Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR
Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment. Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to...
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| description | Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment. Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60-80°C). After a mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over 20% improvement in thermostability (80°C for 10 min), and 6-7°C increases in melting temperatures (T m). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (k cat / K m) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic efficiency of these enzymes was not inversely related to their thermostability. |
| doi_str_mv | 10.1007/s00253-008-1412-7 |
| format | Article |
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Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60-80°C). After a mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over 20% improvement in thermostability (80°C for 10 min), and 6-7°C increases in melting temperatures (T m). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (k cat / K m) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic efficiency of these enzymes was not inversely related to their thermostability.</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-008-1412-7</identifier><identifier>PMID: 18340444</identifier><identifier>CODEN: AMBIDG</identifier><language>eng</language><publisher>Berlin/Heidelberg: Berlin/Heidelberg : Springer-Verlag</publisher><subject>6-Phytase - genetics ; 6-Phytase - metabolism ; Acid Phosphatase - genetics ; Acid Phosphatase - metabolism ; Animal sciences ; Bacteria ; Biological and medical sciences ; Biotechnologically Relevant Enzymes and Proteins ; Biotechnology ; Calorimetry, Differential Scanning ; Catalysis ; Directed Molecular Evolution ; E coli ; Efficiency ; Enzyme ; Enzyme Stability ; Enzymes ; Error-prone PCR ; Escherichia coli ; Escherichia coli - enzymology ; Escherichia coli - genetics ; Escherichia coli Proteins - genetics ; Escherichia coli Proteins - metabolism ; Fundamental and applied biological sciences. Psychology ; Genetic engineering ; Hydrogen Bonding ; Hydrogen bonds ; Hydrogen-Ion Concentration ; Inactivation ; Kinetics ; Life Sciences ; Melting temperature ; Methods. Procedures. Technologies ; Microbial Genetics and Genomics ; Microbiology ; Models, Molecular ; Multienzyme Complexes - genetics ; Multienzyme Complexes - metabolism ; Mutagenesis ; Phosphatase ; Phosphorus ; Phytase ; Pichia pastoris ; Polymerase Chain Reaction ; Protein Engineering ; Protein Structure, Secondary ; Proteins ; Saccharomyces cerevisiae ; Studies ; Temperature ; thermal stability ; Transition Temperature ; Yeast ; Zoology</subject><ispartof>Applied microbiology and biotechnology, 2008-05, Vol.79 (1), p.69-75</ispartof><rights>Springer-Verlag 2008</rights><rights>2008 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c520t-2498417863256b86f6397abfe2f0a7e41970f1419ccf6e113d8ea011c116ed5d3</citedby><cites>FETCH-LOGICAL-c520t-2498417863256b86f6397abfe2f0a7e41970f1419ccf6e113d8ea011c116ed5d3</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/s00253-008-1412-7$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00253-008-1412-7$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=20280856$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18340444$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kim, Moon-Soo</creatorcontrib><creatorcontrib>Lei, Xin Gen</creatorcontrib><title>Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR</title><title>Applied microbiology and biotechnology</title><addtitle>Appl Microbiol Biotechnol</addtitle><addtitle>Appl Microbiol Biotechnol</addtitle><description>Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment. Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60-80°C). After a mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over 20% improvement in thermostability (80°C for 10 min), and 6-7°C increases in melting temperatures (T m). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (k cat / K m) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic efficiency of these enzymes was not inversely related to their thermostability.</description><subject>6-Phytase - genetics</subject><subject>6-Phytase - metabolism</subject><subject>Acid Phosphatase - genetics</subject><subject>Acid Phosphatase - metabolism</subject><subject>Animal sciences</subject><subject>Bacteria</subject><subject>Biological and medical sciences</subject><subject>Biotechnologically Relevant Enzymes and Proteins</subject><subject>Biotechnology</subject><subject>Calorimetry, Differential Scanning</subject><subject>Catalysis</subject><subject>Directed Molecular Evolution</subject><subject>E coli</subject><subject>Efficiency</subject><subject>Enzyme</subject><subject>Enzyme Stability</subject><subject>Enzymes</subject><subject>Error-prone PCR</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Escherichia coli Proteins - metabolism</subject><subject>Fundamental and applied biological sciences. 