Stability and activity improvement of cephalosporin esterase EstB from Burkholderia gladioli by directed evolution and structural interpretation of muteins
Esterase EstB from Burkholderia gladioli, which belongs to a family of esterases related to β-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substituti...
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| Veröffentlicht in: | Journal of biotechnology 2007-03, Vol.129 (1), p.98-108 |
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| creator | Valinger, Goran Hermann, Manuela Wagner, Ulrike G. Schwab, Helmut |
| description | Esterase EstB from
Burkholderia gladioli, which belongs to a family of esterases related to β-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substitutions per gene. A newly designed colony filter-screening assay, which was based on pH change after deacetylation of cephalosporin C in presence of DMF was established. In a first evolution round employing random mutagenesis, which included about 10
6 mutants, a set of interesting mutants was isolated. Distinct mutations identified as significant for stability were combined by a rational recombination step and the resulting recombinant was further evolved by an additional random mutagenesis round. After screening an additional 10
5 clones, it was possible to isolate a variant of EstB having more than 100-fold better activity in reactions containing 35% DMF. This mutant also showed a high increase in temperature stability (
T
m was raised by 13
°C) and retained high activity towards cephalosporin C under standard assay conditions. The molecular effects of mutations found in random mutants are discussed in view of the three-dimensional structure of wild-type EstB. |
| doi_str_mv | 10.1016/j.jbiotec.2006.10.008 |
| format | Article |
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Burkholderia gladioli, which belongs to a family of esterases related to β-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substitutions per gene. A newly designed colony filter-screening assay, which was based on pH change after deacetylation of cephalosporin C in presence of DMF was established. In a first evolution round employing random mutagenesis, which included about 10
6 mutants, a set of interesting mutants was isolated. Distinct mutations identified as significant for stability were combined by a rational recombination step and the resulting recombinant was further evolved by an additional random mutagenesis round. After screening an additional 10
5 clones, it was possible to isolate a variant of EstB having more than 100-fold better activity in reactions containing 35% DMF. This mutant also showed a high increase in temperature stability (
T
m was raised by 13
°C) and retained high activity towards cephalosporin C under standard assay conditions. The molecular effects of mutations found in random mutants are discussed in view of the three-dimensional structure of wild-type EstB.</description><identifier>ISSN: 0168-1656</identifier><identifier>EISSN: 1873-4863</identifier><identifier>DOI: 10.1016/j.jbiotec.2006.10.008</identifier><identifier>PMID: 17137667</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Activity ; Amino Acid Sequence ; Burkholderia gladioli ; Burkholderia gladioli - enzymology ; Calorimetry, Differential Scanning ; Cephalosporin C ; Directed evolution ; Directed Molecular Evolution - methods ; Enzyme Stability ; Esterase ; Esterases - metabolism ; Half-Life ; Kinetics ; Models, Molecular ; Mutagenesis ; Mutant Proteins - chemistry ; Mutant Proteins - isolation & purification ; Mutation - genetics ; Protein Structure, Secondary ; Sequence Analysis, Protein ; Stability ; Transition Temperature</subject><ispartof>Journal of biotechnology, 2007-03, Vol.129 (1), p.98-108</ispartof><rights>2006 Elsevier B.V.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c394t-eeaa7b15d074da551ec6d9d872b579c0c42c6b93a67dabc91c9067645bdb1f683</citedby><cites>FETCH-LOGICAL-c394t-eeaa7b15d074da551ec6d9d872b579c0c42c6b93a67dabc91c9067645bdb1f683</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jbiotec.2006.10.008$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3548,27923,27924,45994</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17137667$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Valinger, Goran</creatorcontrib><creatorcontrib>Hermann, Manuela</creatorcontrib><creatorcontrib>Wagner, Ulrike G.</creatorcontrib><creatorcontrib>Schwab, Helmut</creatorcontrib><title>Stability and activity improvement of cephalosporin esterase EstB from Burkholderia gladioli by directed evolution and structural interpretation of muteins</title><title>Journal of biotechnology</title><addtitle>J Biotechnol</addtitle><description>Esterase EstB from
Burkholderia gladioli, which belongs to a family of esterases related to β-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substitutions per gene. A newly designed colony filter-screening assay, which was based on pH change after deacetylation of cephalosporin C in presence of DMF was established. In a first evolution round employing random mutagenesis, which included about 10
6 mutants, a set of interesting mutants was isolated. Distinct mutations identified as significant for stability were combined by a rational recombination step and the resulting recombinant was further evolved by an additional random mutagenesis round. After screening an additional 10
5 clones, it was possible to isolate a variant of EstB having more than 100-fold better activity in reactions containing 35% DMF. This mutant also showed a high increase in temperature stability (
T
m was raised by 13
