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
Hauptverfasser: SCHÄR, Hans‐Peter, HOLZMANN, Werner, RAMOS TOMBO, Gerardo M., GHISALBA, Oreste
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container_end_page 475
container_issue 3
container_start_page 469
container_title European journal of biochemistry
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creator SCHÄR, Hans‐Peter
HOLZMANN, Werner
RAMOS TOMBO, Gerardo M.
GHISALBA, Oreste
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
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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. 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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. 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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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