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Applied Microbiology and Biotechnology

, Volume 97, Issue 2, pp 633–639 | Cite as

One-step synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid using a multienzymatic system

  • Luo Liu
  • Michael Braun
  • Gabi Gebhardt
  • Dirk Weuster-Botz
  • Ralf Gross
  • Rolf D. SchmidEmail author
Biotechnological Products and Process Engineering

Abstract

12-ketoursodeoxycholic acid (12-keto-UDCA) is a key intermediate for the synthesis of ursodeoxycholic acid (UDCA), an important therapeutic agent for non-surgical treatment of human cholesterol gallstones and various liver diseases. The goal of this study is to develop a new enzymatic route for the synthesis 12-keto-UDCA based on a combination of NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH, EC 1.1.1.201) and NADH-dependent 3α-hydroxysteroid dehydrogenase (3α-HSDH, EC 1.1.1.50). In the presence of NADPH and NADH, the combination of these enzymes has the capacity to reduce the 3-carbonyl- and 7-carbonyl-groups of dehydrocholic acid (DHCA), forming 12-keto-UDCA in a single step. For cofactor regeneration, an engineered formate dehydrogenase, which is able to regenerate NADPH and NADH simultaneously, was used. All three enzymes were overexpressed in an engineered expression host Escherichia coli BL21(DE3)Δ7α-HSDH devoid of 7α-hydroxysteroid dehydrogenase, an enzyme indigenous to E. coli, in order to avoid formation of the undesired by-product 12-chenodeoxycholic acid in the reaction mixture. The stability of enzymes and reaction conditions such as pH value and substrate concentration were evaluated. No significant loss of activity was observed after 5 days under reaction condition. Under the optimal condition (10 mM of DHCA and pH 6), 99 % formation of 12-keto-UDCA with 91 % yield was observed.

Keywords

Cofactor regeneration Dehydrocholic acid Formate dehydrogenase-7β-hydroxysteroid dehydrogenase 12-keto ursodeoxycholic acid Ursodeoxycholic acid 

Notes

Acknowledgments

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

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Luo Liu
    • 1
    • 4
  • Michael Braun
    • 2
  • Gabi Gebhardt
    • 2
  • Dirk Weuster-Botz
    • 2
  • Ralf Gross
    • 3
  • Rolf D. Schmid
    • 1
    • 5
    Email author
  1. 1.Institute of Technical BiochemistryUniversity of StuttgartStuttgartGermany
  2. 2.Institute of Biochemical EngineeringTechnische Universität MünchenGarchingGermany
  3. 3.PharmaZell GmbHRaublingGermany
  4. 4.Beijing Key Laboratory of Bioprocess, College of Life Science and TechnologyBeijing University of Chemical TechnologyBeijingPeople’s Republic of China
  5. 5.StuttgartGermany

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