Effective production of retinal from beta-carotene using recombinant mouse beta-carotene 15,15'-monooxygenase.
The gene encoding beta-carotene 15,15'-monooxygenase from Mus musculus (house mouse), which cleaves beta-carotene into two molecules of retinal, was cloned and expressed in Escherichia coli. The expressed enzyme was purified by His-tag affinity and resource Q ion exchange chromatography columns to a final specific activity of 0.51 U mg(-1). The optimum pH, temperature, substrate and detergent concentrations, and enzyme amount for effective retinal production were determined to be 9.0, 37 degrees C, 200 mg l(-1) beta-carotene, 5% (w/v) Tween 40, and 0.2 U ml(-1) enzyme, respectively. Under optimum conditions, the recombinant enzyme produced 72 mg l(-1) retinal in a 15-h reaction time, with a conversion yield of 36% (w/w). The specific activity of the purified enzyme and retinal production obtained in the present study were the highest results ever reported.
Kim
Yeong-Su
YS
Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, South Korea.
Kim
Nam-Hee
NH
Kim
Hye-Jung
HJ
Lee
Jung-Kul
JK
Kim
Seon-Won
SW
Oh
Deok-Kun
DK
D000818:Animals; D003001:Cloning, Molecular; D003902:Detergents; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D051379:Mice; D011994:Recombinant Proteins; D012172:Retinaldehyde / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D019207:beta Carotene / Q000378:metabolism*; D050559:beta-Carotene 15,15'-Monooxygenase / Q000378:metabolism*
D003902:Detergents; D011994:Recombinant Proteins; D019207:beta Carotene; C493359:Bco1 protein, mouse; D050559:beta-Carotene 15,15'-Monooxygenase; D012172:Retinaldehyde