THE PURIFICATION OF PHYTOENE DEHYDROGENASE FROM PHYCOMYCES-BLAKESLEEANUS

Citation
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
Citations number
41
Categorie Soggetti
Biology,Biophysics
ISSN journal
00052760
Volume
1212
Issue
1
Year of publication
1994
Pages
59 - 66
Database
ISI
SICI code
0005-2760(1994)1212:1<59:TPOPDF>2.0.ZU;2-H
Abstract
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.