Controlled feeding of hydrogen peroxide as oxygen source improves production of 5-ketofructose from L-sorbose using engineered pyranose 2-oxidase from Peniophora gigantea.

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 O(2) as a cosubstrate. As the solubility of O(2) in aqueous solution is low and the affinity of P2Ox for O(2) 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 O(2) from H(2) O(2) with catalase. This kind of oxygen supply required efficient mixing of the bioreactor which was achieved by controlled feeding of H(2) O(2) 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 O(2) from H(2) O(2) lowered the oxygen demand of the process by a factor of 100 compared to gaseous oxygen supply.