Immobilization of the Aspartate Ammonia‐Lyase from Pseudomonas fluorescens R124 on Magnetic Nanoparticles: Characterization and Kinetics
Aspartate ammonia‐lyases (AALs) catalyze the non‐oxidative elimination of ammonia from l‐aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET‐15b expression vector and expressed in E. coli. The pu...
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| creator | Csuka, Pál Molnár, Zsófia Tóth, Veronika Imarah, Ali Obaid Balogh‐Weiser, Diána Vértessy, Beáta G. Poppe, László |
| description | Aspartate ammonia‐lyases (AALs) catalyze the non‐oxidative elimination of ammonia from l‐aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET‐15b expression vector and expressed in E. coli. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis‐Menten kinetics in the ammonia elimination from l‐aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy‐functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL‐MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL‐MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL‐MNP is promising for further biocatalytic applications.
Aspartate ammonia‐lyase from Pseudomonas fluorescens R124 (PfAAL) was isolated, expressed and characterized in this study. PfAAL was covalently immobilized on magnetic nanoparticles. Both the native and immobilized PfAAL were efficient biocatalysts in the ammonia elimination of aspartate. The effect of the viscosity of the reaction medium was investigated with the free and immobilized enzyme. |
| doi_str_mv | 10.1002/cbic.202100708 |
| format | Article |
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Aspartate ammonia‐lyase from Pseudomonas fluorescens R124 (PfAAL) was isolated, expressed and characterized in this study. PfAAL was covalently immobilized on magnetic nanoparticles. Both the native and immobilized PfAAL were efficient biocatalysts in the ammonia elimination of aspartate. The effect of the viscosity of the reaction medium was investigated with the free and immobilized enzyme.</description><identifier>ISSN: 1439-4227</identifier><identifier>ISSN: 1439-7633</identifier><identifier>EISSN: 1439-7633</identifier><identifier>DOI: 10.1002/cbic.202100708</identifier><identifier>PMID: 35114050</identifier><language>eng</language><publisher>Germany: Wiley Subscription Services, Inc</publisher><subject>Ammonia ; aspartases ; Aspartate Ammonia-Lyase - genetics ; Aspartate Ammonia-Lyase - metabolism ; aspartate ammonia-lyases ; biocatalysis ; E coli ; Enzymatic activity ; enzyme kinetics ; Enzymes, Immobilized - metabolism ; Escherichia coli - metabolism ; Glycerol ; Hydrogen-Ion Concentration ; Immobilization ; Kinetics ; magnetic nanoparticles ; Magnetite Nanoparticles - chemistry ; Metal ions ; Nanoparticles ; Pseudomonas fluorescens ; Recyclability ; Shelf life ; Storage stability ; viscosity</subject><ispartof>Chembiochem : a European journal of chemical biology, 2022-04, Vol.23 (7), p.e202100708-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-c4688-3610a66a5d22c813f64f9a1780dfb68d8f57e100b565cc708f7aed01c846448c3</citedby><cites>FETCH-LOGICAL-c4688-3610a66a5d22c813f64f9a1780dfb68d8f57e100b565cc708f7aed01c846448c3</cites><orcidid>0000-0002-9957-1203 ; 0000-0003-4715-4886 ; 0000-0002-9552-7061 ; 0000-0003-0450-2588 ; 0000-0003-3817-376X ; 0000-0002-8358-1378</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.202100708$$EPDF$$P50$$Gwiley$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fcbic.202100708$$EHTML$$P50$$Gwiley$$Hfree_for_read</linktohtml><link.rule.ids>230,314,776,780,881,1411,27901,27902,45550,45551</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/35114050$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Csuka, Pál</creatorcontrib><creatorcontrib>Molnár, Zsófia</creatorcontrib><creatorcontrib>Tóth, Veronika</creatorcontrib><creatorcontrib>Imarah, Ali Obaid</creatorcontrib><creatorcontrib>Balogh‐Weiser, Diána</creatorcontrib><creatorcontrib>Vértessy, Beáta G.</creatorcontrib><creatorcontrib>Poppe, László</creatorcontrib><title>Immobilization of the Aspartate Ammonia‐Lyase from Pseudomonas fluorescens R124 on Magnetic Nanoparticles: Characterization and Kinetics</title><title>Chembiochem : a European journal of chemical biology</title><addtitle>Chembiochem</addtitle><description>Aspartate ammonia‐lyases (AALs) catalyze the non‐oxidative elimination of ammonia from l‐aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET‐15b expression vector and expressed in E. coli. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis‐Menten kinetics in the ammonia elimination from l‐aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy‐functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL‐MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL‐MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL‐MNP is promising for further biocatalytic applications.
