Regioselective hydroxylation of isoflavones by Streptomyces avermitilis MA-4680

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Abstract

Screening of bacterial whole cells was performed for regioselective hydroxylation of daidzein and genistein. Among the strains examined, Streptomyces avermitilis MA-4680 showed high ortho-dihydroxylation activity to produce 3′,4′,7-trihydroxyisoflavone and 3′,4′,5,7-tetrahydroxyisoflavone from daidzein (4′,7-dihydroxyisoflavone) and genistein (4′,5,7-trihydroxyisoflavone), respectively. Using 100 mg cells (wet wt.) and 1% (v/v) Triton X100 in 1 ml of total reaction volume, where 100 μl of the substrate solution (0.5 mM in 10% (v/v) mixed solvent of DMSO:MeOH = 3:7) was added to 900 μl of potassium phosphate buffer (100 mM, pH 7.2), a 16% molar conversion yield of 3′,4′,7-trihydroxyisoflavone was obtained from 0.5 mM daidzein after 24 h of reaction time at 28 °C and 200 rpm. Ketoconazole significantly (ca. 90%) inhibited the ortho-hydroxylation activity of daidzein, suggesting that cytochrome P450 enzymes putatively play roles in regiospecific daidzein hydroxylation. The analysis of the reaction products was determined by gas chromatography/mass spectrometry (GC/MS) and 1H NMR.

Section snippets

Chemicals

Daidzein, genistein, 3′,4′,7-trihydroxyisoflavone, quinidine, coumarin, and erythromycin were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Ketoconazole and N,O-bis(trimethylsilyl)trifluoroacetamide were purchased from Fluka Chemical Co. (Buchs, Switzerland). All other chemicals were of the highest grade.

Culture conditions of type culture strains

Actinomycete strains including S. avermitilis MA-4680 were obtained from the Korea Collection for Type Cultures (KCTC, Daejeon, South Korea). Various kinds of Bacillus spp. and

Hydroxylation of daidzein and genistein by the S. avermitilis strain

Recently completed genome sequences of a couple of Actinomycete strains revealed that more than a dozen cytochrome P450 monooxygenases are present in each strain, and that they are involved in various hydroxylation steps of primary and secondary metabolites 24., 25., 26., 27., 28.. Based on this observation, various available Actinomyces, Bacillus spp., and fungal strains were screened for their ability to convert daidzein and genistein into their corresponding hydroxylated products. Among the

Acknowledgments

This research was partially supported by the Brain Korea 21 program of the Ministry of Education, Republic of Korea (Grant No. A05-0572-A11101-06A2-00020B) and by the National Research Laboratory Program (Grant No. R0A-2007-000-10007-0), Korea Ministry of Science and Technology. We thank Prof. Yoongho Lim and Younghee Park from Konkuk University for their help in the interpretation of the NMR analysis.

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    The first two authors contributed equally to this work.

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