Utilization of the recombinant human beta-carotene-15,15'-monooxygenase gene in Escherichia coli and mammalian cells.
In animals, beta-carotene 15,15'-monooxygenase (BCMO) is the key enzyme involved in the metabolism of plant beta-carotene to retinal. In the present study, we utilized beta-carotene-producing Escherichia coli to screen for mutants with higher BCMO activity which was monitored by color changes derived from beta-carotene cleavage. Recombinant wild-type and T381L mutant BCMO proteins were purified to near homogeneity in E. coli, and their enzymatic activities were determined by HPLC analysis. The catalytic efficiency for beta-carotene and retinal production of the mutant were 1.5-fold and 1.7-fold higher than those of wild-type, respectively. Further BCMO function in mammalian cells was analyzed by a retinoic acid receptor reporter assay, which responds to the metabolic conversion of beta-carotene to retinoic acid in vivo. Overall, these tools can be used to screen more active BCMO for the industrial and pharmacological purpose of retinal production from beta-carotene.
Park
Chan-Soo
CS
Department of Bioscience & Biotechnology/Institute of Bioscience, Sejong University, Seoul 143-747, Korea.
Lee
Sang-Wang
SW
Kim
Yeong-Su
YS
Kim
Eun-Joo
EJ
Sin
Hong-Sig
HS
Oh
Deok-Kun
DK
Kim
Seon-Won
SW
Um
Soo-Jong
SJ
D015153:Blotting, Western; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D014212:Tretinoin / Q000378:metabolism; D019207:beta Carotene / Q000378:metabolism; D050559:beta-Carotene 15,15'-Monooxygenase / Q000235:genetics / Q000378:metabolism*
D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D019207:beta Carotene; D014212:Tretinoin; D050559:beta-Carotene 15,15'-Monooxygenase