Aspartate ammonia‐lyase from Pseudomonas fluorescens R124 (PfAAL) was isolated, expressed and characterized in this study. PfAAL was covalently immobilized on magnetic nanoparticles. Both the native and immobilized PfAAL were efficient biocatalysts in the ammonia elimination of aspartate. The effect of the viscosity of the reaction medium was investigated with the free and immobilized enzyme.</description><subject>Ammonia</subject><subject>aspartases</subject><subject>Aspartate Ammonia-Lyase - genetics</subject><subject>Aspartate Ammonia-Lyase - metabolism</subject><subject>aspartate ammonia-lyases</subject><subject>biocatalysis</subject><subject>E coli</subject><subject>Enzymatic activity</subject><subject>enzyme kinetics</subject><subject>Enzymes, Immobilized - metabolism</subject><subject>Escherichia coli - metabolism</subject><subject>Glycerol</subject><subject>Hydrogen-Ion Concentration</subject><subject>Immobilization</subject><subject>Kinetics</subject><subject>magnetic nanoparticles</subject><subject>Magnetite Nanoparticles - chemistry</subject><subject>Metal ions</subject><subject>Nanoparticles</subject><subject>Pseudomonas fluorescens</subject><subject>Recyclability</subject><subject>Shelf life</subject><subject>Storage stability</subject><subject>viscosity</subject><issn>1439-4227</issn><issn>1439-7633</issn><issn>1439-7633</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>24P</sourceid><sourceid>EIF</sourceid><recordid>eNqFkUtv1DAUhSMEoqWwZYkssWEzgx-J47BAKhHQUYeHEKytG8fuuEriwU5Aw4o1K34jv6R3OtPhsWHla93Px_ecm2UPGZ0zSvlT03gz55TjpaTqVnbMclHNSinE7X2dc14eZfdSuqSUVlKwu9mRKBjLaUGPsx-Lvg-N7_w3GH0YSHBkXFlymtYQRxixwv7g4df3n8sNJEtcDD15n-zUBmxAIq6bQrTJ2CGRD4znBFXewMVgR2_IWxjCVsmbzqZnpF5BBDPaePMdDC0599dsup_dcdAl-2B_nmSfXr38WJ_Nlu9eL-rT5czkUqmZkIyClFC0nBvFhJO5q4CViraukapVrigtxtEUsjAGQ3El2JYyo3KZ58qIk-z5Tnc9Nb1tcfAxQqfX0fcQNzqA1393Br_SF-GLrgRmzAQKPNkLxPB5smnUvUf_XQeDDVPSXHJJMV2qEH38D3oZpjigPaTQTkVLViA131EmhpSidYdhGNXbNevtmvVhzfjg0Z8WDvjNXhGodsBX39nNf-R0_WJR_xa_AnpFtzQ</recordid><startdate>20220405</startdate><enddate>20220405</enddate><creator>Csuka, Pál</creator><creator>Molnár, Zsófia</creator><creator>Tóth, Veronika</creator><creator>Imarah, Ali Obaid</creator><creator>Balogh‐Weiser, Diána</creator><creator>Vértessy, Beáta G.</creator><creator>Poppe, László</creator><general>Wiley Subscription Services, Inc</general><general>John Wiley and Sons Inc</general><scope>24P</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/0000-0002-9957-1203</orcidid><orcidid>https://orcid.org/0000-0003-4715-4886</orcidid><orcidid>https://orcid.org/0000-0002-9552-7061</orcidid><orcidid>https://orcid.org/0000-0003-0450-2588</orcidid><orcidid>https://orcid.org/0000-0003-3817-376X</orcidid><orcidid>https://orcid.org/0000-0002-8358-1378</orcidid></search><sort><creationdate>20220405</creationdate><title>Immobilization of the Aspartate Ammonia‐Lyase from Pseudomonas fluorescens R124 on Magnetic Nanoparticles: Characterization and Kinetics</title><author>Csuka, Pál ; 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In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET‐15b expression vector and expressed in E. coli. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis‐Menten kinetics in the ammonia elimination from l‐aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy‐functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL‐MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL‐MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL‐MNP is promising for further biocatalytic applications.
Aspartate ammonia‐lyase from Pseudomonas fluorescens R124 (PfAAL) was isolated, expressed and characterized in this study. PfAAL was covalently immobilized on magnetic nanoparticles. Both the native and immobilized PfAAL were efficient biocatalysts in the ammonia elimination of aspartate. The effect of the viscosity of the reaction medium was investigated with the free and immobilized enzyme.</abstract><cop>Germany</cop><pub>Wiley Subscription Services, Inc</pub><pmid>35114050</pmid><doi>10.1002/cbic.202100708</doi><tpages>9</tpages><orcidid>https://orcid.org/0000-0002-9957-1203</orcidid><orcidid>https://orcid.org/0000-0003-4715-4886</orcidid><orcidid>https://orcid.org/0000-0002-9552-7061</orcidid><orcidid>https://orcid.org/0000-0003-0450-2588</orcidid><orcidid>https://orcid.org/0000-0003-3817-376X</orcidid><orcidid>https://orcid.org/0000-0002-8358-1378</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Ammonia aspartases Aspartate Ammonia-Lyase - genetics Aspartate Ammonia-Lyase - metabolism aspartate ammonia-lyases biocatalysis E coli Enzymatic activity enzyme kinetics Enzymes, Immobilized - metabolism Escherichia coli - metabolism Glycerol Hydrogen-Ion Concentration Immobilization Kinetics magnetic nanoparticles Magnetite Nanoparticles - chemistry Metal ions Nanoparticles Pseudomonas fluorescens Recyclability Shelf life Storage stability viscosity |
| title | Immobilization of the Aspartate Ammonia‐Lyase from Pseudomonas fluorescens R124 on Magnetic Nanoparticles: Characterization and Kinetics |
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