Direct electron transfer type disposable sensor strip for glucose sensing employing an engineered FAD glucose dehydrogenase
► FADGDH is great for glucose sensing, but suffers from broad substrate specificity. ► 3D model of FADGDH was generated by homology modeling. ► Engineered FADGDH has no activity with maltose, retains high activity with glucose. ► Developed first direct electron transfer-type, disposable glucose sens...
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| Veröffentlicht in: | Enzyme and microbial technology 2013-02, Vol.52 (2), p.123-128 |
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| creator | Yamashita, Yuki Ferri, Stefano Huynh, Mai Linh Shimizu, Hitomi Yamaoka, Hideaki Sode, Koji |
| description | ► FADGDH is great for glucose sensing, but suffers from broad substrate specificity. ► 3D model of FADGDH was generated by homology modeling. ► Engineered FADGDH has no activity with maltose, retains high activity with glucose. ► Developed first direct electron transfer-type, disposable glucose sensor strip.
The FAD-dependent glucose dehydrogenase (FADGDH) from Burkholderia cepacia has several attractive features for glucose sensing. However, expanding the application of this enzyme requires improvement of its substrate specificity, especially decreasing its high activity toward maltose. A three-dimensional structural model of the FADGDH catalytic subunit was generated by homology modeling. By comparing the predicted active site with that of glucose oxidase, the two amino acid residues serine 326 and serine 365 were targeted for site-directed mutagenesis. The single mutations that produced the highest glucose specificity were combined, leading to the creation of the S326Q/S365Y double mutant, which was virtually nonreactive to maltose while retaining high glucose dehydrogenase activity. The engineered FADGDH was used to develop a direct electron transfer-type, disposable glucose sensor strip by immobilizing the enzyme complex onto a carbon screen-printed electrode. While the electrode employing wild-type FADGDH provided dangerously flawed results in the presence of maltose, the sensor employing our engineered FADGDH showed a clear glucose concentration-dependent response that was not affected by the presence of maltose. |
| doi_str_mv | 10.1016/j.enzmictec.2012.11.002 |
| format | Article |
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The FAD-dependent glucose dehydrogenase (FADGDH) from Burkholderia cepacia has several attractive features for glucose sensing. However, expanding the application of this enzyme requires improvement of its substrate specificity, especially decreasing its high activity toward maltose. A three-dimensional structural model of the FADGDH catalytic subunit was generated by homology modeling. By comparing the predicted active site with that of glucose oxidase, the two amino acid residues serine 326 and serine 365 were targeted for site-directed mutagenesis. The single mutations that produced the highest glucose specificity were combined, leading to the creation of the S326Q/S365Y double mutant, which was virtually nonreactive to maltose while retaining high glucose dehydrogenase activity. The engineered FADGDH was used to develop a direct electron transfer-type, disposable glucose sensor strip by immobilizing the enzyme complex onto a carbon screen-printed electrode. While the electrode employing wild-type FADGDH provided dangerously flawed results in the presence of maltose, the sensor employing our engineered FADGDH showed a clear glucose concentration-dependent response that was not affected by the presence of maltose.