Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia
2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity...
Ausführliche Beschreibung
Gespeichert in:
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
1991 |
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| Reproduktion: |
Elsevier Journal Backfiles on ScienceDirect 1907 - 2002 |
|---|---|
| Übergeordnetes Werk: |
in: Archives of Biochemistry and Biophysics - Amsterdam : Elsevier, 288(1991), 1, Seite 169-176 |
| Übergeordnetes Werk: |
volume:288 ; year:1991 ; number:1 ; pages:169-176 |
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| 245 | 1 | 0 | |a Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia |
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| 520 | |a 2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had K"m value of 14 μm for 2,4-dichlorophenol, and 100 μm for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg^2^+ and Zn^2^+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity. | ||
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(DE-627)NLEJ183404440 (DE-599)GBVNLZ183404440 DE-627 ger DE-627 rakwb eng Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia 1991 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier 2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had K"m value of 14 μm for 2,4-dichlorophenol, and 100 μm for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg^2^+ and Zn^2^+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity. Elsevier Journal Backfiles on ScienceDirect 1907 - 2002 Radjendirane, V. oth Bhat, M.A. oth Vaidyanathan, C.S. oth in Archives of Biochemistry and Biophysics Amsterdam : Elsevier 288(1991), 1, Seite 169-176 (DE-627)NLEJ177020539 (DE-600)1461378-5 0003-9861 nnns volume:288 year:1991 number:1 pages:169-176 http://dx.doi.org/10.1016/0003-9861(91)90180-Q GBV_USEFLAG_H ZDB-1-SDJ GBV_NL_ARTICLE AR 288 1991 1 169-176 |
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(DE-627)NLEJ183404440 (DE-599)GBVNLZ183404440 DE-627 ger DE-627 rakwb eng Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia 1991 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier 2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had K"m value of 14 μm for 2,4-dichlorophenol, and 100 μm for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg^2^+ and Zn^2^+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity. Elsevier Journal Backfiles on ScienceDirect 1907 - 2002 Radjendirane, V. oth Bhat, M.A. oth Vaidyanathan, C.S. oth in Archives of Biochemistry and Biophysics Amsterdam : Elsevier 288(1991), 1, Seite 169-176 (DE-627)NLEJ177020539 (DE-600)1461378-5 0003-9861 nnns volume:288 year:1991 number:1 pages:169-176 http://dx.doi.org/10.1016/0003-9861(91)90180-Q GBV_USEFLAG_H ZDB-1-SDJ GBV_NL_ARTICLE AR 288 1991 1 169-176 |
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(DE-627)NLEJ183404440 (DE-599)GBVNLZ183404440 DE-627 ger DE-627 rakwb eng Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia 1991 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier 2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had K"m value of 14 μm for 2,4-dichlorophenol, and 100 μm for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg^2^+ and Zn^2^+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity. Elsevier Journal Backfiles on ScienceDirect 1907 - 2002 Radjendirane, V. oth Bhat, M.A. oth Vaidyanathan, C.S. oth in Archives of Biochemistry and Biophysics Amsterdam : Elsevier 288(1991), 1, Seite 169-176 (DE-627)NLEJ177020539 (DE-600)1461378-5 0003-9861 nnns volume:288 year:1991 number:1 pages:169-176 http://dx.doi.org/10.1016/0003-9861(91)90180-Q GBV_USEFLAG_H ZDB-1-SDJ GBV_NL_ARTICLE AR 288 1991 1 169-176 |
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(DE-627)NLEJ183404440 (DE-599)GBVNLZ183404440 DE-627 ger DE-627 rakwb eng Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia 1991 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier 2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had K"m value of 14 μm for 2,4-dichlorophenol, and 100 μm for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg^2^+ and Zn^2^+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity. Elsevier Journal Backfiles on ScienceDirect 1907 - 2002 Radjendirane, V. oth Bhat, M.A. oth Vaidyanathan, C.S. oth in Archives of Biochemistry and Biophysics Amsterdam : Elsevier 288(1991), 1, Seite 169-176 (DE-627)NLEJ177020539 (DE-600)1461378-5 0003-9861 nnns volume:288 year:1991 number:1 pages:169-176 http://dx.doi.org/10.1016/0003-9861(91)90180-Q GBV_USEFLAG_H ZDB-1-SDJ GBV_NL_ARTICLE AR 288 1991 1 169-176 |
