwY08-DoObYprJam2kkMXgf6BAURsNR8d3f4G7NOeypsIIU7egdFsgXCCfE1GOnd3nfnT5wSLYY1x2icuWuOwxLmuK5s3JeN6MMTwqTvkU4P0JcOTd0GU3-Z7-cAJRtdIcjcTCUXmatjE__vBf3d8uos4l67a5GN_eCEBZmhsttJC_AUIQtUI</recordid><startdate>20090301</startdate><enddate>20090301</enddate><creator>Doukyu, Noriyuki</creator><creator>Shibata, Kanpei</creator><creator>Ogino, Hiroyasu</creator><creator>Sagermann, Martin</creator><general>Berlin/Heidelberg : Springer-Verlag</general><general>Springer Berlin Heidelberg</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>7T7</scope><scope>7WY</scope><scope>7WZ</scope><scope>7X7</scope><scope>7XB</scope><scope>87Z</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8FL</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BEZIV</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FRNLG</scope><scope>FYUFA</scope><scope>F~G</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K60</scope><scope>K6~</scope><scope>K9.</scope><scope>L.-</scope><scope>LK8</scope><scope>M0C</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQBIZ</scope><scope>PQBZA</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7QO</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20090301</creationdate><title>Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. 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DS-1 cholesterol oxidase</atitle><jtitle>Applied microbiology and biotechnology</jtitle><stitle>Appl Microbiol Biotechnol</stitle><addtitle>Appl Microbiol Biotechnol</addtitle><date>2009-03-01</date><risdate>2009</risdate><volume>82</volume><issue>3</issue><spage>479</spage><epage>490</epage><pages>479-490</pages><issn>0175-7598</issn><eissn>1432-0614</eissn><coden>AMBIDG</coden><abstract>Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53-62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. K m and V max values of the cholesterol oxidase were estimated from Lineweaver-Burk plots. The V max/K m ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures.</abstract><cop>Berlin/Heidelberg</cop><pub>Berlin/Heidelberg : Springer-Verlag</pub><pmid>19015844</pmid><doi>10.1007/s00253-008-1775-9</doi><tpages>12</tpages></addata></record> |
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| subjects | Amino Acid Sequence Amino acids Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - isolation & purification Bacterial Proteins - metabolism Base Sequence Biological and medical sciences Biotechnologically Relevant Enzymes and Proteins Biotechnology Burkholderia cepacia Chemicals Cholesterol Cholesterol oxidase Cholesterol Oxidase - chemistry Cholesterol Oxidase - genetics Cholesterol Oxidase - isolation & purification Cholesterol Oxidase - metabolism Chromobacterium Chromobacterium - chemistry Chromobacterium - enzymology Chromobacterium - genetics Cloning Cloning, Molecular Detergent Detergents E coli Enzyme Stability Enzymes Escherichia coli Escherichia coli - genetics Escherichia coli - metabolism Fundamental and applied biological sciences. Psychology Gene Expression Genetic research High temperature Kinetics Laboratories Life Sciences Microbial Genetics and Genomics Microbiology Molecular Sequence Data Molecular Weight Organic solvent Organic solvents Peptides Physicochemical properties Pseudomonas aeruginosa Sequence Analysis Sodium Solvents Spectral analysis Studies thermal stability |
| title | Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase |
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