Production of a lactose-free galacto-oligosaccharide mixture by using selective enzymatic oxidation of lactose into lactobionic acid

We report a novel and efficient way of producing lactose-derived galacto-ol igosaccharides (GOS) that do not contain remaining lactose and monosacchari des. The initial sugar mixture was obtained by enzymatic transformation at 70 degreesC of a lactose solution of 270 g/liter using recombinant P-glycos idase from the Archaeon Sulfolobus solfataricus. At the optimum reaction ti me for kinetically controlled transgalactosylation, it contained 46% monosa ccharides, 13% lactose and 41% GOS. Lactose was selectively oxidised into l actobionic acid by using fungal cellobiose dehydrogenase which displays a a pproximate to 100-fold preference for reaction with lactose compared to rea ction with GOS. Oxidation of lactose was coupled to reduction of 2,6-dichlo ro-indophenol which was added in catalytic concentrations. The oxidised red ox mediator was regenerated continuously by fungal laccase-catalysed reduct ion of molecular oxygen into water. Ion exchange chromatographies were empl oyed to remove lactobionic acid, other ions and monosaccharides. The final product contained 97% GOS, 1.2% lactose and 2.1% monosaccharides. The yield accounted for 25% of original lactose. An enzymatic assay for lactose has been developed. It is robust and allows sensitive quantification of the ana lyte in complex sugar mixtures containing large excesses of monosaccharides and GOS. (C) 2001 Elsevier Science Inc. All rights reserved.