The purification of phytoene dehydrogenase from Phycomyces blakesleeanus.
The carotenogenic enzyme phytoene dehydrogenase has been purified from the C9carR21(-) (lycopene-accumulating) mutant of the filamentous fungus Phycomyces blakesleeanus. Solubilization of the membrane-bound enzyme with 1% Tween-60 was followed by a 250-fold purification to homogeneity using polyethylene glycol precipitation, CM-Sepharose, gel filtration and isoelectric focusing. Multiple peaks of enzymic activity were found in eluates from ion-exchange and gel filtration chromatography, with the lowest molecular weight fraction having an apparent molecular mass of approx. 14 kDa. All active fractions catalyzed the dehydrogenation of 15-cis phytoene into all-trans lycopene, with a cis-trans isomerization occurring at phytofluene. Both NADP+ and FAD were required for the dehydrogenation reaction. The presence of > 0.5% Tween-60 was necessary to maintain enzymic activity, although in its absence lipids restored some activity. The enzyme could be stored for at least 6 weeks at -70 degrees C in the presence of 20% (v/v) glycerol.
Fraser
P D
PD
Department of Biochemistry, University of London, Egham, Surrey, UK.
Bramley
P M
PM
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D010800:Phycomyces / Q000201:enzymology*; D012995:Solubility; D013379:Substrate Specificity
D010088:Oxidoreductases; C022398:phytoene dehydrogenase