new thermostable peroxidase from garlic Allium sativum: Purification, biochemical properties, immobilization, and use in H₂O₂ detection in milk
Analysis of peroxidase activity by native polyacrylamide gel electrophoresis (PAGE) from a garlic bulb (Allium sativum L) extract showed two major activities (designated POX1 and POX2). The POX2 isoenzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and cation-excha...
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| Veröffentlicht in: | Applied biochemistry and biotechnology 2005-12, Vol.127 (3), p.201-214 |
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| creator | Marzouki, S.M Limam, F Smaali, M.I Ulber, R Marzouki, M.N |
| description | Analysis of peroxidase activity by native polyacrylamide gel electrophoresis (PAGE) from a garlic bulb (Allium sativum L) extract showed two major activities (designated POX1 and POX2). The POX2 isoenzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. The purified enzyme was found to be monomeric with a molecular mass of 36.5 kDa, as determined by sodium dodecyl sulfate-PAGE. The optimum temperature ranged from 25 to 40°C and optimum pH was about 5.0. The apparent Km values for guaiacol and H2O2 were 9.5 and 2 mM, respectively. POX2 appeared highly stable since 50% of its activity was conserved at 50°C for 5 h. Moreover POX2 was stable over a pH range of 3.5-11.0. Immobilization of POX2 was achieved by covalent binding of the enzyme to an epoxy-Sepharose matrix. The immobilized enzyme showed great stability toward heat and storage when compared with soluble enzyme. These properties permit the use of this enzyme as a biosensor to detect H2O2 in some food components such as milk or its derivatives. |
| doi_str_mv | 10.1385/ABAB:127:3:201 |
| format | Article |
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The POX2 isoenzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. The purified enzyme was found to be monomeric with a molecular mass of 36.5 kDa, as determined by sodium dodecyl sulfate-PAGE. The optimum temperature ranged from 25 to 40°C and optimum pH was about 5.0. The apparent Km values for guaiacol and H2O2 were 9.5 and 2 mM, respectively. POX2 appeared highly stable since 50% of its activity was conserved at 50°C for 5 h. Moreover POX2 was stable over a pH range of 3.5-11.0. Immobilization of POX2 was achieved by covalent binding of the enzyme to an epoxy-Sepharose matrix. The immobilized enzyme showed great stability toward heat and storage when compared with soluble enzyme. These properties permit the use of this enzyme as a biosensor to detect H2O2 in some food components such as milk or its derivatives.</description><identifier>ISSN: 0273-2289</identifier><identifier>EISSN: 1559-0291</identifier><identifier>EISSN: 0273-2289</identifier><identifier>DOI: 10.1385/ABAB:127:3:201</identifier><identifier>PMID: 16377850</identifier><language>eng</language><publisher>United States: Springer Nature B.V</publisher><subject>Ammonium ; Animals ; Biochemistry ; Biosensing Techniques ; Biosensors ; enzyme kinetics ; Enzymes ; Garlic ; Garlic - enzymology ; Hydrogen Peroxide - analysis ; immobilized enzymes ; Milk - chemistry ; peroxidases ; Peroxidases - chemistry ; Peroxidases - isolation & purification ; Plant Proteins - chemistry ; Plant Proteins - isolation & purification ; Plant Roots - enzymology ; Studies ; Sulfates</subject><ispartof>Applied biochemistry and biotechnology, 2005-12, Vol.127 (3), p.201-214</ispartof><rights>Humana Press Inc. 2005</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c259t-4aa3e10e7ab753ab3e3b1e4d49fbbeeb4d88a30ba1fc3e3b4d2fbda419eabacb3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16377850$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Marzouki, S.M</creatorcontrib><creatorcontrib>Limam, F</creatorcontrib><creatorcontrib>Smaali, M.I</creatorcontrib><creatorcontrib>Ulber, R</creatorcontrib><creatorcontrib>Marzouki, M.N</creatorcontrib><title>new