Chemical biotechnology for the specific oxyfunctionalization of hydrocarbons on a technical scale.

Oxygenases catalyze, among other interesting reactions, highly selective hydrocarbon oxyfunctionalizations, which are important in industrial organic synthesis but difficult to achieve by chemical means. Many enzymatic oxygenations have been described, but few of these have been scaled up to industrial scales, due to the complexity of oxygenase based biocatalysts and demanding process implementation. We have combined recombinant whole-cell catalysis in a two-liquid phase system with fed-batch cultivation in an optimized medium and developed an industrially feasible process for the kinetically controlled and complex multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde using the xylene monooxygenase of Pseudomonas putida mt-2 in Escherichia coli. Successful scale up to 30 L working volume using downscaled industrial equipment allowed a productivity of 31 g L(-1) d(-1) and a product concentration of 37 g L(-1). These performance characteristics meet present industry requirements. Product purification resulted in the recovery of 469 g of 3,4-dimethyl- benzaldehyde at a purity of 97% and an overall yield of 65%. This process illustrates the general feasibility of industrial biocatalytic oxyfunctionalization.