Improving Biocatalytic Properties of an Azoreductase via the N‐Terminal Fusion of Formate Dehydrogenase
Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows NAD(P)+ recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreduc...
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| Veröffentlicht in: | Chembiochem : a European journal of chemical biology 2022-03, Vol.23 (6), p.e202100643-n/a |
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| creator | Ngo, Anna Christina R. Schultes, Fabian Peter Josef Maier, Artur Hadewig, Simon Niklas Hermann Tischler, Dirk |
| description | Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows NAD(P)+ recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreductase (AzoRo) of Rhodococcus opacus 1CP and FDH (FDHC23S and FDHC23SD195QY196H) of Candida boidinii in different positions with His‐tag as the linker. The position affected enzyme activities as AzoRo activity decreased by 20‐fold when it is in the N‐terminus of the fusion protein. FDHC23S+AzoRo was the most active construct and was further characterized. Enzymatic activities of FDHC23S+AzoRo decreased compared to parental enzymes but showed improved substrate scope – accepting bulkier dyes. Moreover, pH has an influence on the stability and activity of the fusion protein because at pH 6 (pH that is suboptimal for FDH), the dye reduction decreased to more than 50 % and this could be attributed to the impaired NADH supply for the AzoRo part.
A fusion protein comprised of formate dehydrogenase and azoreductase was constructed to allow the simultaneous activity of dye reduction and NAD+ regeneration. The fusion protein showed improved substrate scope, but it only showed partial degradation for the bulkier azo dyes. |
| doi_str_mv | 10.1002/cbic.202100643 |
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A fusion protein comprised of formate dehydrogenase and azoreductase was constructed to allow the simultaneous activity of dye reduction and NAD+ regeneration. The fusion protein showed improved substrate scope, but it only showed partial degradation for the bulkier azo dyes.</description><identifier>ISSN: 1439-4227</identifier><identifier>EISSN: 1439-7633</identifier><identifier>DOI: 10.1002/cbic.202100643</identifier><identifier>PMID: 35080802</identifier><language>eng</language><publisher>Germany: Wiley Subscription Services, Inc</publisher><subject>Azo dyes ; Azoreductase ; azoreductases ; Biocatalysis ; Biocatalysts ; Brilliant Black BN ; Coloring Agents ; Dehydrogenase ; Dehydrogenases ; diazo dyes ; Dyes ; Enzymatic activity ; Formate dehydrogenase ; Formate Dehydrogenases - chemistry ; Fusion protein ; fusion proteins ; NAD ; NAD - metabolism ; NADH ; NADH regeneration systems ; Nicotinamide adenine dinucleotide ; Nitroreductases - metabolism ; pH effects ; Protein folding ; Proteins ; Rhodococcus ; Substrates</subject><ispartof>Chembiochem : a European journal of chemical biology, 2022-03, Vol.23 (6), p.e202100643-n/a</ispartof><rights>2022 The Authors. ChemBioChem published by Wiley-VCH GmbH</rights><rights>2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.</rights><rights>2022. This article is published under http://creativecommons.org/licenses/by-nc-nd/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4683-1bb0c6e1125915b678dc7af0dd792b5d4364ebe13db213763f075866c22805ed3</citedby><cites>FETCH-LOGICAL-c4683-1bb0c6e1125915b678dc7af0dd792b5d4364ebe13db213763f075866c22805ed3</cites><orcidid>0000-0002-6288-2403</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fcbic.202100643$$EPDF$$P50$$Gwiley$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fcbic.202100643$$EHTML$$P50$$Gwiley$$Hfree_for_read</linktohtml><link.rule.ids>230,314,780,784,885,1417,27924,27925,45574,45575</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/35080802$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ngo, Anna Christina R.</creatorcontrib><creatorcontrib>Schultes, Fabian Peter Josef</creatorcontrib><creatorcontrib>Maier, Artur</creatorcontrib><creatorcontrib>Hadewig, Simon Niklas Hermann</creatorcontrib><creatorcontrib>Tischler, Dirk</creatorcontrib><title>Improving Biocatalytic Properties of an Azoreductase via the N‐Terminal Fusion of Formate Dehydrogenase</title><title>Chembiochem : a European journal of chemical biology</title><addtitle>Chembiochem</addtitle><description>Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows NAD(P)+ recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreductase (AzoRo) of Rhodococcus opacus 1CP and FDH (FDHC23S and FDHC23SD195QY196H) of Candida boidinii in different positions with His‐tag as the linker. The position affected enzyme activities as AzoRo activity decreased by 20‐fold when it is in the N‐terminus of the fusion protein. FDHC23S+AzoRo was the most active construct and was further characterized. Enzymatic activities of FDHC23S+AzoRo decreased compared to parental enzymes but showed improved substrate scope – accepting bulkier dyes. Moreover, pH has an influence on the stability and activity of the fusion protein because at pH 6 (pH that is suboptimal for FDH), the dye reduction decreased to more than 50 % and this could be attributed to the impaired NADH supply for the AzoRo part.
