NAD+‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

Epimerization of cholic and chenodeoxycholic acid (CA and CDCA, respectively) is a notable conversion for the production of ursodeoxycholic acid (UDCA). Two enantiocomplementary hydroxysteroid dehydrogenases (7α‐ and 7β‐HSDHs) can carry out this transformation fully selectively by specific oxidation of the 7α‐OH group of the substrate and subsequent reduction of the keto intermediate to the final product (7β‐OH). With a view to developing robust and active biocatalysts, novel NADH‐active 7β‐HSDH species are necessary to enable a solely NAD+‐dependent redox‐neutral cascade for UDCA production. A wild‐type NADH‐dependent 7β‐HSDH from Lactobacillus spicheri (Ls7β‐HSDH) was identified, recombinantly expressed, purified, and biochemically characterized. Using this novel NAD+‐dependent 7β‐HSDH enzyme in combination with 7α‐HSDH from Stenotrophomonas maltophilia permitted the biotransformations of CA and CDCA in the presence of catalytic amounts of NAD+, resulting in high yields (>90 %) of UCA and UDCA. Biotransformation: By employing a compatible set of newly designed and stable enzymes, the epimerization of hydroxysteroids was carried out in a redox‐neutral environment by using a NAD+‐dependent cascade. The racemization of chenodeoxycholic acid (CDCA) is substantially favored towards the products, as governed by the thermodynamic and solubility properties of the compounds involved.