</description><identifier>ISSN: 0141-0229</identifier><identifier>EISSN: 1879-0909</identifier><identifier>DOI: 10.1016/j.enzmictec.2012.11.002</identifier><identifier>PMID: 23273282</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Amino Acid Substitution ; Amino acids ; Aspergillus niger - enzymology ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Biosensing Techniques ; Blood Glucose - analysis ; Blood Glucose Self-Monitoring - instrumentation ; Blood Glucose Self-Monitoring - methods ; Burkholderia cepacia ; Burkholderia cepacia - enzymology ; Burkholderia cepacia - genetics ; Carbon ; Catalytic Domain ; Catalytic subunits ; Chemoreception ; Computer Simulation ; Diabetes ; Direct electron transfer ; Electrochemical Techniques ; Electrodes ; Electron transfer ; Electron Transport ; Enzymes ; Escherichia coli ; flavin-adenine dinucleotide ; Flavin-Adenine Dinucleotide - metabolism ; Fungal Proteins - metabolism ; Glucose ; Glucose - metabolism ; Glucose 1-Dehydrogenase - genetics ; Glucose 1-Dehydrogenase - metabolism ; Glucose dehydrogenase ; Glucose Dehydrogenases - metabolism ; Glucose oxidase ; Glucose Oxidase - metabolism ; Glucose sensor ; Homology ; Humans ; Maltose ; Maltose - metabolism ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation ; Protein Binding ; Protein Conformation ; Reagent Strips ; Recombinant Proteins - metabolism ; Sensitivity and Specificity ; Serine ; Site-directed mutagenesis ; SMBG ; Substrate Specificity</subject><ispartof>Enzyme and microbial technology, 2013-02, Vol.52 (2), p.123-128</ispartof><rights>2012 Elsevier Inc.</rights><rights>Copyright © 2012 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c404t-421806b5773e91108e0095362cf3b2fbd095057efcb4fec6372d93fa7f816d403</citedby><cites>FETCH-LOGICAL-c404t-421806b5773e91108e0095362cf3b2fbd095057efcb4fec6372d93fa7f816d403</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0141022912001810$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23273282$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Yamashita, Yuki</creatorcontrib><creatorcontrib>Ferri, Stefano</creatorcontrib><creatorcontrib>Huynh, Mai Linh</creatorcontrib><creatorcontrib>Shimizu, Hitomi</creatorcontrib><creatorcontrib>Yamaoka, Hideaki</creatorcontrib><creatorcontrib>Sode, Koji</creatorcontrib><title>Direct electron transfer type disposable sensor strip for glucose sensing employing an engineered FAD glucose dehydrogenase</title><title>Enzyme and microbial technology</title><addtitle>Enzyme Microb Technol</addtitle><description>► FADGDH is great for glucose sensing, but suffers from broad substrate specificity. ► 3D model of FADGDH was generated by homology modeling. ► Engineered FADGDH has no activity with maltose, retains high activity with glucose. ► Developed first direct electron transfer-type, disposable glucose sensor strip.
The FAD-dependent glucose dehydrogenase (FADGDH) from Burkholderia cepacia has several attractive features for glucose sensing. However, expanding the application of this enzyme requires improvement of its substrate specificity, especially decreasing its high activity toward maltose. A three-dimensional structural model of the FADGDH catalytic subunit was generated by homology modeling. By comparing the predicted active site with that of glucose oxidase, the two amino acid residues serine 326 and serine 365 were targeted for site-directed mutagenesis. The single mutations that produced the highest glucose specificity were combined, leading to the creation of the S326Q/S365Y double mutant, which was virtually nonreactive to maltose while retaining high glucose dehydrogenase activity. The engineered FADGDH was used to develop a direct electron transfer-type, disposable glucose sensor strip by immobilizing the enzyme complex onto a carbon screen-printed electrode. While the electrode employing wild-type FADGDH provided dangerously flawed results in the presence of maltose, the sensor employing our engineered FADGDH showed a clear glucose concentration-dependent response that was not affected by the presence of maltose.</description><subject>Amino Acid Sequence</subject><subject>Amino Acid Substitution</subject><subject>Amino acids</subject><subject>Aspergillus niger - enzymology</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Biosensing Techniques</subject><subject>Blood Glucose - analysis</subject><subject>Blood Glucose Self-Monitoring - instrumentation</subject><subject>Blood Glucose Self-Monitoring - methods</subject><subject>Burkholderia cepacia</subject><subject>Burkholderia cepacia - enzymology</subject><subject>Burkholderia cepacia - genetics</subject><subject>Carbon</subject><subject>Catalytic Domain</subject><subject>Catalytic subunits</subject><subject>Chemoreception</subject><subject>Computer Simulation</subject><subject>Diabetes</subject><subject>Direct electron transfer</subject><subject>Electrochemical