Purification and characterization of N,N‐dimethylformamidase from Pseudomonas DMF 3/3
The N,N‐dimethylformamide‐hydrolyzing enzyme (DMFase) from Pseudomonas DMF 3/3 has been purified to apparent electrophoretic homogeneity with an overall 49‐fold purification, a 24% yield and a final specific activity of 1.98 μmol N,N‐dimethylformamide (DMF) hydrolyzed min−1 (mg protein)−1. The nativ...
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Veröffentlicht in: | European journal of biochemistry 1986-08, Vol.158 (3), p.469-475 |
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description | The N,N‐dimethylformamide‐hydrolyzing enzyme (DMFase) from Pseudomonas DMF 3/3 has been purified to apparent electrophoretic homogeneity with an overall 49‐fold purification, a 24% yield and a final specific activity of 1.98 μmol N,N‐dimethylformamide (DMF) hydrolyzed min−1 (mg protein)−1. The native DMFase has a relative molecular mass of 250000 and is composed of two light‐chain (Mr= 15000) and two heavy‐chain (Mr= 105000) subunits. The stability of DMFase is optimal at pH values above 7.5 and at temperatures below 20°C. The activity of the enzyme is inhibited by metal‐chelating agents such as EDTA and 2,2′‐dipyridyl. Emission and atomic absorption spectroscopy measurements showed that iron is present in significant amounts in DMFase, indicating that it is an iron‐containing amidohydrolase. In the ultraviolet/visible spectrum prominent bands were observed at 224 nm, 280 nm and 396 nm and shoulders are present at 418 nm and 467 nm.
DMFase from Ps. DMF 3/3 has an isoelectric point of 7.7. The enzyme exhibits optimal activity between pH 5 and 6 and at 40°C. The substrate spectrum is rather narrow. The enzyme hydrolyzes preferentially substituted short‐chain aliphatic amides such as DMF, N‐ethylformamide and N‐methylformamide. N,N‐diethylformamide, N,N‐dimethylacetamide and unsubstituted amides, e.g. formamide, prolinamide, acetamide, acrylamide and butyramide are substrates as well, but are hydrolysed at significantly lower rates. DMFase obeys Michaelis‐Menten kinetics and its Km and Vmax values for DMF are 13.8 mM and 1.89 U/mg, respectively, as determined from a Lineweaver‐Burk plot. |
doi_str_mv | 10.1111/j.1432-1033.1986.tb09778.x |
format | Article |
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DMFase from Ps. DMF 3/3 has an isoelectric point of 7.7. The enzyme exhibits optimal activity between pH 5 and 6 and at 40°C. The substrate spectrum is rather narrow. The enzyme hydrolyzes preferentially substituted short‐chain aliphatic amides such as DMF, N‐ethylformamide and N‐methylformamide. N,N‐diethylformamide, N,N‐dimethylacetamide and unsubstituted amides, e.g. formamide, prolinamide, acetamide, acrylamide and butyramide are substrates as well, but are hydrolysed at significantly lower rates. DMFase obeys Michaelis‐Menten kinetics and its Km and Vmax values for DMF are 13.8 mM and 1.89 U/mg, respectively, as determined from a Lineweaver‐Burk plot.</description><identifier>ISSN: 0014-2956</identifier><identifier>EISSN: 1432-1033</identifier><identifier>DOI: 10.1111/j.1432-1033.1986.tb09778.x</identifier><identifier>PMID: 3732281</identifier><identifier>CODEN: EJBCAI</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>Amidohydrolases - analysis ; Amidohydrolases - isolation & purification ; Applied sciences ; Dimethylformamide - metabolism ; Exact sciences and technology ; Hot Temperature ; Hydrogen-Ion Concentration ; Isoelectric Point ; Kinetics ; Metals - analysis ; Molecular Weight ; N,N-dimethylformamidase ; Other techniques and industries ; Pseudomonas ; Pseudomonas - enzymology ; Substrate Specificity</subject><ispartof>European journal of biochemistry, 1986-08, Vol.158 (3), p.469-475</ispartof><rights>1987 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4969-2de872804ce0fafef76e0151f8643e7f6f9f50b3f8c1e54c9aadf7f2321e02293</citedby><cites>FETCH-LOGICAL-c4969-2de872804ce0fafef76e0151f8643e7f6f9f50b3f8c1e54c9aadf7f2321e02293</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=8270803$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3732281$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>SCHÄR, Hans‐Peter</creatorcontrib><creatorcontrib>HOLZMANN, Werner</creatorcontrib><creatorcontrib>RAMOS TOMBO, Gerardo M.