Pd. Fraser et Pm. Bramley, THE PURIFICATION OF PHYTOENE DEHYDROGENASE FROM PHYCOMYCES-BLAKESLEEANUS, Biochimica et biophysica acta, L. Lipids and lipid metabolism, 1212(1), 1994, pp. 59-66
The carotenogenic enzyme phytoene dehydrogenase has been purified from
the C9carR21(-) (lycopene-accumulating) mutant of the filamentous fun
gus Phycomyces blakesleeanus. Solubilization of the membrane-bound enz
yme with 1% Tween-60 was followed by a 250-fold purification to homoge
neity using polyethylene glycol precipitation, CM-Sepharose, gel filtr
ation and isoelectric focusing. Multiple peaks of enzymic activity wer
e found in eluates from ion-exchange and gel filtration chromatography
, with the lowest molecular weight fraction having an apparent molecul
ar mass of approx. 14 kDa. All active fractions catalyzed the dehydrog
enation of 15-cis phytoene into all-trans lycopene, with a cis-trans i
somerization occurring at phytofluene. Both NADP(+) and FAD were requi
red for the dehydrogenation reaction. The presence of > 0.5% Tween-60
was necessary to maintain enzymic activity, although in its absence li
pids 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.