A fusion protein comprised of formate dehydrogenase and azoreductase was constructed to allow the simultaneous activity of dye reduction and NAD+ regeneration. The fusion protein showed improved substrate scope, but it only showed partial degradation for the bulkier azo dyes.</description><subject>Azo dyes</subject><subject>Azoreductase</subject><subject>azoreductases</subject><subject>Biocatalysis</subject><subject>Biocatalysts</subject><subject>Brilliant Black BN</subject><subject>Coloring Agents</subject><subject>Dehydrogenase</subject><subject>Dehydrogenases</subject><subject>diazo dyes</subject><subject>Dyes</subject><subject>Enzymatic activity</subject><subject>Formate dehydrogenase</subject><subject>Formate Dehydrogenases - chemistry</subject><subject>Fusion protein</subject><subject>fusion proteins</subject><subject>NAD</subject><subject>NAD - metabolism</subject><subject>NADH</subject><subject>NADH regeneration systems</subject><subject>Nicotinamide adenine dinucleotide</subject><subject>Nitroreductases - metabolism</subject><subject>pH effects</subject><subject>Protein folding</subject><subject>Proteins</subject><subject>Rhodococcus</subject><subject>Substrates</subject><issn>1439-4227</issn><issn>1439-7633</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>24P</sourceid><sourceid>WIN</sourceid><sourceid>EIF</sourceid><recordid>eNqFkc1uEzEURi0EoqWwZYkssWGT4P-Z2SC1oWkjVYVFWVse-07iamYc7JmgsOoj8Iw8CY4SAmWDvLAtn3t0rz-EXlMypYSw97b2dsoIyxcl-BN0SgWvJoXi_OnhLBgrTtCLlO4JIZXi9Dk64ZKUebFT5BfdOoaN75f4wgdrBtNuB2_x5xjWEAcPCYcGmx6ffw8R3GgHkwBvvMHDCvDtz4cfdxA735sWz8fkQ7_D5yF2ZgD8EVZbF8MS-lz0Ej1rTJvg1WE_Q1_ml3ez68nNp6vF7PxmYoUq-YTWNbEKKGWyorJWRelsYRriXFGxWjrBlYAaKHc1ozwP2pBClkpZxkoiwfEz9GHvXY91B85CP0TT6nX0nYlbHYzXj196v9LLsNEVJ1LyMgveHQQxfB0hDbrzyULbmh7CmDRTjFWKEUEz-vYf9D6MMX_GjhJEFEoVMlPTPWVjSClCc2yGEr1LUe9S1McUc8Gbv0c44r9jy0C1B775Frb_0enZxWL2R_4L1Jaq5w</recordid><startdate>20220318</startdate><enddate>20220318</enddate><creator>Ngo, Anna Christina R.</creator><creator>Schultes, Fabian Peter Josef</creator><creator>Maier, Artur</creator><creator>Hadewig, Simon Niklas Hermann</creator><creator>Tischler, Dirk</creator><general>Wiley Subscription Services, Inc</general><general>John Wiley and Sons Inc</general><scope>24P</scope><scope>WIN</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>7QL</scope><scope>7QO</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-6288-2403</orcidid></search><sort><creationdate>20220318</creationdate><title>Improving Biocatalytic Properties of an Azoreductase via the N‐Terminal Fusion of Formate Dehydrogenase</title><author>Ngo, Anna Christina R. ; Schultes, Fabian Peter Josef ; Maier, Artur ; Hadewig, Simon Niklas Hermann ; Tischler, Dirk</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4683-1bb0c6e1125915b678dc7af0dd792b5d4364ebe13db213763f075866c22805ed3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>Azo dyes</topic><topic>Azoreductase</topic><topic>azoreductases</topic><topic>Biocatalysis</topic><topic>Biocatalysts</topic><topic>Brilliant Black BN</topic><topic>Coloring