Abstract of PhD Thesis

Novel Alcohol dehydrogenases for the synthesis of chiral alcohols

Gabriele Gucciardo, University College Dublin (2010)

The aim of this project was to investigate the practical use of alcohol dehydrogenase enzymes as biocatalysts for the production of chiral alcohols. The investigation was oriented on the study of halophilic enzymes, which have been found to be active in buffer/organic solvent system.

During the project the genes aad (aryl alcohol dehydrogenase) and adh2 (alchol dehydrogenase 2) from the Halobacterium sp. NRC-1 were cloned. The expression system E. coli was found no to be appropriate for the expression of these genes. The genes were expressed using the organism Haloferax volcanii DS70.

ADH2 showed alcohol dehydrogenase activity against primary alcohols, the enzyme has a double cofactor specificity. ADH2 activity was strongly dependent on the salt concentration (3M KCl and 4M NaCl) and basic pH. For the protein purification ADH2 was cloned with His-tag, and partially purified by using Ni2+ IMAC sepharose. A 33-fold purification was obtained.