Synthesis of keto-sugars using pyranose dehydrogenase

Lactose from cheese whey is a readily available raw material, which is underutilized at present, as a large part of world whey production is disposed of as effluent. As direct utilization of lactose in food and pharmaceuticals is limited, enzymatic sugar transformations can be an interesting alternative that would aid in turning the by-product lactose into a value-added product. Potential products include the isomerization product lactulose, a proven prebiotic sugar and widely used medicinal drug for treatment of chronic constipation or hepatic encephalopathy, the galactose isomer tagatose (a prebiotic, low-calory sugar and potential food ingredient), and lactobionic acid, an ingredient of organ stabilizing solutions (for transplantations) and potential component of washing powder. Pyranose dehydrogenase (PDH) is a sugar oxidoreductase from litter-decomposing basidiomycetous fungi such as Agaricaceae and Coprinaceae. Compared to the catalytically related pyranose-2-oxidase it displays a broader substrate spectrum, is active against a number of disaccharides including lactose, and shows variable regioselectivity depending on the substrate and the source of the enzyme. PDH does not use oxygen as electron acceptor and therefore does not produce hydrogen peroxide, which rapidly inactivates enzyme proteins unless efficiently removed from the system. We plan to establish an expression system for - already available - pyranose dehydrogenase genes in Pichia pastoris, and develop an enzymatic process for conversion of lactose to 2-keto-lactose (a precursor of lactulose) and lactobionic acid, as well as galactose to 2-keto-galactose (a precursor of tagatose). A laccase-based enzymatic regeneration system for redox mediators, which has been recently developed at our Department for a similar process, will be adapted to the specific requirements of PDH as biocatalyst. Furthermore, the reaction engineering of the process will be developed, including studies on immobilization or derivatization of the redox mediators. This process should be suitable for a range of other carbohydrate substrates such as maltose, xylose, arabinose and others, with minimal modifications such as use of a PDH protein from an alternative fungal source, and should be widely applicable for a number of processes of industrial interest.