Techniques</subject><subject>Electrodes</subject><subject>Electron transfer</subject><subject>Electron Transport</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>flavin-adenine dinucleotide</subject><subject>Flavin-Adenine Dinucleotide - metabolism</subject><subject>Fungal Proteins - metabolism</subject><subject>Glucose</subject><subject>Glucose - metabolism</subject><subject>Glucose 1-Dehydrogenase - genetics</subject><subject>Glucose 1-Dehydrogenase - metabolism</subject><subject>Glucose dehydrogenase</subject><subject>Glucose Dehydrogenases - metabolism</subject><subject>Glucose oxidase</subject><subject>Glucose Oxidase - metabolism</subject><subject>Glucose sensor</subject><subject>Homology</subject><subject>Humans</subject><subject>Maltose</subject><subject>Maltose - metabolism</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Mutation</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Reagent Strips</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sensitivity and Specificity</subject><subject>Serine</subject><subject>Site-directed mutagenesis</subject><subject>SMBG</subject><subject>Substrate Specificity</subject><issn>0141-0229</issn><issn>1879-0909</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1v3CAQQFHVqNkm_Qstx17sDthr7OMqH22kSL0kZ2TDsGVlg8t4K23658t2071WSDDAmxnxYOyTgFKAaL7sSgwvkzcLmlKCkKUQJYB8w1aiVV0BHXRv2QpELQqQsrtk74l2APmghnfsUlZSVbKVK_b71ic0C8cxzykGvqQ-kMPEl8OM3HqaI_XDiJwwUEycluRn7nK0Hfcm0unChy3HaR7j4Rj1gWPY-oCY0PL7ze2ZtfjjYFPcYugJr9mF60fCD6_rFXu-v3u6-VY8fv_6cLN5LEwN9VLUUrTQDGulKuyEgBYBunXVSOOqQbrB5h2sFToz1A5NUylpu8r1yrWisTVUV-zzqe6c4s890qInTwbHsQ8Y96SFVMfRNG1G1Qk1KRIldHpOfurTQQvQR_N6p8_m9dG8FkJn8znz42uT_TChPef9U52BzQnA_NRfHpMm4zEYtH9_QNvo_9vkD9tymy4</recordid><startdate>20130205</startdate><enddate>20130205</enddate><creator>Yamashita, Yuki</creator><creator>Ferri, Stefano</creator><creator>Huynh, Mai Linh</creator><creator>Shimizu, Hitomi</creator><creator>Yamaoka, Hideaki</creator><creator>Sode, Koji</creator><general>Elsevier Inc</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>7QR</scope><scope>7T7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>20130205</creationdate><title>Direct electron transfer type disposable sensor strip for glucose sensing employing an engineered FAD glucose dehydrogenase</title><author>Yamashita, Yuki ; Ferri, Stefano ; Huynh, Mai Linh ; Shimizu, Hitomi ; Yamaoka, Hideaki ; Sode, Koji</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c404t-421806b5773e91108e0095362cf3b2fbd095057efcb4fec6372d93fa7f816d403</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Amino Acid Sequence</topic><topic>Amino Acid Substitution</topic><topic>Amino acids</topic><topic>Aspergillus niger - enzymology</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Biosensing Techniques</topic><topic>Blood Glucose - analysis</topic><topic>Blood Glucose Self-Monitoring - instrumentation</topic><topic>Blood Glucose Self-Monitoring - methods</topic><topic>Burkholderia cepacia</topic><topic>Burkholderia cepacia - enzymology</topic><topic>Burkholderia cepacia - genetics</topic><topic>Carbon</topic><topic>Catalytic Domain</topic><topic>Catalytic subunits</topic><topic>Chemoreception</topic><topic>Computer Simulation</topic><topic>Diabetes</topic><topic>Direct electron transfer</topic><topic>Electrochemical Techniques</topic><topic>Electrodes</topic><topic>Electron transfer</topic><topic>Electron Transport</topic><topic>Enzymes</topic><topic>Escherichia coli</topic><topic>flavin-adenine dinucleotide</topic><topic>Flavin-Adenine Dinucleotide - metabolism</topic><topic>Fungal Proteins - metabolism</topic><topic>Glucose</topic><topic>Glucose - metabolism</topic><topic>Glucose 1-Dehydrogenase - genetics</topic><topic>Glucose 1-Dehydrogenase - metabolism</topic><topic>Glucose dehydrogenase</topic><topic>Glucose Dehydrogenases - metabolism</topic><topic>Glucose oxidase</topic><topic>Glucose Oxidase - metabolism</topic><topic>Glucose sensor</topic><topic>Homology</topic><topic>Humans</topic><topic>Maltose</topic><topic>Maltose - metabolism</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Mutation</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Reagent Strips</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sensitivity and Specificity</topic><topic>Serine</topic><topic>Site-directed