Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene
A Klebsiella ozaenae nitrilase which converts the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) to 3,5-dibromo-4-hydroxybenzoic acid has been expressed at 5-10% of the total protein in Escherichia coli from a cloned K. ozaenae DNA segment and purified 10.3-fold to homogeneity. The purifie...
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| Veröffentlicht in: | The Journal of biological chemistry 1988-05, Vol.263 (13), p.6310-6314 |
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| creator | Stalker, D M Malyj, L D McBride, K E |
| description | A Klebsiella ozaenae nitrilase which converts the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) to 3,5-dibromo-4-hydroxybenzoic acid has been expressed at 5-10% of the total protein in Escherichia coli from a cloned K. ozaenae DNA segment and purified 10.3-fold to homogeneity. The purified polypeptide is molecular weight 37,000 in size, but the active form of the enzyme is composed of two identical subunits. The purified enzyme exhibits a pH optimum of 9.2 and a temperature optimum of 35 degrees C. The purified enzyme is also quite sensitive to thiol-specific reagents. The nitrilase is highly specific for bromoxynil as substrate with a Km of 0.31 mM and Vmax of 15 mumol of NH3 released/min/mg protein. Analysis of bromoxynil-related substrates indicates the enzyme exhibits preference for compounds containing two meta-positioned halogen atoms. Nucleotide sequence analysis of a 1,212-base pair PstI-HincII DNA segment containing the locus (bxn) encoding the bromoxynil-specific nitrilase reveals a single open reading frame encoding a polypeptide 349 amino acids in length. The predicted sequence of the purified enzyme was derived from the nucleotide sequence of the bxn gene. |
| doi_str_mv | 10.1016/S0021-9258(18)68787-3 |
| format | Article |
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The purified polypeptide is molecular weight 37,000 in size, but the active form of the enzyme is composed of two identical subunits. The purified enzyme exhibits a pH optimum of 9.2 and a temperature optimum of 35 degrees C. The purified enzyme is also quite sensitive to thiol-specific reagents. The nitrilase is highly specific for bromoxynil as substrate with a Km of 0.31 mM and Vmax of 15 mumol of NH3 released/min/mg protein. Analysis of bromoxynil-related substrates indicates the enzyme exhibits preference for compounds containing two meta-positioned halogen atoms. Nucleotide sequence analysis of a 1,212-base pair PstI-HincII DNA segment containing the locus (bxn) encoding the bromoxynil-specific nitrilase reveals a single open reading frame encoding a polypeptide 349 amino acids in length. The predicted sequence of the purified enzyme was derived from the nucleotide sequence of the bxn gene.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(18)68787-3</identifier><identifier>PMID: 2834373</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Amino Acids - analysis ; Aminohydrolases - isolation & purification ; Analytical, structural and metabolic biochemistry ; Base Sequence ; Biological and medical sciences ; Biotechnology ; bromoxynil ; Chromosome Mapping ; cloning ; DNA Restriction Enzymes - metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzymes and enzyme inhibitors ; Fundamental and applied biological sciences. Psychology ; gene products ; Genetic engineering ; Genetic technics ; Herbicides - metabolism ; Hydrolases ; Klebsiella - enzymology ; Klebsiella ozaenae ; Methods. Procedures. Technologies ; Molecular Sequence Data ; Molecular Weight ; nitrilase ; Substrate Specificity ; Vectors (cloning, transfer, expression). Insertion sequences and transposons</subject><ispartof>The Journal of biological chemistry, 1988-05, Vol.263 (13), p.6310-6314</ispartof><rights>1988 © 1988 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>1988 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c561t-a49954a1878d343f90319c9e4dd730d9dfcdca889bc2fddd0a411488b76458483</citedby><cites>FETCH-LOGICAL-c561t-a49954a1878d343f90319c9e4dd730d9dfcdca889bc2fddd0a411488b76458483</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,781,785,27929,27930</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=7824466$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2834373$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Stalker, D M</creatorcontrib><creatorcontrib>Malyj, L D</creatorcontrib><creatorcontrib>McBride, K E</creatorcontrib><title>Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>A Klebsiella ozaenae nitrilase which converts the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) to 3,5-dibromo-4-hydroxybenzoic acid has been expressed at 5-10% of the total protein in Escherichia coli from a cloned K. ozaenae DNA segment and purified 10.3-fold to homogeneity. The purified polypeptide is molecular weight 37,000 in size, but the active form of the enzyme is composed of two identical subunits. The purified enzyme exhibits a pH optimum of 9.2 and a temperature optimum of 35 degrees C. The purified enzyme is also quite sensitive to thiol-specific reagents. The nitrilase is highly specific for bromoxynil as substrate with a Km of 0.31 mM and Vmax of 15 mumol of NH3 released/min/mg protein. Analysis of bromoxynil-related substrates indicates the enzyme exhibits preference for compounds containing two meta-positioned halogen atoms. Nucleotide sequence analysis of a 1,212-base pair PstI-HincII DNA segment containing the locus (bxn) encoding the bromoxynil-specific nitrilase reveals a single open reading frame encoding a polypeptide 349 amino acids in length. The predicted sequence of the purified enzyme was derived from the nucleotide sequence of the bxn gene.