Pennacchio, Angela (2013) New Dehydrogenase/Reductase as Biocatalysts for Stereoselective Reduction of Prochiral Ketones. [Tesi di dottorato]

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Testo PhD Thesis Dr.ssa Angela Pennacchio.pdf Download (21MB) | Anteprima

Abstract

Abstract Biocatalysis is well-matched to chemical synthesis in the pharmaceutical as well as the agrochemical and flavours industry. A significant number of products are chiral in at least one center, and many of them have multiple chiral centers. These compounds are usually manufactured in single isomer form since the current Food and Drug Administration regulations demand proof that the non-therapeutic isomer is non-toxic. The synthesis of a chiral center requires enantio and regioselective catalysts, and enzymes are consistently the most selective catalysts available being able to perform reactions under mild conditions of pH value, temperature, and pressure in aqueous solutions, a greener approach to chemistry, with remarkable chemo-, regio-, and stereoselectivity. Chiral alcohols are very important precursors for a large number of drugs, agrochemical and flavours. Their production by asymmetric bioreduction of a prochiral carbonyl precursor is becoming well-established in the field of biocatalysis. Enzymes that catalyze ketone reductions (known as ketoreductases) are a reliable source of high enantiomeric excess chiral alcohols. The aim of the present PhD research is to characterize new alcohol dehydrogenases (ADHs) for the synthesis of chiral secondary alcohols and the structural study of the isolated enzymes for the understanding of the structure-function relationships. The basic approach was to identify oxidoreductases with these distinctive features, operational stability and NAD dependency, by looking for in the genome of thermophilic microorganisms for genes coding putative short-chain dehydrogenases/reductases (SDR) with an acidic residue which determines NADH specificity in a particular position of the sequence. In the present work an applicative development of Thermus termophilus ADH in the asymmetric synthesis of two valuable chiral building blocks methyl (R)-mandelato and methyl (R)-o-chloromandelate at lab scale was carried out, and adaption of two different enzyme-coupled systems for the cofactor regeneration system was studied. Two novel NADH dependent-ADHs identified in the crenarchaeon Sulfolobus acidocaldarius SaADH and SaADH2 were biochemically characterized, a suitable cofactor regeneration system for the analytical study of the stereoselectivity both enzymes was utilized, moreover, the solved SaADH2 3D structure was discussed. Finally, an applicative utilization of Bacillus stearothermophilus ADH both as an isolated enzyme, and in recombinant E. coli whole cells in the synthesis of cinnamyl alcohol, a versatile fine chemical, by chemoselective reduction of cinnamaldehyde at gram scale was described.

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