earticle

영어

Lignan, one of phytoestogens, has attracted great attention due to their estrogenic, anticarcinogenic, antioxidant effects. Among lignans, enterodiol (END) is converted by intestinal bacteria from the plant lignan secoisolariciresinol, which exist in flaxseed mainly. Among the 20 Actinomycetes strains examined, Streptomyces avermitilis MA-4680 and Nocardia facinica IFM10152 showed the highest hydroxylation activity for enterodiol. Reaction products profiling using GC/MS revealed that, five products of aliphatic and three of aromatic monohydroxylation were found from Streptomyces avermitilis MA-4680, whereas only two aromatic monohydroxylated products were detected from Nocardia facinica IFM10152.To identify which cytochrome P450s are related to hydroxylation reaction toward enterodiol, we constructed 27 P450s from Nocardia facinica IFM10152 in pET vector and putidaredoxin (camB) and putidaredoxin reductase (camA) from Pseudomonas putida in pET duet vector to use different antibiotic system. P450s are co-expressed with camA and camB as redox partner in E.coli. Eventually, we found that CYP154 were able to catalyze hydroxylation reaction of enterodiol at ortho position in vivo. Finally, we purified CYP154, camA and camB by using Ni-NTA his tag purification system and confirmed hydroxylation activity with enterodiol.