thermostable peroxidase from garlic Allium sativum: Purification, biochemical properties, immobilization, and use in H₂O₂ detection in milk</title><title>Applied biochemistry and biotechnology</title><addtitle>Appl Biochem Biotechnol</addtitle><description>Analysis of peroxidase activity by native polyacrylamide gel electrophoresis (PAGE) from a garlic bulb (Allium sativum L) extract showed two major activities (designated POX1 and POX2). The POX2 isoenzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. The purified enzyme was found to be monomeric with a molecular mass of 36.5 kDa, as determined by sodium dodecyl sulfate-PAGE. The optimum temperature ranged from 25 to 40°C and optimum pH was about 5.0. The apparent Km values for guaiacol and H2O2 were 9.5 and 2 mM, respectively. POX2 appeared highly stable since 50% of its activity was conserved at 50°C for 5 h. Moreover POX2 was stable over a pH range of 3.5-11.0. Immobilization of POX2 was achieved by covalent binding of the enzyme to an epoxy-Sepharose matrix. The immobilized enzyme showed great stability toward heat and storage when compared with soluble enzyme. These properties permit the use of this enzyme as a biosensor to detect H2O2 in some food components such as milk or its derivatives.</description><subject>Ammonium</subject><subject>Animals</subject><subject>Biochemistry</subject><subject>Biosensing Techniques</subject><subject>Biosensors</subject><subject>enzyme kinetics</subject><subject>Enzymes</subject><subject>Garlic</subject><subject>Garlic - enzymology</subject><subject>Hydrogen Peroxide - analysis</subject><subject>immobilized enzymes</subject><subject>Milk - chemistry</subject><subject>peroxidases</subject><subject>Peroxidases - chemistry</subject><subject>Peroxidases - isolation & purification</subject><subject>Plant Proteins - chemistry</subject><subject>Plant Proteins - isolation & purification</subject><subject>Plant Roots - enzymology</subject><subject>Studies</subject><subject>Sulfates</subject><issn>0273-2289</issn><issn>1559-0291</issn><issn>0273-2289</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNpdkctuFDEQRS1ERIbAliVYLFilgx_98uwmEUmQIgUJsrbK3dWJg90e7G5ey_xC_pAvwa0ZCYmFZVXVqauSDiGvODvhsq3eb043p2sumrVcC8afkBWvKlUwofhTsmKikYUQrTokz1O6Z4yLtmqekUNey6ZpK7YijyP-oNMdRh_SBMYh3WIMP20PCekQg6e3EJ3t6MY5O3uaYLLfZ7-mn-ZoB9vlMozH1NjQ3aHPtaPbGHLGZDEdU-t9MNbZ33sOxp7OOdmO9PLPw8N1frTHCbtlvHS9dV9fkIMBXMKX-_-I3Jx_-HJ2WVxdX3w821wVnajUVJQAEjnDBkxTSTASpeFY9qUajEE0Zd-2IJkBPnTLrOzFYHoouUIw0Bl5RN7tcvPF32ZMk_Y2degcjBjmpOtWCabaOoNv_wPvwxzHfJvmquG8qXmVoZMd1MWQUsRBb6P1EH9pzvTiSi-udHalpc6u8sLrfepsPPb_8L2cDLzZAQMEDbfRJn3zedlknFWyrJX8Cx5tnl0</recordid><startdate>200512</startdate><enddate>200512</enddate><creator>Marzouki, S.M</creator><creator>Limam, F</creator><creator>Smaali, M.I</creator><creator>Ulber, R</creator><creator>Marzouki, M.N</creator><general>Springer Nature B.V</general><scope>FBQ</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>7ST</scope><scope>7T7</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</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>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>RC3</scope><scope>SOI</scope><scope>7X8</scope></search><sort><creationdate>200512</creationdate><title>new thermostable peroxidase from garlic Allium sativum: Purification, biochemical properties, immobilization, and use in H₂O₂ detection in milk</title><author>Marzouki, S.M ; Limam, F ; Smaali, M.I ; Ulber, R ; Marzouki, M.N</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c259t-4aa3e10e7ab753ab3e3b1e4d49fbbeeb4d88a30ba1fc3e3b4d2fbda419eabacb3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Ammonium</topic><topic>Animals</topic><topic>Biochemistry</topic><topic>Biosensing Techniques</topic><topic>Biosensors</topic><topic>enzyme kinetics</topic><topic>Enzymes</topic><topic>Garlic</topic><topic>Garlic - enzymology</topic><topic>Hydrogen Peroxide - analysis</topic><topic>immobilized enzymes</topic><topic>Milk - chemistry</topic><topic>peroxidases</topic><topic>Peroxidases - chemistry</topic><topic>Peroxidases - isolation & purification</topic><topic>Plant Proteins - chemistry</topic><topic>Plant Proteins - isolation & purification</topic><topic>Plant Roots - enzymology</topic><topic>Studies</topic><topic>Sulfates</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Marzouki, S.M</creatorcontrib><creatorcontrib>Limam, F</creatorcontrib><creatorcontrib>Smaali, M.I</creatorcontrib><creatorcontrib>Ulber, R</creatorcontrib><creatorcontrib>Marzouki, M.N</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Environment Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>Environment Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Applied biochemistry and biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Marzouki, S.M</au><au>Limam, F</au><au>Smaali, M.I</au><au>Ulber, R</au><au>Marzouki, M.N</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>new thermostable peroxidase from garlic Allium sativum: Purification, biochemical properties, immobilization, and use in H₂O₂ detection in milk</atitle><jtitle>Applied biochemistry and biotechnology</jtitle><addtitle>Appl Biochem Biotechnol</addtitle><date>2005-12</date><risdate>2005</risdate><volume>127</volume><issue>3</issue><spage>201</spage><epage>214</epage><pages>201-214</pages><issn>0273-2289</issn><eissn>1559-0291</eissn><eissn>0273-2289</eissn><abstract>Analysis of peroxidase activity by native polyacrylamide gel electrophoresis (PAGE) from a garlic bulb (Allium sativum L) extract showed two major activities (designated POX1 and POX2). The POX2 isoenzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. The purified enzyme was found to be monomeric with a molecular mass of 36.5 kDa, as determined by sodium dodecyl sulfate-PAGE. The optimum temperature ranged from 25 to 40°C and optimum pH was about 5.0. The apparent Km values for guaiacol and H2O2 were 9.5 and 2 mM, respectively. POX2 appeared highly stable since 50% of its activity was conserved at 50°C for 5 h. Moreover POX2 was stable over a pH range of 3.5-11.0. Immobilization of POX2 was achieved by covalent binding of the enzyme to an epoxy-Sepharose matrix. The immobilized enzyme showed great stability toward heat and storage when compared with soluble enzyme. These properties permit the use of this enzyme as a biosensor to detect H2O2 in some food components such as milk or its derivatives.</abstract><cop>United States</cop><pub>Springer Nature B.V</pub><pmid>16377850</pmid><doi>10.1385/ABAB:127:3:201</doi><tpages>14</tpages></addata></record> |
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| ispartof | Applied biochemistry and biotechnology, 2005-12, Vol.127 (3), p.201-214 |
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| subjects | Ammonium Animals Biochemistry Biosensing Techniques Biosensors enzyme kinetics Enzymes Garlic Garlic - enzymology Hydrogen Peroxide - analysis immobilized enzymes Milk - chemistry peroxidases Peroxidases - chemistry Peroxidases - isolation & purification Plant Proteins - chemistry Plant Proteins - isolation & purification Plant Roots - enzymology Studies Sulfates |
| title | new thermostable peroxidase from garlic Allium sativum: Purification, biochemical properties, immobilization, and use in H₂O₂ detection in milk |
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