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Identification, cloning, heterologous expression, and characterization of a NADPH-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens

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Abstract

A gene encoding an NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH) from Collinsella aerofaciens DSM 3979 (ATCC 25986, formerly Eubacterium aerofaciens) was identified and cloned in this study. Sequence comparison of the translated amino acid sequence suggests that the enzyme belongs to the short-chain dehydrogenase superfamily. This enzyme was expressed in Escherichia coli with a yield of 330 mg (5,828 U) per liter of culture. The enzyme catalyzes both the oxidation of ursodeoxycholic acid (UDA) forming 7-keto-lithocholic acid (KLA) and the reduction of KLA forming UDA acid in the presence of NADP+ or NADPH, respectively. In the presence of NADPH, 7β-HSDH can also reduce dehydrocholic acid. SDS-PAGE and gel filtration of the expressed and purified enzyme revealed a dimeric nature of 7β-HSDH with a size of 30 kDa for each subunit. If used for the oxidation of UDA, its pH optimum is between 9 and 10 whereas for the reduction of KLA and dehydrocholic acid it shows an optimum between pH 4 to 6. Usage of the enzyme for the biotransformation of KLA in a 0.5-g scale showed that this 7β-HSDH is a useful biocatalyst for producing UDA from suitable precursors in a preparative scale.

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Acknowledgments

We thank the BMBF (German Federal Ministry of Education and Research), Grant No. FKZ 0315269, for financial support.

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Author notes

  1. Luo Liu

    Present address: Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beisanhuan East Road 15#, Beijing, 100029, People’s Republic of China

  2. Rolf D. Schmid

    Present address: , Jagdweg 3, 70569, Stuttgart, Germany

Authors and Affiliations

  1. Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

    Luo Liu & Rolf D. Schmid

  2. PharmaZell GmbH, Rosenheimerstr, 43, 83064, Raubling, Germany

    Arno Aigner

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  1. Luo Liu
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Correspondence to Luo Liu.

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Liu, L., Aigner, A. & Schmid, R.D. Identification, cloning, heterologous expression, and characterization of a NADPH-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens . Appl Microbiol Biotechnol 90, 127–135 (2011). https://doi.org/10.1007/s00253-010-3052-y

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  • DOI: https://doi.org/10.1007/s00253-010-3052-y

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