Knowledge-guided laboratory evolution of protein thermolability
In rare but nevertheless important cases it is of practical interest to decrease the thermostability of an enzyme, that is, to increase thermolability in a controlled manner. In the present model study, this unconventional goal has been reached by applying directed evolution to the lipase from Pseud...
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| Veröffentlicht in: | Biotechnology and bioengineering 2009-04, Vol.102 (6), p.1712-1717 |
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| creator | Reetz, Manfred T Soni, Pankaj Fernández, Layla |
| description | In rare but nevertheless important cases it is of practical interest to decrease the thermostability of an enzyme, that is, to increase thermolability in a controlled manner. In the present model study, this unconventional goal has been reached by applying directed evolution to the lipase from Pseudomonas aeruginosa (PAL). By utilizing the B-factor iterative test (B-FIT), previously developed to increase the thermostability of enzymes, it was possible to reduce the $T_{50}^{15} $ value from 71.6°C in the case of wild type (WT-PAL) to 35.6°C (best mutant) without affecting the catalytic profile in terms of substrate acceptance or enantioselectivity at room temperature. Accordingly, saturation mutagenesis was performed at sites in PAL, which on the basis of its X-ray structure, have the lowest B-factors indicative of high rigidity. Focused mutations were introduced which can be expected to decrease rigidity, the ensuing increased flexibility leading to higher thermolability without changing the actual catalytic profile. Biotechnol. Bioeng. 2009;102: 1712-1717. |
| doi_str_mv | 10.1002/bit.22202 |
| format | Article |
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In the present model study, this unconventional goal has been reached by applying directed evolution to the lipase from Pseudomonas aeruginosa (PAL). By utilizing the B-factor iterative test (B-FIT), previously developed to increase the thermostability of enzymes, it was possible to reduce the $T_{50}^{15} $ value from 71.6°C in the case of wild type (WT-PAL) to 35.6°C (best mutant) without affecting the catalytic profile in terms of substrate acceptance or enantioselectivity at room temperature. Accordingly, saturation mutagenesis was performed at sites in PAL, which on the basis of its X-ray structure, have the lowest B-factors indicative of high rigidity. Focused mutations were introduced which can be expected to decrease rigidity, the ensuing increased flexibility leading to higher thermolability without changing the actual catalytic profile. Biotechnol. Bioeng. 2009;102: 1712-1717.</description><identifier>ISSN: 0006-3592</identifier><identifier>EISSN: 1097-0290</identifier><identifier>DOI: 10.1002/bit.22202</identifier><identifier>PMID: 19072845</identifier><identifier>CODEN: BIBIAU</identifier><language>eng</language><publisher>Hoboken: Wiley Subscription Services, Inc., A Wiley Company</publisher><subject>B-factors ; Bacteria ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Biological and medical sciences ; Biotechnology ; Catalysis ; directed evolution ; Directed Molecular Evolution - methods ; Enzyme Stability - genetics ; enzyme thermolability ; Enzymes ; Evolution & development ; Fundamental and applied biological sciences. Psychology ; Gene Library ; Lipase - chemistry ; Lipase - genetics ; lipases ; Methods. Procedures. Technologies ; Models, Molecular ; Mutagenesis ; Mutation ; Protein engineering ; Proteins ; Pseudomonas aeruginosa ; Pseudomonas aeruginosa - enzymology ; Pseudomonas aeruginosa - genetics ; saturation mutagenesis ; Substrate Specificity - genetics ; Temperature</subject><ispartof>Biotechnology and bioengineering, 2009-04, Vol.102 (6), p.1712-1717</ispartof><rights>Copyright © 2008 Wiley Periodicals, Inc.</rights><rights>2009 INIST-CNRS</rights><rights>2008 Wiley Periodicals, Inc.</rights><rights>Copyright John Wiley and Sons, Limited Apr 15, 2009</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5702-79db4f714538e21a8690f86f09e16a70891ab8ce2df01648db7e4c085b4828813</citedby><cites>FETCH-LOGICAL-c5702-79db4f714538e21a8690f86f09e16a70891ab8ce2df01648db7e4c085b4828813</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fbit.22202$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fbit.22202$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1417,27924,27925,45574,45575</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=21295915$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19072845$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Reetz, Manfred T</creatorcontrib><creatorcontrib>Soni, Pankaj</creatorcontrib><creatorcontrib>Fernández, Layla</creatorcontrib><title>Knowledge-guided laboratory evolution of protein thermolability</title><title>Biotechnology and bioengineering</title><addtitle>Biotechnol. Bioeng</addtitle><description>In rare but nevertheless important cases it is of practical interest to decrease the thermostability of an enzyme, that is, to increase thermolability in a controlled manner. In the present model study, this unconventional goal has been reached by applying directed evolution to the lipase from Pseudomonas aeruginosa (PAL). By utilizing the B-factor iterative test (B-FIT), previously developed to increase the thermostability of enzymes, it was possible to reduce the $T_{50}^{15} $ value from 71.6°C in the case of wild type (WT-PAL) to 35.6°C (best mutant) without affecting the catalytic profile in terms of substrate acceptance or enantioselectivity at room temperature. Accordingly, saturation mutagenesis was performed at sites in PAL, which on the basis of its X-ray structure, have the lowest B-factors indicative of high rigidity. Focused mutations were introduced which can be expected to decrease rigidity, the ensuing increased flexibility leading to higher thermolability without changing the actual catalytic profile. Biotechnol. Bioeng. 