</description><subject>Amino Acid Sequence</subject><subject>Amino Acids - analysis</subject><subject>Aminohydrolases - isolation & purification</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>bromoxynil</subject><subject>Chromosome Mapping</subject><subject>cloning</subject><subject>DNA Restriction Enzymes - metabolism</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>gene products</subject><subject>Genetic engineering</subject><subject>Genetic technics</subject><subject>Herbicides - metabolism</subject><subject>Hydrolases</subject><subject>Klebsiella - enzymology</subject><subject>Klebsiella ozaenae</subject><subject>Methods. Procedures. Technologies</subject><subject>Molecular Sequence Data</subject><subject>Molecular Weight</subject><subject>nitrilase</subject><subject>Substrate Specificity</subject><subject>Vectors (cloning, transfer, expression). Insertion sequences and transposons</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1988</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkdGK1DAUhoso6-zqIywEFNGLatKkbXIlsrgqLCio4F1Ik9PpkTYZk1Z3nsJXNp0Z5tbc5OJ85__P-U9RXDP6mlHWvPlKacVKVdXyJZOvGtnKtuQPig2jkpe8Zj8eFpsz8ri4TOknzU8odlFcVJIL3vJN8ffLErFHa2YMnhjvyC6GHcQZIZHQE0M8zhFHk4CkHdiVJX2IZB6ADBA7tOiAdDFM4X7vcTxo2BAjpF3wDv2W-MWOEOaVS_BrAW8hU2bcJzx4rFLdvSdb8PCkeNSbMcHT039VfL99_-3mY3n3-cOnm3d3pa0bNpdGKFULw_LWLm_SK8qZsgqEcy2nTrneOmukVJ2teuccNYIxIWXXNqKWQvKr4sVRN6-bR0qznjBZGEfjISxJM6EqylSTwfoI2hhSitDrXcTJxL1mVK-H0IdD6DVlzaQ-HELz3Hd9Mli6Cdy565R8rj8_1U2yZuyj8RbTGWtlJUSz2j87YgNuhz8YQXcY7ACTrhquGdcNZzRTb48U5Mh-I0SdLK45u9xhZ-0C_mfcfz5ms4k</recordid><startdate>19880505</startdate><enddate>19880505</enddate><creator>Stalker, D M</creator><creator>Malyj, L D</creator><creator>McBride, K E</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>IQODW</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>7T7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>19880505</creationdate><title>Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene</title><author>Stalker, D M ; Malyj, L D ; McBride, K E</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c561t-a49954a1878d343f90319c9e4dd730d9dfcdca889bc2fddd0a411488b76458483</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1988</creationdate><topic>Amino Acid Sequence</topic><topic>Amino Acids - analysis</topic><topic>Aminohydrolases - isolation & purification</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>bromoxynil</topic><topic>Chromosome Mapping</topic><topic>cloning</topic><topic>DNA Restriction Enzymes - metabolism</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>gene products</topic><topic>Genetic engineering</topic><topic>Genetic technics</topic><topic>Herbicides - metabolism</topic><topic>Hydrolases</topic><topic>Klebsiella - enzymology</topic><topic>Klebsiella ozaenae</topic><topic>Methods. Procedures. Technologies</topic><topic>Molecular Sequence Data</topic><topic>Molecular Weight</topic><topic>nitrilase</topic><topic>Substrate Specificity</topic><topic>Vectors (cloning, transfer, expression). Insertion sequences and transposons</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Stalker, D M</creatorcontrib><creatorcontrib>Malyj, L D</creatorcontrib><creatorcontrib>McBride, K E</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Pascal-Francis</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>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>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Stalker, D M</au><au>Malyj, L D</au><au>McBride, K E</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1988-05-05</date><risdate>1988</risdate><volume>263</volume><issue>13</issue><spage>6310</spage><epage>6314</epage><pages>6310-6314</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>A Klebsiella ozaenae nitrilase which converts the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) to 3,5-dibromo-4-hydroxybenzoic acid has been expressed at 5-10% of the total protein in Escherichia coli from a cloned K. ozaenae DNA segment and purified 10.3-fold to homogeneity. The purified polypeptide is molecular weight 37,000 in size, but the active form of the enzyme is composed of two identical subunits. The purified enzyme exhibits a pH optimum of 9.2 and a temperature optimum of 35 degrees C. The purified enzyme is also quite sensitive to thiol-specific reagents. The nitrilase is highly specific for bromoxynil as substrate with a Km of 0.31 mM and Vmax of 15 mumol of NH3 released/min/mg protein. Analysis of bromoxynil-related substrates indicates the enzyme exhibits preference for compounds containing two meta-positioned halogen atoms. Nucleotide sequence analysis of a 1,212-base pair PstI-HincII DNA segment containing the locus (bxn) encoding the bromoxynil-specific nitrilase reveals a single open reading frame encoding a polypeptide 349 amino acids in length. The predicted sequence of the purified enzyme was derived from the nucleotide sequence of the bxn gene.</abstract><cop>Bethesda, MD</cop><pub>Elsevier Inc</pub><pmid>2834373</pmid><doi>10.1016/S0021-9258(18)68787-3</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amino Acid Sequence Amino Acids - analysis Aminohydrolases - isolation & purification Analytical, structural and metabolic biochemistry Base Sequence Biological and medical sciences Biotechnology bromoxynil Chromosome Mapping cloning DNA Restriction Enzymes - metabolism Electrophoresis, Polyacrylamide Gel Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology gene products Genetic engineering Genetic technics Herbicides - metabolism Hydrolases Klebsiella - enzymology Klebsiella ozaenae Methods. Procedures. Technologies Molecular Sequence Data Molecular Weight nitrilase Substrate Specificity Vectors (cloning, transfer, expression). Insertion sequences and transposons |
| title | Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene |
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