Development and characterization of cyclodextrin glucanotransferase as a maltoheptaose-producing enzyme using site-directed mutagenesis
Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) mainly produces cyclodextrins (CDs) using linear maltooligosaccharides. We performed site-directed saturation mutagenesis on the +1 substrate-binding residue, H233, of CGTase from alkalophilic Bacillus sp. I-5 to prepare specific-length oligosacc...
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| Veröffentlicht in: | Protein engineering, design and selection design and selection, 2015-11, Vol.28 (11), p.531-537 |
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| container_title | Protein engineering, design and selection |
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| creator | Koo, Ye-Seul Lee, Hye-Won Jeon, Hye-Yeon Choi, Hye-Jeong Choung, Woo-Jae Shim, Jae-Hoon |
| description | Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) mainly produces cyclodextrins (CDs) using linear maltooligosaccharides. We performed site-directed saturation mutagenesis on the +1 substrate-binding residue, H233, of CGTase from alkalophilic Bacillus sp. I-5 to prepare specific-length oligosaccharides. The obtained mutant CGTase, H233Y, primarily produced maltoheptaose (G7) using β-CD via a hydrolysis reaction. A kinetic study of H233Y showed that the kcat/Km value of β-CD was 7-fold greater than that of G7, which accounts for the accumulation of G7 during the H233Y enzyme reaction. A structure comparison of CGTases with H233Y modeling suggests that the substitution of H233Y may alter the position of the +1 subsite and slow the further hydrolysis of G7 after the ring-opening reaction. |
| doi_str_mv | 10.1093/protein/gzv044 |
| format | Article |
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We performed site-directed saturation mutagenesis on the +1 substrate-binding residue, H233, of CGTase from alkalophilic Bacillus sp. I-5 to prepare specific-length oligosaccharides. The obtained mutant CGTase, H233Y, primarily produced maltoheptaose (G7) using β-CD via a hydrolysis reaction. A kinetic study of H233Y showed that the kcat/Km value of β-CD was 7-fold greater than that of G7, which accounts for the accumulation of G7 during the H233Y enzyme reaction. A structure comparison of CGTases with H233Y modeling suggests that the substitution of H233Y may alter the position of the +1 subsite and slow the further hydrolysis of G7 after the ring-opening reaction.</description><identifier>ISSN: 1741-0126</identifier><identifier>EISSN: 1741-0134</identifier><identifier>DOI: 10.1093/protein/gzv044</identifier><identifier>PMID: 26359254</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Amino Acid Sequence ; Bacillus ; Bacillus - enzymology ; Bacillus - genetics ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Glucans - metabolism ; Glucosyltransferases - chemistry ; Glucosyltransferases - genetics ; Glucosyltransferases - metabolism ; Molecular Sequence Data ; Mutagenesis, Site-Directed - methods ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Sequence Alignment</subject><ispartof>Protein engineering, design and selection, 2015-11, Vol.28 (11), p.531-537</ispartof><rights>The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com 2015</rights><rights>The Author 2015. Published by Oxford University Press. All rights reserved. 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A structure comparison of CGTases with H233Y modeling suggests that the substitution of H233Y may alter the position of the +1 subsite and slow the further hydrolysis of G7 after the ring-opening reaction.</description><subject>Amino Acid Sequence</subject><subject>Bacillus</subject><subject>Bacillus - enzymology</subject><subject>Bacillus - genetics</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Glucans - metabolism</subject><subject>Glucosyltransferases - chemistry</subject><subject>Glucosyltransferases - genetics</subject><subject>Glucosyltransferases - metabolism</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed - methods</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sequence