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Please use this identifier to cite or link to this item: http://hdl.handle.net/10277/445

Authors: Ferrandi, Erica Elisa
Internal Tutor: POLLEGIONI, LOREDANO
Co-tutor: MONTI, DANIELA
Title: New microbial hydroxysteroid dehydrogenases and their synthetic application for the selective modification of bile acids.
Abstract: Hydroxysteroid dehydrogenases (HSDHs), mainly obtained from bacterial sources, have been widely employed for the regio- and stereoselective oxidoreduction of the hydroxyl-keto groups of steroids, bile acids and their derivatives. In particular they might be suitable biocatalysts for the industrial synthesis of bile acids derivatives of pharmacological interest such as ursodeoxycholic acid (UDCA; 3α,7β-dihydroxy-5β-cholan-24-oic acid). Aim of this work was i) to investigate a new enzymatic process for the synthesis of UDCA; ii) to develop new cofactor regeneration systems to be coupled to HSDHs-catalyzed reactions; iii) to clone and overexpress in E. coli new HSDHs suitable for the modification of bile acids up to an industrial level. Specifically, the preparative-scale HSDHs-catalyzed one-pot enzymatic synthesis of 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-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 and employing HSDHs with different cofactor specificity. To provide the necessary driving force to opposite reactions (i.e., oxidation and reduction) acting concurrently on different sites of the same substrate molecule, suitable cofactor regeneration systems were coupled to these reactions. However, due to a limited cofactor specificity of some of the enzymes used, an undesired reaction equilibrium was established resulting in by-products formation. This problem was overcome by uncoupling the oxidative and reductive biocatalysts (Paper I). Moreover, a new laccase/mediator system for NAD(P)+ cofactor regeneration in HSDHs-catalyzed oxidations has been developed. This system has been successfully applied in aqueous, buffered reactions (space-time yield of 2.35 g L-1 h-1 ) and in biphasic systems (space-time yields of up to 27.47 g L-1 h-1), demonstrating to be very efficient, to have high stability, to tolerate solvents, and to be simple to employ (Paper II). Finally, NADPH-dependent 7α- and 7β-hydroxysteroid dehydrogenases (7α-HSDH and 7β-HSDH) from Clostridium absonum were cloned and overexpressed in recombinant form in E. coli. The enzymes were further characterized from a functional and a kinetic point of view, demonstrating that both of them, in suitable bioconversion conditions, could be promising candidates for further applications in the epimerization reaction of bile acids at the C-7 position (Paper III).
Keywords: bile acids, hydroxy steroid dehydrogenasis, cloning, biochemical characterization, cofactor regeneration systems.
Subject MIUR : BIO/04 FISIOLOGIA VEGETALE
Issue Date: 2011
Language: eng
Doctoral course: Biotecnologie
Academic cycle: 24
Publisher: Università degli Studi dell'Insubria
Other information: Istituto di Chimica del Riconoscimento Molecolare - CNR, Milano
Citation: Ferrandi, E.E.New microbial hydroxysteroid dehydrogenases and their synthetic application for the selective modification of bile acids. (Doctoral Thesis, Università degli Studi dell'Insubria, 2011).

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