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

Abstract

Dedicated to Professor Heribert Offermanns on the occasion of his 75 th birthday12-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.

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

“…Moreover, for each one of these positions, HSDHs usually show a practically absolute stereoselectivity by oxidizing only either the hydroxyl group above (β configuration) or below (α configuration) the plane of the steroid molecule . The reactions catalyzed by HSDHs are reversible, thus these enzymes can be applied in the regioselective reduction of the corresponding keto derivatives as well …”

“…[4,5] The reactions catalyzed by HSDHs are reversible, thus these enzymes can be applied in the regioselective reduction of the corresponding keto derivatives as well. [4,6,7] Thanks to these interesting features, HSDHs have been widely studied during the last years for their exploitation in the biocatalyzed synthesis of key intermediates of the drug ursodeoxycholic acid (UDCA), a largely applied therapeutic agent used for the dissolution of cholesterol gallstones and for the treatment of different hepatic diseases. These efforts have led to the characterization of different enzymes showing either 7α-, 7β-or 12α-HSDH activity, [2,3] as well as to the development of biocatalyzed processes for the preparation of UDCA intermediates.…”

Continuous Production of Ursodeoxycholic Acid by Using Two Cascade Reactors with Co‐immobilized Enzymes

“…However, the yield of TUDCA was only 62.5 %. Similar works on the production of 12‐ketoursodeoxycholic acid have been reported by the groups of Weuster‐Botz, Riva, and Hummel . In our work, UDCA was continuously produced in two cascade PBRs, with nearly 100 % yield and the space–time yield (88.5 g L −1 d −1 ) was higher than that of the other reports (Table ).…”

Chemoenzymatic Synthesis of Steroidal Products: Recent Advances

“…The synthesis of steroids often involves multiple biocatalytic pathways and enzyme cascade strategy expected to be designed for the efficiently production of steroids. Ursodeoxycholic Acid (UDCA) is an important pharmacologically active natural product and approved by the FDA for the treatment of primary biliary cirrhosis (PBC) [27a–d] . Production of UDCA is usually achieved from cholic acid (CA) by using chemical methods with poor selectivity and is accompanied by a large number of by‐products and low yield [27] .…”