Substrate-based protein engineering of a flavoprotein oxidase for improved alcohol over-oxidation
September 24-28, 2017
The oxidation of alcohols to the corresponding carbonyl compounds represents a convenient strategy for the selective introduction of carbon-acceptor units into carbohydrate-based starting materials from renewable resources. A simple system to accomplish this transformation is by using flavin-containing alcohol oxidases. However, with prim-alcohols, the oxidation does not necessarily stop at the aldehyde stage, but may furnish the carboxylic acid via 'over'-oxidation of the aldehyde hydrate. In order to develop an alcohol oxidase for the efficient transformation of alcohols into carboxylic acids, we chose the recently discovered (5-hydroxymethyl)furfural oxidase (HMFO), which converts not only the eponymous (5-hydroxymethyl)furfural, but also a range of aromatic and allylic alcohols (Figure 1).In order to improve the performance of HMFO for over-oxidation, we anticipated an improved stabilisation of the aldehyde hydrate in the active site to be a crucial factor. After inspection of the HMFO crystal structure, two positions were identified, where hydrogen bond donating and accepting amino acids were introduced, in order to stabilize the gem-diol moiety. Indeed, one of the new HMFO variants exhibited a significantly increased activity for the formation of carboxylic acids from benzylic alcohols.
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Mathias Pickl, Silvia M. Glueck, and Kurt Faber, "Substrate-based protein engineering of a flavoprotein oxidase for improved alcohol over-oxidation" in "Enzyme Engineering XXIV", Pierre Monsan, Toulouse White Biotechnology, France Magali Remaud-Simeon, LISBP-INSA, University of Toulouse, France Eds, ECI Symposium Series, (2017). http://dc.engconfintl.org/enzyme_xxiv/76