Large-scale Enzymatic Synthesis of 12-Ketoursodeoxycholic Acid from Dehydrocholic Acid by Simultaneous Combination of 3-Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni and 7-Hydroxysteroid Dehydrogenase from Collinsella aerofaciens

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 stereoand 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.