caa a22 4500 397558236 CHVBK 20180308164809.0 cr unu---uuuuu 161202e199603 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021278 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021278 Purification in an Active State and Properties of the 3-Step Phytoene Desaturase from Rhodobacter capsulatus Overexpressed in Escherichia coil [Elektronische Daten] [Axel Raisig, Glenn Bartley, Pablo Scolnik, Gerhard Sandmann] The phytoene desaturase gene from Rhodobacter capsulatus was expressed in Escherichia coli and the resulting protein was purified. The purification steps involved were ammonium sulfate precipitation and ion exchange chromatography, leading to a homogenous protein of 57 kDa with high specific enzymatic activity. The purified enzyme was characterized with respect to substrate specificity and product formation. In addition to phytoene, the intermediates, phytofluene and ζ-carotene, were both converted to neurosporene, the end product of the reaction. Furthermore, 1,2-epoxy phytoene was a suitable substrate whereas the C30 diapophytoene was not. The Km values for phytoene and ζ-carotene were determined to be 33.3 and 16.6 /iM, respectively. The desaturation reaction is dependent on the cofactor FAD. Oxidized nicotine nucleotides or ATP had no positive effect. The Km value for FAD was 4.9 μM. Inhibition of the desaturation reaction was observed with diphenyl-amine © by the Journal of Biochemistry Regular Papers nationallicence ζ-carotene nationallicence neurosporene nationallicence overexpression of protein nationallicence phytoene desaturase nationallicence Rhodobacter capsulatus nationallicence Raisig Axel Biosynthesis Group, Botanisches Institut, J.W. Goethe Universitat 60054 Frankfurt, Germany aut Bartley Glenn DuPont Central Research and Development Wilmington, DE 19880, USA aut Scolnik Pablo DuPont Central Research and Development Wilmington, DE 19880, USA aut Sandmann Gerhard Biosynthesis Group, Botanisches Institut, J.W. Goethe Universitat 60054 Frankfurt, Germany aut The Journal of Biochemistry Oxford University Press 119/3(1996-03), 559-564 0021-924X 119:3<559 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021278 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021278 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Raisig Axel Biosynthesis Group, Botanisches Institut, J.W. Goethe Universitat 60054 Frankfurt, Germany aut NATIONALLICENCE 700 1- Bartley Glenn DuPont Central Research and Development Wilmington, DE 19880, USA aut NATIONALLICENCE 700 1- Scolnik Pablo DuPont Central Research and Development Wilmington, DE 19880, USA aut NATIONALLICENCE 700 1- Sandmann Gerhard Biosynthesis Group, Botanisches Institut, J.W. Goethe Universitat 60054 Frankfurt, Germany aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/3(1996-03), 559-564 0021-924X 119:3<559 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford