New Biocatalytic Approaches for Alcohol Oxidations and Ketone Reductions Using (Deaza)Flavoenzymes

Biocatalysis is increasing in popularity compared to chemical approaches to produce the most diverse chemical components. This popularity is often the consequence of the lower environmental impact and/or of the selectivity of biocatalysts. Despite this, to compete with chemical processes, biocatalysts must fulfill many requirements. Therefore, there is a strong demand for stable, fast, and easy to produce biocatalysts. The research described in this thesis focused on the exploration of new or engineered redox enzymes that can be used for selective alcohol oxidations or ketone reductions

Production of Hydroxy Acids:Selective Double Oxidation of Diols by Flavoprotein Alcohol Oxidase

Flavoprotein oxidases can catalyze oxidations of alcohols and amines by merely using molecular oxygen as the oxidant, making this class of enzymes appealing for biocatalysis. The FAD-containing (FAD=flavin adenine dinucleotide) alcohol oxidase from P. chrysosporium facilitated double and triple oxidations for a range of aliphatic diols. Interestingly, depending on the diol substrate, these reactions result in formation of either lactones or hydroxy acids. For example, diethylene glycol could be selectively and fully converted into 2-(2-hydroxyethoxy)acetic acid. Such a facile cofactor-independent biocatalytic route towards hydroxy acids opens up new avenues for the preparation of polyester building blocks

Means and methods for selective double diol oxidation.

The invention relates to the field of enzyme engineering and biocatalysis, in particular to alcohol oxidases and their application in the selective oxidation of diols for the production of industrially useful oxidation products thereof, such as precursors for biodegradable polyesters. Provided is a method for the selective oxidation of a diol substrate, comprising subjecting a diol substrate of the formula HO-CH2-CH2-X-CH2-CH2-OH, wherein X = O, S or CH2, to a FAD-containing alcohol oxidase (AOX) enzyme of the class EC 1.1.3.13 or EC 1.1.3.17. or a mutant thereof, under conditions allowing lor the double oxidation of one hydroxyl moiety of the diol substrate into an oxidation product

Enantioselective oxidation of secondary alcohols by the flavoprotein alcohol oxidase from Phanerochaete chrysosporium

The enantioselective oxidation of secondary alcohols represents a valuable approach for the synthesis of optically pure compounds. Flavoprotein oxidases can catalyse such selective transformations by merely using oxygen as electron acceptor. While many flavoprotein oxidases preferably act on primary alcohols, the FAD-containing alcohol oxidase from Phanerochaete chrysosporium was found to be able to perform kinetic resolutions of several secondary alcohols. By selective oxidation of the (S)-alcohols, the (R)-alcohols were obtained in high enantiopurity. In silico docking studies were carried out in order to substantiate the observed (S)-selectivity. Several hydrophobic and aromatic residues in the substrate binding site create a cavity in which the substrates can comfortably undergo van der Waals and pi-stacking interactions. Consequently, oxidation of the secondary alcohols is restricted to one of the two enantiomers. This study has uncovered the ability of an FAD-containing alcohol oxidase, that is known for oxidizing small primary alcohols, to perform enantioselective oxidations of various secondary alcohols

Rapid enzyme stabilization by computationally designed libraries of HMF oxidase

HMF oxidase (HMFO) from Methylovorus sp. is a recently characterized flavoprotein oxidase [1]. HMFO is able to oxidize 5-(hydroxymethyl)furfural (HMF) into 2,5-furandicarboxylic acid (FDCA). Because HMF can be formed from fructose or other sugars and FDCA is a polymer building block, the oxidase has attracted attention as industrially relevant biocatalyst. The dicarboxylic acid FDCA can be polymerized with ethylene glycol to produce polyethylene furanoate (PEF). This renewable and bio-based polyester can be a valid alternative to the petroleum-based polyethylene terephthalate (PET) thanks to its similar characteristics. HMFO is a promising biocatalyst for various oxidations and not only for the production of FDCA. The first step to the development of an HMFO with improved catalytic properties is the engineering of the enzyme to enhance its thermostability using the recently developed FRESCO method Please click Additional Files below to see the full abstract