Agents</topic><topic>Dehydrogenase</topic><topic>Dehydrogenases</topic><topic>diazo dyes</topic><topic>Dyes</topic><topic>Enzymatic activity</topic><topic>Formate dehydrogenase</topic><topic>Formate Dehydrogenases - chemistry</topic><topic>Fusion protein</topic><topic>fusion proteins</topic><topic>NAD</topic><topic>NAD - metabolism</topic><topic>NADH</topic><topic>NADH regeneration systems</topic><topic>Nicotinamide adenine dinucleotide</topic><topic>Nitroreductases - metabolism</topic><topic>pH effects</topic><topic>Protein folding</topic><topic>Proteins</topic><topic>Rhodococcus</topic><topic>Substrates</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ngo, Anna Christina R.</creatorcontrib><creatorcontrib>Schultes, Fabian Peter Josef</creatorcontrib><creatorcontrib>Maier, Artur</creatorcontrib><creatorcontrib>Hadewig, Simon Niklas Hermann</creatorcontrib><creatorcontrib>Tischler, Dirk</creatorcontrib><collection>Wiley Online Library (Open Access Collection)</collection><collection>Wiley Online Library (Open Access Collection)</collection><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>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Chembiochem : a European journal of chemical biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ngo, Anna Christina R.</au><au>Schultes, Fabian Peter Josef</au><au>Maier, Artur</au><au>Hadewig, Simon Niklas Hermann</au><au>Tischler, Dirk</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Improving Biocatalytic Properties of an Azoreductase via the N‐Terminal Fusion of Formate Dehydrogenase</atitle><jtitle>Chembiochem : a European journal of chemical biology</jtitle><addtitle>Chembiochem</addtitle><date>2022-03-18</date><risdate>2022</risdate><volume>23</volume><issue>6</issue><spage>e202100643</spage><epage>n/a</epage><pages>e202100643-n/a</pages><issn>1439-4227</issn><eissn>1439-7633</eissn><abstract>Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. 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A fusion protein comprised of formate dehydrogenase and azoreductase was constructed to allow the simultaneous activity of dye reduction and NAD+ regeneration. The fusion protein showed improved substrate scope, but it only showed partial degradation for the bulkier azo dyes.</abstract><cop>Germany</cop><pub>Wiley Subscription Services, Inc</pub><pmid>35080802</pmid><doi>10.1002/cbic.202100643</doi><tpages>10</tpages><orcidid>https://orcid.org/0000-0002-6288-2403</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Azo dyes Azoreductase azoreductases Biocatalysis Biocatalysts Brilliant Black BN Coloring Agents Dehydrogenase Dehydrogenases diazo dyes Dyes Enzymatic activity Formate dehydrogenase Formate Dehydrogenases - chemistry Fusion protein fusion proteins NAD NAD - metabolism NADH NADH regeneration systems Nicotinamide adenine dinucleotide Nitroreductases - metabolism pH effects Protein folding Proteins Rhodococcus Substrates |
| title | Improving Biocatalytic Properties of an Azoreductase via the N‐Terminal Fusion of Formate Dehydrogenase |
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