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Technologies</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Models, Molecular</subject><subject>Multienzyme Complexes - genetics</subject><subject>Multienzyme Complexes - metabolism</subject><subject>Mutagenesis</subject><subject>Phosphatase</subject><subject>Phosphorus</subject><subject>Phytase</subject><subject>Pichia pastoris</subject><subject>Polymerase Chain Reaction</subject><subject>Protein Engineering</subject><subject>Protein Structure, Secondary</subject><subject>Proteins</subject><subject>Saccharomyces cerevisiae</subject><subject>Studies</subject><subject>Temperature</subject><subject>thermal stability</subject><subject>Transition Temperature</subject><subject>Yeast</subject><subject>Zoology</subject><issn>0175-7598</issn><issn>1432-0614</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</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>eNp9kMFuEzEURS0EomnhA9iAhQQ7w3u2x_YsoygUpKpFQNeWx7GTqSYzgz1Z5O9xNBGVuujKkn3ue9eHkHcIXxBAf80AvBIMwDCUyJl-QRYoBWegUL4kC0BdMV3V5oJc5vwAgNwo9ZpcoBESpJQLcrvud673bb-l0y6k_ZAn17RdOx3pEOk6-3LZ-l3rqB-6lo674-RyoMtxXHLaHGlIaUhsTEMf6M_VrzfkVXRdDm_P5xW5_7b-s_rObu6uf6yWN8xXHCbGZW0kaqMEr1RjVFSi1q6JgUdwOkisNcTypdr7qAKi2JjgANEjqrCpNuKKfJ7nls1_DyFPdt9mH7rO9WE4ZMsBoMRVAT8-AR-GQ-pLN8t5XRllKlkgnCGfhpxTiHZM7d6lo0WwJ9N2Nm2LaXsybXXJvD8PPjT7sHlMnNUW4NMZcNm7LqaT5vyf48ANmOrUkM9cLk_9NqTHhs9t_zCHohus26Yy-P43BxSFMVpqI_4B_MqcuQ</recordid><startdate>20080501</startdate><enddate>20080501</enddate><creator>Kim, Moon-Soo</creator><creator>Lei, Xin Gen</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>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></search><sort><creationdate>20080501</creationdate><title>Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR</title><author>Kim, Moon-Soo ; Lei, Xin Gen</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c520t-2498417863256b86f6397abfe2f0a7e41970f1419ccf6e113d8ea011c116ed5d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>6-Phytase - genetics</topic><topic>6-Phytase - metabolism</topic><topic>Acid Phosphatase - genetics</topic><topic>Acid Phosphatase - metabolism</topic><topic>Animal sciences</topic><topic>Bacteria</topic><topic>Biological and medical sciences</topic><topic>Biotechnologically Relevant Enzymes and Proteins</topic><topic>Biotechnology</topic><topic>Calorimetry, Differential Scanning</topic><topic>Catalysis</topic><topic>Directed Molecular Evolution</topic><topic>E coli</topic><topic>Efficiency</topic><topic>Enzyme</topic><topic>Enzyme Stability</topic><topic>Enzymes</topic><topic>Error-prone PCR</topic><topic>Escherichia coli</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Escherichia coli Proteins - metabolism</topic><topic>Fundamental and applied biological sciences. 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Microbiol Biotechnol</stitle><addtitle>Appl Microbiol Biotechnol</addtitle><date>2008-05-01</date><risdate>2008</risdate><volume>79</volume><issue>1</issue><spage>69</spage><epage>75</epage><pages>69-75</pages><issn>0175-7598</issn><eissn>1432-0614</eissn><coden>AMBIDG</coden><abstract>Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment. Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60-80°C). After a mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over 20% improvement in thermostability (80°C for 10 min), and 6-7°C increases in melting temperatures (T m). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (k cat / K m) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic efficiency of these enzymes was not inversely related to their thermostability.</abstract><cop>Berlin/Heidelberg</cop><pub>Berlin/Heidelberg : Springer-Verlag</pub><pmid>18340444</pmid><doi>10.1007/s00253-008-1412-7</doi><tpages>7</tpages></addata></record> |
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| subjects | 6-Phytase - genetics 6-Phytase - metabolism Acid Phosphatase - genetics Acid Phosphatase - metabolism Animal sciences Bacteria Biological and medical sciences Biotechnologically Relevant Enzymes and Proteins Biotechnology Calorimetry, Differential Scanning Catalysis Directed Molecular Evolution E coli Efficiency Enzyme Enzyme Stability Enzymes Error-prone PCR Escherichia coli Escherichia coli - enzymology Escherichia coli - genetics Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Fundamental and applied biological sciences. Psychology Genetic engineering Hydrogen Bonding Hydrogen bonds Hydrogen-Ion Concentration Inactivation Kinetics Life Sciences Melting temperature Methods. Procedures. Technologies Microbial Genetics and Genomics Microbiology Models, Molecular Multienzyme Complexes - genetics Multienzyme Complexes - metabolism Mutagenesis Phosphatase Phosphorus Phytase Pichia pastoris Polymerase Chain Reaction Protein Engineering Protein Structure, Secondary Proteins Saccharomyces cerevisiae Studies Temperature thermal stability Transition Temperature Yeast Zoology |
| title | Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR |
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