THE PURIFICATION OF PHYTOENE DEHYDROGENASE FROM PHYCOMYCES-BLAKESLEEANUS

被引:9
作者
FRASER, PD
BRAMLEY, PM
机构
[1] Department of Biochemistry, Royal Holloway, University of London, Egham
来源
BIOCHIMICA ET BIOPHYSICA ACTA-LIPIDS AND LIPID METABOLISM | 1994年 / 1212卷 / 01期
关键词
PHYTOENE DEHYDROGENASE; CAROTENOID; PURIFICATION; (P-BLAKESLEEANUS);
D O I
10.1016/0005-2760(94)90189-9
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:59 / 66
页数:8
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