</creatorcontrib><creatorcontrib>GHISALBA, Oreste</creatorcontrib><title>Purification and characterization of N,N‐dimethylformamidase from Pseudomonas DMF 3/3</title><title>European journal of biochemistry</title><addtitle>Eur J Biochem</addtitle><description>The N,N‐dimethylformamide‐hydrolyzing enzyme (DMFase) from Pseudomonas DMF 3/3 has been purified to apparent electrophoretic homogeneity with an overall 49‐fold purification, a 24% yield and a final specific activity of 1.98 μmol N,N‐dimethylformamide (DMF) hydrolyzed min−1 (mg protein)−1. The native DMFase has a relative molecular mass of 250000 and is composed of two light‐chain (Mr= 15000) and two heavy‐chain (Mr= 105000) subunits. The stability of DMFase is optimal at pH values above 7.5 and at temperatures below 20°C. The activity of the enzyme is inhibited by metal‐chelating agents such as EDTA and 2,2′‐dipyridyl. Emission and atomic absorption spectroscopy measurements showed that iron is present in significant amounts in DMFase, indicating that it is an iron‐containing amidohydrolase. In the ultraviolet/visible spectrum prominent bands were observed at 224 nm, 280 nm and 396 nm and shoulders are present at 418 nm and 467 nm.
DMFase from Ps. DMF 3/3 has an isoelectric point of 7.7. The enzyme exhibits optimal activity between pH 5 and 6 and at 40°C. The substrate spectrum is rather narrow. The enzyme hydrolyzes preferentially substituted short‐chain aliphatic amides such as DMF, N‐ethylformamide and N‐methylformamide. N,N‐diethylformamide, N,N‐dimethylacetamide and unsubstituted amides, e.g. formamide, prolinamide, acetamide, acrylamide and butyramide are substrates as well, but are hydrolysed at significantly lower rates. DMFase obeys Michaelis‐Menten kinetics and its Km and Vmax values for DMF are 13.8 mM and 1.89 U/mg, respectively, as determined from a Lineweaver‐Burk plot.</description><subject>Amidohydrolases - analysis</subject><subject>Amidohydrolases - isolation & purification</subject><subject>Applied sciences</subject><subject>Dimethylformamide - metabolism</subject><subject>Exact sciences and technology</subject><subject>Hot Temperature</subject><subject>Hydrogen-Ion Concentration</subject><subject>Isoelectric Point</subject><subject>Kinetics</subject><subject>Metals - analysis</subject><subject>Molecular Weight</subject><subject>N,N-dimethylformamidase</subject><subject>Other techniques and industries</subject><subject>Pseudomonas</subject><subject>Pseudomonas - enzymology</subject><subject>Substrate Specificity</subject><issn>0014-2956</issn><issn>1432-1033</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1986</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqVkM1q3DAQx0VpSbbbPkLBhJJT7ejD1kcvIUmzaSFNA23pUWjlEdFiW4lk02xOeYQ8Y54kXtbstXQugvn_Zkb8EDoguCBjHa0KUjKaE8xYQZTkRb_ESghZ3L9Cs130Gs0wJmVOVcX30duUVhhjrrjYQ3tMMEolmaE_10P0zlvT-9Blpqsze2OisT1E_7BtBpddfbp6fnyqfQv9zbpxIbam9bVJkLkY2uw6wVCHNnQmZV--LzJ2xN6hN840Cd5P7xz9Xpz_OvuaX_64-HZ2cpnbUnGV0xqkoBKXFrAzDpzggElFnOQlA-G4U67CS-akJVCVVhlTO-EoowQwpYrN0eF2720MdwOkXrc-WWga00EYkhZcVUzgf4Ok5ISMqkbw8xa0MaQUwenb6FsT15pgvdGvV3rjWG8c641-PenX9-Pwh-nKsGyh3o1Ovsf845SbZE3joumsTztMUoHluHaOjrfYX9_A-j8-oBfnpz9LrtgL2hyi1A</recordid><startdate>198608</startdate><enddate>198608</enddate><creator>SCHÄR, Hans‐Peter</creator><creator>HOLZMANN, Werner</creator><creator>RAMOS TOMBO, Gerardo M.