5-Keto-D-fructose is a useful starting material for the synthesis of pyrrolidine iminosugars. It can be prepared by regioselective oxidation of L-sorbose using pyranose 2-oxidase (P2Ox) and O2 as a cosubstrate. As the solubility of O2 in aqueous solution is low and the affinity of P2Ox for O2 is poor, we developed a new and efficient process for the production of 5-keto-D-fructose based on engineered P2Ox from Peniophora gigantea and in situ generation of O2 from H2O2 with catalase. This kind of oxygen supply required efficient mixing of the bioreactor which was achieved by controlled feeding of H2O2 close to the impeller tip where energy dissipation rate is highest. Thus bubbling, known to affect enzyme stability, was largely avoided, and the process could be run up to 145% oxygen super-saturation which speeds-up P2Ox activity. Under these conditions quantitative oxidation of 180?g?L-1 L-sorbose to 5-keto-D-fructose could be achieved within 4?h, resulting in a threefold higher overall productivity of the process compared to a process using gaseous oxygen supply. In addition, in situ generation of O2 from H2O2 lowered the oxygen demand of the process by a factor of 100 compared to gaseous oxygen supply. Biotechnol. Bioeng. 2012; 109: 29412945. (c) 2012 Wiley Periodicals, Inc.