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(DE-627)NLEJ183404440 (DE-599)GBVNLZ183404440 DE-627 ger DE-627 rakwb eng Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia 1991 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier 2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had K"m value of 14 μm for 2,4-dichlorophenol, and 100 μm for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg^2^+ and Zn^2^+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity. Elsevier Journal Backfiles on ScienceDirect 1907 - 2002 Radjendirane, V. oth Bhat, M.A. oth Vaidyanathan, C.S. oth in Archives of Biochemistry and Biophysics Amsterdam : Elsevier 288(1991), 1, Seite 169-176 (DE-627)NLEJ177020539 (DE-600)1461378-5 0003-9861 nnns volume:288 year:1991 number:1 pages:169-176 http://dx.doi.org/10.1016/0003-9861(91)90180-Q GBV_USEFLAG_H ZDB-1-SDJ GBV_NL_ARTICLE AR 288 1991 1 169-176 |
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Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia |
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2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had K"m value of 14 μm for 2,4-dichlorophenol, and 100 μm for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg^2^+ and Zn^2^+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity. |
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2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had K"m value of 14 μm for 2,4-dichlorophenol, and 100 μm for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg^2^+ and Zn^2^+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity. |
| abstract_unstemmed |
2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had K"m value of 14 μm for 2,4-dichlorophenol, and 100 μm for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg^2^+ and Zn^2^+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity. |
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<?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01000caa a22002652 4500</leader><controlfield tag="001">NLEJ183404440</controlfield><controlfield tag="003">DE-627</controlfield><controlfield tag="005">20210706202117.0</controlfield><controlfield tag="007">cr uuu---uuuuu</controlfield><controlfield tag="008">070506s1991 xx |||||o 00| ||eng c</controlfield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-627)NLEJ183404440</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-599)GBVNLZ183404440</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-627</subfield><subfield code="b">ger</subfield><subfield code="c">DE-627</subfield><subfield code="e">rakwb</subfield></datafield><datafield tag="041" ind1=" " ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">1991</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">nicht spezifiziert</subfield><subfield code="b">zzz</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">nicht spezifiziert</subfield><subfield code="b">z</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">nicht spezifiziert</subfield><subfield code="b">zu</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had K"m value of 14 μm for 2,4-dichlorophenol, and 100 μm for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg^2^+ and Zn^2^+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.</subfield></datafield><datafield tag="533" ind1=" " ind2=" "><subfield code="f">Elsevier Journal Backfiles on ScienceDirect 1907 - 2002</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Radjendirane, V.</subfield><subfield code="4">oth</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Bhat, M.A.</subfield><subfield code="4">oth</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Vaidyanathan, C.S.</subfield><subfield code="4">oth</subfield></datafield><datafield tag="773" ind1="0" ind2="8"><subfield code="i">in</subfield><subfield code="t">Archives of Biochemistry and Biophysics</subfield><subfield code="d">Amsterdam : Elsevier</subfield><subfield code="g">288(1991), 1, Seite 169-176</subfield><subfield code="w">(DE-627)NLEJ177020539</subfield><subfield code="w">(DE-600)1461378-5</subfield><subfield code="x">0003-9861</subfield><subfield code="7">nnns</subfield></datafield><datafield tag="773" ind1="1" ind2="8"><subfield code="g">volume:288</subfield><subfield code="g">year:1991</subfield><subfield code="g">number:1</subfield><subfield code="g">pages:169-176</subfield></datafield><datafield tag="856" ind1="4" ind2="0"><subfield code="u">http://dx.doi.org/10.1016/0003-9861(91)90180-Q</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_USEFLAG_H</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">ZDB-1-SDJ</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_NL_ARTICLE</subfield></datafield><datafield tag="951" ind1=" " ind2=" "><subfield code="a">AR</subfield></datafield><datafield tag="952" ind1=" " ind2=" "><subfield code="d">288</subfield><subfield code="j">1991</subfield><subfield code="e">1</subfield><subfield code="h">169-176</subfield></datafield></record></collection>
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7.39822 |