</creator><creator>GHISALBA, Oreste</creator><general>Blackwell Publishing Ltd</general><general>Blackwell</general><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>C1K</scope><scope>7X8</scope></search><sort><creationdate>198608</creationdate><title>Purification and characterization of N,N‐dimethylformamidase from Pseudomonas DMF 3/3</title><author>SCHÄR, Hans‐Peter ; HOLZMANN, Werner ; RAMOS TOMBO, Gerardo M. ; GHISALBA, Oreste</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4969-2de872804ce0fafef76e0151f8643e7f6f9f50b3f8c1e54c9aadf7f2321e02293</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1986</creationdate><topic>Amidohydrolases - analysis</topic><topic>Amidohydrolases - isolation & purification</topic><topic>Applied sciences</topic><topic>Dimethylformamide - metabolism</topic><topic>Exact sciences and technology</topic><topic>Hot Temperature</topic><topic>Hydrogen-Ion Concentration</topic><topic>Isoelectric Point</topic><topic>Kinetics</topic><topic>Metals - analysis</topic><topic>Molecular Weight</topic><topic>N,N-dimethylformamidase</topic><topic>Other techniques and industries</topic><topic>Pseudomonas</topic><topic>Pseudomonas - enzymology</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>SCHÄR, Hans‐Peter</creatorcontrib><creatorcontrib>HOLZMANN, Werner</creatorcontrib><creatorcontrib>RAMOS TOMBO, Gerardo M.</creatorcontrib><creatorcontrib>GHISALBA, Oreste</creatorcontrib><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>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>European journal of biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>SCHÄR, Hans‐Peter</au><au>HOLZMANN, Werner</au><au>RAMOS TOMBO, Gerardo M.</au><au>GHISALBA, Oreste</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and characterization of N,N‐dimethylformamidase from Pseudomonas DMF 3/3</atitle><jtitle>European journal of biochemistry</jtitle><addtitle>Eur J Biochem</addtitle><date>1986-08</date><risdate>1986</risdate><volume>158</volume><issue>3</issue><spage>469</spage><epage>475</epage><pages>469-475</pages><issn>0014-2956</issn><eissn>1432-1033</eissn><coden>EJBCAI</coden><abstract>The N,N‐dimethylformamide‐hydrolyzing enzyme (DMFase) from Pseudomonas DMF 3/3 has been purified to apparent electrophoretic homogeneity with an overall 49‐fold purification, a 24% yield and a final specific activity of 1.98 μmol N,N‐dimethylformamide (DMF) hydrolyzed min−1 (mg protein)−1. The native DMFase has a relative molecular mass of 250000 and is composed of two light‐chain (Mr= 15000) and two heavy‐chain (Mr= 105000) subunits. The stability of DMFase is optimal at pH values above 7.5 and at temperatures below 20°C. The activity of the enzyme is inhibited by metal‐chelating agents such as EDTA and 2,2′‐dipyridyl. Emission and atomic absorption spectroscopy measurements showed that iron is present in significant amounts in DMFase, indicating that it is an iron‐containing amidohydrolase. In the ultraviolet/visible spectrum prominent bands were observed at 224 nm, 280 nm and 396 nm and shoulders are present at 418 nm and 467 nm.
DMFase from Ps. DMF 3/3 has an isoelectric point of 7.7. The enzyme exhibits optimal activity between pH 5 and 6 and at 40°C. The substrate spectrum is rather narrow. The enzyme hydrolyzes preferentially substituted short‐chain aliphatic amides such as DMF, N‐ethylformamide and N‐methylformamide. N,N‐diethylformamide, N,N‐dimethylacetamide and unsubstituted amides, e.g. formamide, prolinamide, acetamide, acrylamide and butyramide are substrates as well, but are hydrolysed at significantly lower rates. DMFase obeys Michaelis‐Menten kinetics and its Km and Vmax values for DMF are 13.8 mM and 1.89 U/mg, respectively, as determined from a Lineweaver‐Burk plot.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>3732281</pmid><doi>10.1111/j.1432-1033.1986.tb09778.x</doi><tpages>7</tpages></addata></record> |
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subjects | Amidohydrolases - analysis Amidohydrolases - isolation & purification Applied sciences Dimethylformamide - metabolism Exact sciences and technology Hot Temperature Hydrogen-Ion Concentration Isoelectric Point Kinetics Metals - analysis Molecular Weight N,N-dimethylformamidase Other techniques and industries Pseudomonas Pseudomonas - enzymology Substrate Specificity |
title | Purification and characterization of N,N‐dimethylformamidase from Pseudomonas DMF 3/3 |
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