2009;102: 1712-1717.</description><subject>B-factors</subject><subject>Bacteria</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Catalysis</subject><subject>directed evolution</subject><subject>Directed Molecular Evolution - methods</subject><subject>Enzyme Stability - genetics</subject><subject>enzyme thermolability</subject><subject>Enzymes</subject><subject>Evolution & development</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Library</subject><subject>Lipase - chemistry</subject><subject>Lipase - genetics</subject><subject>lipases</subject><subject>Methods. Procedures. Technologies</subject><subject>Models, Molecular</subject><subject>Mutagenesis</subject><subject>Mutation</subject><subject>Protein engineering</subject><subject>Proteins</subject><subject>Pseudomonas aeruginosa</subject><subject>Pseudomonas aeruginosa - enzymology</subject><subject>Pseudomonas aeruginosa - genetics</subject><subject>saturation mutagenesis</subject><subject>Substrate Specificity - genetics</subject><subject>Temperature</subject><issn>0006-3592</issn><issn>1097-0290</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0E1rFDEYB_Agil2rB7-ADoJCD9M-SSaT5FR0sdvWoge3CF5CZiZZU7OTmsxY99s366wVCuIpBH7P2x-h5xgOMQA5atxwSAgB8gDNMEheApHwEM0AoC4pk2QPPUnpKn-5qOvHaA9L4ERUbIaOP_ThxptuZcrV6DrTFV43IeohxE1hfgY_Di70RbDFdQyDcX0xfDNxHbJy3g2bp-iR1T6ZZ7t3H12evF_OT8uLT4uz-duLsmUcSMll11SW44pRYQjWopZgRW1BGlxrDkJi3YjWkM4CrivRNdxULQjWVIIIgek-ejP1zWv8GE0a1Nql1nivexPGpCjllIEk_4UEMK0whQxf3YNXYYx9PkIRTHmNBRUZHUyojSGlaKy6jm6t40ZhUNvsVc5e_c4-2xe7hmOzNt1fuQs7g9c7oFOrvY26b126cwQTySTeuqPJ3ThvNv-eqN6dLf-MLqcKlwbz665Cx--q5pQz9eXjQp2y869zcrJQy-xfTt7qoPQq5i0uP2-TyeEDYMHpLYpvsOM</recordid><startdate>20090415</startdate><enddate>20090415</enddate><creator>Reetz, Manfred T</creator><creator>Soni, Pankaj</creator><creator>Fernández, Layla</creator><general>Wiley Subscription Services, Inc., A Wiley Company</general><general>Wiley</general><general>Wiley Subscription Services, Inc</general><scope>FBQ</scope><scope>BSCLL</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>7QF</scope><scope>7QO</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7T7</scope><scope>7TA</scope><scope>7TB</scope><scope>7U5</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7QL</scope></search><sort><creationdate>20090415</creationdate><title>Knowledge-guided laboratory evolution of protein thermolability</title><author>Reetz, Manfred T ; Soni, Pankaj ; Fernández, Layla</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5702-79db4f714538e21a8690f86f09e16a70891ab8ce2df01648db7e4c085b4828813</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>B-factors</topic><topic>Bacteria</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Catalysis</topic><topic>directed evolution</topic><topic>Directed Molecular Evolution - methods</topic><topic>Enzyme Stability - genetics</topic><topic>enzyme thermolability</topic><topic>Enzymes</topic><topic>Evolution & development</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Library</topic><topic>Lipase - chemistry</topic><topic>Lipase - genetics</topic><topic>lipases</topic><topic>Methods. Procedures. Technologies</topic><topic>Models, Molecular</topic><topic>Mutagenesis</topic><topic>Mutation</topic><topic>Protein engineering</topic><topic>Proteins</topic><topic>Pseudomonas aeruginosa</topic><topic>Pseudomonas aeruginosa - enzymology</topic><topic>Pseudomonas aeruginosa - genetics</topic><topic>saturation mutagenesis</topic><topic>Substrate Specificity - genetics</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Reetz, Manfred T</creatorcontrib><creatorcontrib>Soni, Pankaj</creatorcontrib><creatorcontrib>Fernández, Layla</creatorcontrib><collection>AGRIS</collection><collection>Istex</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>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><jtitle>Biotechnology and bioengineering</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Reetz, Manfred T</au><au>Soni, Pankaj</au><au>Fernández, Layla</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Knowledge-guided laboratory evolution of protein thermolability</atitle><jtitle>Biotechnology and bioengineering</jtitle><addtitle>Biotechnol. 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Accordingly, saturation mutagenesis was performed at sites in PAL, which on the basis of its X-ray structure, have the lowest B-factors indicative of high rigidity. Focused mutations were introduced which can be expected to decrease rigidity, the ensuing increased flexibility leading to higher thermolability without changing the actual catalytic profile. Biotechnol. Bioeng. 2009;102: 1712-1717.</abstract><cop>Hoboken</cop><pub>Wiley Subscription Services, Inc., A Wiley Company</pub><pmid>19072845</pmid><doi>10.1002/bit.22202</doi><tpages>6</tpages></addata></record> |
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| subjects | B-factors Bacteria Bacterial Proteins - genetics Bacterial Proteins - metabolism Biological and medical sciences Biotechnology Catalysis directed evolution Directed Molecular Evolution - methods Enzyme Stability - genetics enzyme thermolability Enzymes Evolution & development Fundamental and applied biological sciences. Psychology Gene Library Lipase - chemistry Lipase - genetics lipases Methods. Procedures. Technologies Models, Molecular Mutagenesis Mutation Protein engineering Proteins Pseudomonas aeruginosa Pseudomonas aeruginosa - enzymology Pseudomonas aeruginosa - genetics saturation mutagenesis Substrate Specificity - genetics Temperature |
| title | Knowledge-guided laboratory evolution of protein thermolability |
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