mutagenesis</topic><topic>SMBG</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Yamashita, Yuki</creatorcontrib><creatorcontrib>Ferri, Stefano</creatorcontrib><creatorcontrib>Huynh, Mai Linh</creatorcontrib><creatorcontrib>Shimizu, Hitomi</creatorcontrib><creatorcontrib>Yamaoka, Hideaki</creatorcontrib><creatorcontrib>Sode, Koji</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>Chemoreception 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>Enzyme and microbial technology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Yamashita, Yuki</au><au>Ferri, Stefano</au><au>Huynh, Mai Linh</au><au>Shimizu, Hitomi</au><au>Yamaoka, Hideaki</au><au>Sode, Koji</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Direct electron transfer type disposable sensor strip for glucose sensing employing an engineered FAD glucose dehydrogenase</atitle><jtitle>Enzyme and microbial technology</jtitle><addtitle>Enzyme Microb Technol</addtitle><date>2013-02-05</date><risdate>2013</risdate><volume>52</volume><issue>2</issue><spage>123</spage><epage>128</epage><pages>123-128</pages><issn>0141-0229</issn><eissn>1879-0909</eissn><abstract>► FADGDH is great for glucose sensing, but suffers from broad substrate specificity. ► 3D model of FADGDH was generated by homology modeling. ► Engineered FADGDH has no activity with maltose, retains high activity with glucose. ► Developed first direct electron transfer-type, disposable glucose sensor strip.
The FAD-dependent glucose dehydrogenase (FADGDH) from Burkholderia cepacia has several attractive features for glucose sensing. However, expanding the application of this enzyme requires improvement of its substrate specificity, especially decreasing its high activity toward maltose. A three-dimensional structural model of the FADGDH catalytic subunit was generated by homology modeling. By comparing the predicted active site with that of glucose oxidase, the two amino acid residues serine 326 and serine 365 were targeted for site-directed mutagenesis. The single mutations that produced the highest glucose specificity were combined, leading to the creation of the S326Q/S365Y double mutant, which was virtually nonreactive to maltose while retaining high glucose dehydrogenase activity. The engineered FADGDH was used to develop a direct electron transfer-type, disposable glucose sensor strip by immobilizing the enzyme complex onto a carbon screen-printed electrode. While the electrode employing wild-type FADGDH provided dangerously flawed results in the presence of maltose, the sensor employing our engineered FADGDH showed a clear glucose concentration-dependent response that was not affected by the presence of maltose.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>23273282</pmid><doi>10.1016/j.enzmictec.2012.11.002</doi><tpages>6</tpages></addata></record> |
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| subjects | Amino Acid Sequence Amino Acid Substitution Amino acids Aspergillus niger - enzymology Bacterial Proteins - genetics Bacterial Proteins - metabolism Biosensing Techniques Blood Glucose - analysis Blood Glucose Self-Monitoring - instrumentation Blood Glucose Self-Monitoring - methods Burkholderia cepacia Burkholderia cepacia - enzymology Burkholderia cepacia - genetics Carbon Catalytic Domain Catalytic subunits Chemoreception Computer Simulation Diabetes Direct electron transfer Electrochemical Techniques Electrodes Electron transfer Electron Transport Enzymes Escherichia coli flavin-adenine dinucleotide Flavin-Adenine Dinucleotide - metabolism Fungal Proteins - metabolism Glucose Glucose - metabolism Glucose 1-Dehydrogenase - genetics Glucose 1-Dehydrogenase - metabolism Glucose dehydrogenase Glucose Dehydrogenases - metabolism Glucose oxidase Glucose Oxidase - metabolism Glucose sensor Homology Humans Maltose Maltose - metabolism Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Mutation Protein Binding Protein Conformation Reagent Strips Recombinant Proteins - metabolism Sensitivity and Specificity Serine Site-directed mutagenesis SMBG Substrate Specificity |
| title | Direct electron transfer type disposable sensor strip for glucose sensing employing an engineered FAD glucose dehydrogenase |
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