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

Abstract

Ursodeoxycholic acid (UDCA) is an effective drug for the treatment of hepatitis. In this study, 7α-hydroxysteroid dehydrogenase (7α-HSDH) and lactate dehydrogenase (LDH), as well as 7β-hydroxysteroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH), were co-immobilized onto an epoxy-functionalized resin (ES-103) to catalyze the synthesis of UDCA from chenodeoxycholic acid (CDCA). Through optimizing the immobilization pH, time, and loading ratio of enzymes to resin, the specific activities of immobilized LDH-7αHSDH@ES-103 and 7βHSDH-GDH@ES-103 were 43.2 and 25.8 U g , respectively, which were 12- and 516-fold higher than that under the initial immobilization conditions. Continuous production of UDCA from CDCA was subsequently achieved by using immobilized LDH-7αHSDH@ES-103 and 7βHSDH-GDH@ES-103 in two serial packed-bed reactors. The yield of UDCA reached nearly 100 % and lasted for at least 12 h in the packed-bed reactors, which was superior to that of the batchwise reaction. This efficient continuous approach developed herein might provide a feasible route for large-scale biotransformation of CDCA into UDCA.

NAD+‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

“…A solely NAD + ‐dependent cascade for CDCA and CA epimerization, in which NAD + is present in catalytic amounts, represents a promising method for a more efficient process in terms of atom economy and number of reagents required. To achieve this goal it is fundamental to employ robust, active, and selective enzymes: Sm 7α‐HSDH shows kinetic parameters better or comparable to its E. coli or Bacteroides homologues, widely used by researchers in these kinds of processes . This enzyme selectively oxidizes the 7α‐OH group of CA and CDCA.…”

“…In particular, Sm 7α‐HSDH and Ls 7β‐HSDH are a compatible, stable set of enzymes and promising candidates as biocatalysts in the synthesis of hydroxysteroid derivatives. Further investigations will be carried out to develop a flow system or a membrane reactor similar to the ones described in literature for our newly developed biocatalytic UDCA synthesis procedure and to increase the substrate loading in the process.…”

“…HSDHs are enzymes of the class of alcohol dehydrogenases (ADH) that use NAD(P) + and NAD(P)H as the electron acceptor and donor, respectively. In the literature, several cascades that use these enzymes are reported; however, since known enzymes have different cofactor specificities, that is, 7α‐HSDHs are usually NAD + ‐dependent and 7β‐HSDHs NADPH‐dependent, the oxidative and reductive reaction steps are decoupled in currently reported procedures (Figure —state of art). The cofactors, in this case, are regenerated by the addition of two other enzymes and two sacrificial substrates .…”

“…In the literature, several cascades that use these enzymes are reported; however, since known enzymes have different cofactor specificities, that is, 7α‐HSDHs are usually NAD + ‐dependent and 7β‐HSDHs NADPH‐dependent, the oxidative and reductive reaction steps are decoupled in currently reported procedures (Figure —state of art). The cofactors, in this case, are regenerated by the addition of two other enzymes and two sacrificial substrates . In this specific case, the reaction is driven towards completion by the addition of sacrificial substrate (in great surplus): Although good yields were achieved (>80 %), large amounts of side products and waste were formed that have to be removed during the downstream processing steps, increasing the overall cost and the environmental burden of UDCA production.…”

The Impact of Recent Developments in Technologies which Enable the Increased Use of Biocatalysts

“…Another example of a biocatalytic process which is improved by transfer to continuous flow is the synthesis of ursodeoxycholic acid 26 (Scheme ) . The process in batch involved the use of four enzymes in a two‐step one‐pot system.…”