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One‐Pot Multienzymatic Synthesis of 12‐Ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule the Reaction Equilibria of Five Biocatalysts Working in a Row
Author(s) -
Monti Daniela,
Ferrandi Erica Elisa,
Zanellato Ilaria,
Hua Ling,
Polentini Fausto,
Carrea Giacomo,
Riva Sergio
Publication year - 2009
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.200800727
Subject(s) - chemistry , cholic acid , cofactor , catalysis , combinatorial chemistry , substrate (aquarium) , enzyme , hydroxysteroid dehydrogenases , biocatalysis , redox , stereochemistry , organic chemistry , reaction mechanism , biochemistry , bile acid , dehydrogenase , oceanography , geology
The hydroxysteroid dehydrogenases (HSDHs)‐catalyzed one‐pot enzymatic synthesis of 12‐ketoursodeoxycholic acid (3α,7β‐dihydroxy‐12‐oxo‐5β‐cholanoic acid), a key intermediate for the synthesis of ursodeoxycholic acid, from cholic acid has been investigated. This goal has been achieved by alternating oxidative and reductive steps in a one‐pot system employing HSDHs with different cofactor specificity, namely NADH‐dependent HSDHs in the oxidative step and an NADPH‐dependent 7β‐HSDH in the reductive one. Coupled in situ regeneration systems have been exploited not only to allow the use of catalytic amounts of the cofactors, but also to provide the necessary driving force to opposite reactions (i.e., oxidation and reduction) acting on different sites of the substrate molecule. Biocatalysts suitable for the set‐up of this process have been selected and their kinetic behaviour in respect of the reactions of interest has been evaluated. Finally, the process has been studied employing the enzymes both in free and compartmentalized form.