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Z. Naturforsch. 2012,
67b, 1037 – 1044
doi:10.5560/ZNB.2012-0165
Large-scale Enzymatic Synthesis of 12-Ketoursodeoxycholic Acid from Dehydrocholic Acid by Simultaneous Combination of 3α-Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni and 7β-Hydroxysteroid Dehydrogenase from Collinsella aerofaciens
Institut für Molekulare Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, Forschungszentrum Jülich, Stetternicher Forst, 52426 Jülich, Germany
Reprint requests to Prof. Dr. Werner Hummel. Fax: +49 2461 61-2490. E-mail:
w.hummel@fz-juelich.de
Received June 17, 2012 / published online October 8, 2012
Dedicated to Professor Heribert Offermanns on the occasion of his 75th birthday
12-Keto-UDCA is an important optically active component for the drug ursodeoxycholic acid (UDCA). Starting from the three-keto compound dehydrocholic acid, the carbonyl groups at position 3 and 7 have to be reduced stereo- and regioselectively. In this case we applied two hydroxysteroid dehydrogenases for this purpose, the NAD-dependent 3α-HSDH from Pseudomonas testosteroni and the NADP-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens. Both enzymes can be produced in high yields by an Escherichia coli strain as recombinant proteins. In order to avoid impurities by the 7α-hydroxysteroid dehydrogenase of Escherichia coli, a mutant strain with an inactivated 7α-enzyme was applied for producing the three enzymes. For bioconversion, the dehydrogenases can be used as crude enzyme samples and are
applied simultaneously. A 1.8 L batch of 100 mm DHCA incubated at pH = 8.0 and 25 °C resulted in 80 g crude product with a quite high purity of ≥% 99.5 as judged by HPLC analysis.
Key words: Bile Acid, Hydroxysteroid Dehydrogenase, Short-chain Dehydrogenase, Ursodeoxycholic Acid