°C) and retained high activity towards cephalosporin C under standard assay conditions. The molecular effects of mutations found in random mutants are discussed in view of the three-dimensional structure of wild-type EstB.</description><subject>Activity</subject><subject>Amino Acid Sequence</subject><subject>Burkholderia gladioli</subject><subject>Burkholderia gladioli - enzymology</subject><subject>Calorimetry, Differential Scanning</subject><subject>Cephalosporin C</subject><subject>Directed evolution</subject><subject>Directed Molecular Evolution - methods</subject><subject>Enzyme Stability</subject><subject>Esterase</subject><subject>Esterases - metabolism</subject><subject>Half-Life</subject><subject>Kinetics</subject><subject>Models, Molecular</subject><subject>Mutagenesis</subject><subject>Mutant Proteins - chemistry</subject><subject>Mutant Proteins - isolation & purification</subject><subject>Mutation - genetics</subject><subject>Protein Structure, Secondary</subject><subject>Sequence Analysis, Protein</subject><subject>Stability</subject><subject>Transition Temperature</subject><issn>0168-1656</issn><issn>1873-4863</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcuO1DAQRS0EYpqBTwB5xS6N3UnseIWY0fCQRmIBrC0_Kkw1Thxsp6X-Fn4W93RLLFmVqnyrrq4PIa8523LGxbv9dm8xFnDbHWOizraMDU_Ihg-ybbpBtE_JpuqGhoteXJEXOe8ZY53q-XNyxSVvpRByQ_58K8ZiwHKkZvbUuIKHU4PTkuIBJpgLjSN1sDyYEPMSE84UcoFkMtC7XG7omOJEb9b06yEGDwkN_RmMxxiQ2iP1mMAV8BQOMawF4_xolEtaXVmTCRTnem1JUMzja3Wb1gI455fk2WhChleXek1-fLz7fvu5uf_66cvth_vGtaorDYAx0vLeM9l50_ccnPDKD3Jne6kcc93OCataI6Q31inuFBNSdL31lo9iaK_J2_PdGvn3WsPpCbODEMwMcc2aKyX7Vsoq7M9Cl2LOCUa9JJxMOmrO9ImK3usLFX2ichpXKnXvzcVgtRP4f1sXDFXw_iyAGvOAkHR2CLOD8-9pH_E_Fn8BBWimig</recordid><startdate>20070330</startdate><enddate>20070330</enddate><creator>Valinger, Goran</creator><creator>Hermann, Manuela</creator><creator>Wagner, Ulrike G.</creator><creator>Schwab, Helmut</creator><general>Elsevier 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>7QL</scope><scope>7QO</scope><scope>7T7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>20070330</creationdate><title>Stability and activity improvement of cephalosporin esterase EstB from Burkholderia gladioli by directed evolution and structural interpretation of muteins</title><author>Valinger, Goran ; Hermann, Manuela ; Wagner, Ulrike G. ; Schwab, Helmut</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c394t-eeaa7b15d074da551ec6d9d872b579c0c42c6b93a67dabc91c9067645bdb1f683</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Activity</topic><topic>Amino Acid Sequence</topic><topic>Burkholderia gladioli</topic><topic>Burkholderia gladioli - enzymology</topic><topic>Calorimetry, Differential Scanning</topic><topic>Cephalosporin C</topic><topic>Directed evolution</topic><topic>Directed Molecular Evolution - methods</topic><topic>Enzyme Stability</topic><topic>Esterase</topic><topic>Esterases - metabolism</topic><topic>Half-Life</topic><topic>Kinetics</topic><topic>Models, Molecular</topic><topic>Mutagenesis</topic><topic>Mutant Proteins - chemistry</topic><topic>Mutant Proteins - isolation & purification</topic><topic>Mutation - genetics</topic><topic>Protein Structure, Secondary</topic><topic>Sequence Analysis, Protein</topic><topic>Stability</topic><topic>Transition Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Valinger, Goran</creatorcontrib><creatorcontrib>Hermann, Manuela</creatorcontrib><creatorcontrib>Wagner, Ulrike G.</creatorcontrib><creatorcontrib>Schwab, Helmut</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Journal of biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Valinger, Goran</au><au>Hermann, Manuela</au><au>Wagner, Ulrike G.</au><au>Schwab, Helmut</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Stability and activity improvement of cephalosporin esterase EstB from Burkholderia gladioli by directed evolution and structural interpretation of muteins</atitle><jtitle>Journal of biotechnology</jtitle><addtitle>J Biotechnol</addtitle><date>2007-03-30</date><risdate>2007</risdate><volume>129</volume><issue>1</issue><spage>98</spage><epage>108</epage><pages>98-108</pages><issn>0168-1656</issn><eissn>1873-4863</eissn><abstract>Esterase EstB from
Burkholderia gladioli, which belongs to a family of esterases related to β-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substitutions per gene. A newly designed colony filter-screening assay, which was based on pH change after deacetylation of cephalosporin C in presence of DMF was established. In a first evolution round employing random mutagenesis, which included about 10
6 mutants, a set of interesting mutants was isolated. Distinct mutations identified as significant for stability were combined by a rational recombination step and the resulting recombinant was further evolved by an additional random mutagenesis round. After screening an additional 10
5 clones, it was possible to isolate a variant of EstB having more than 100-fold better activity in reactions containing 35% DMF. This mutant also showed a high increase in temperature stability (
T
m was raised by 13
°C) and retained high activity towards cephalosporin C under standard assay conditions. The molecular effects of mutations found in random mutants are discussed in view of the three-dimensional structure of wild-type EstB.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>17137667</pmid><doi>10.1016/j.jbiotec.2006.10.008</doi><tpages>11</tpages></addata></record> |
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| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Activity Amino Acid Sequence Burkholderia gladioli Burkholderia gladioli - enzymology Calorimetry, Differential Scanning Cephalosporin C Directed evolution Directed Molecular Evolution - methods Enzyme Stability Esterase Esterases - metabolism Half-Life Kinetics Models, Molecular Mutagenesis Mutant Proteins - chemistry Mutant Proteins - isolation & purification Mutation - genetics Protein Structure, Secondary Sequence Analysis, Protein Stability Transition Temperature |
| title | Stability and activity improvement of cephalosporin esterase EstB from Burkholderia gladioli by directed evolution and structural interpretation of muteins |
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