Alignment</subject><issn>1741-0126</issn><issn>1741-0134</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkEFP3DAQha2KqizQa4_IR3oIeBzH2RwraEullXqBczS1J4urxE5tZ8XuH-jfJqtduHKaGc03b54eY19AXINoypsxhkzO36x3G6HUB7aAWkEhoFQnb73Up-wspb9CSF0DfGKnUpdVIyu1YP_vaEN9GAfymaO33DxhRJMpuh1mFzwPHTdb0wdLzzk6z9f9ZNCHHNGnjiIm4pg48gH7HJ5ozBgSFbMvOxnn15z8bjsQn9J-SC5TYV2k-YPlw5RxTZ6SSxfsY4d9os_Hes4ef3x_uL0vVr9__rr9tiqMEjIXTQUCVFVaC7YBUB1WDdRWSRLYaK0R9VLJqpZWgVXaCNURyXkryw6XsinP2dVBdzb4b6KU28ElQ32PnsKUWqiXoiphWekZvT6gJoaUInXtGN2AcduCaPfht8fw20P488HlUXv6M5B9w1_TnoGvByBM43tiL-UylN4</recordid><startdate>201511</startdate><enddate>201511</enddate><creator>Koo, Ye-Seul</creator><creator>Lee, Hye-Won</creator><creator>Jeon, Hye-Yeon</creator><creator>Choi, Hye-Jeong</creator><creator>Choung, Woo-Jae</creator><creator>Shim, Jae-Hoon</creator><general>Oxford University Press</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>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>201511</creationdate><title>Development and characterization of cyclodextrin glucanotransferase as a maltoheptaose-producing enzyme using site-directed mutagenesis</title><author>Koo, Ye-Seul ; Lee, Hye-Won ; Jeon, Hye-Yeon ; Choi, Hye-Jeong ; Choung, Woo-Jae ; Shim, Jae-Hoon</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c402t-95101453dd1d9114fa5917d42e0a9666aa6842572d41d46c04fee22e023fa8293</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Amino Acid Sequence</topic><topic>Bacillus</topic><topic>Bacillus - enzymology</topic><topic>Bacillus - genetics</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Glucans - metabolism</topic><topic>Glucosyltransferases - chemistry</topic><topic>Glucosyltransferases - genetics</topic><topic>Glucosyltransferases - metabolism</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed - methods</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sequence Alignment</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Koo, Ye-Seul</creatorcontrib><creatorcontrib>Lee, Hye-Won</creatorcontrib><creatorcontrib>Jeon, Hye-Yeon</creatorcontrib><creatorcontrib>Choi, Hye-Jeong</creatorcontrib><creatorcontrib>Choung, Woo-Jae</creatorcontrib><creatorcontrib>Shim, Jae-Hoon</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Protein engineering, design and selection</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Koo, Ye-Seul</au><au>Lee, Hye-Won</au><au>Jeon, Hye-Yeon</au><au>Choi, Hye-Jeong</au><au>Choung, Woo-Jae</au><au>Shim, Jae-Hoon</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development and characterization of cyclodextrin glucanotransferase as a maltoheptaose-producing enzyme using site-directed mutagenesis</atitle><jtitle>Protein engineering, design and selection</jtitle><addtitle>Protein Eng Des Sel</addtitle><date>2015-11</date><risdate>2015</risdate><volume>28</volume><issue>11</issue><spage>531</spage><epage>537</epage><pages>531-537</pages><issn>1741-0126</issn><eissn>1741-0134</eissn><abstract>Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) mainly produces cyclodextrins (CDs) using linear maltooligosaccharides. We performed site-directed saturation mutagenesis on the +1 substrate-binding residue, H233, of CGTase from alkalophilic Bacillus sp. I-5 to prepare specific-length oligosaccharides. The obtained mutant CGTase, H233Y, primarily produced maltoheptaose (G7) using β-CD via a hydrolysis reaction. A kinetic study of H233Y showed that the kcat/Km value of β-CD was 7-fold greater than that of G7, which accounts for the accumulation of G7 during the H233Y enzyme reaction. A structure comparison of CGTases with H233Y modeling suggests that the substitution of H233Y may alter the position of the +1 subsite and slow the further hydrolysis of G7 after the ring-opening reaction.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>26359254</pmid><doi>10.1093/protein/gzv044</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Oxford University Press Journals All Titles (1996-Current); Alma/SFX Local Collection |
| subjects | Amino Acid Sequence Bacillus Bacillus - enzymology Bacillus - genetics Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism Glucans - metabolism Glucosyltransferases - chemistry Glucosyltransferases - genetics Glucosyltransferases - metabolism Molecular Sequence Data Mutagenesis, Site-Directed - methods Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Sequence Alignment |
| title | Development and characterization of cyclodextrin glucanotransferase as a maltoheptaose-producing enzyme using site-directed mutagenesis |
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