Intensifying the O2-dependent heterogeneous biocatalysis: Superoxygenation of solid support from H2O2 by a catalase tailor-made for effective immobilization

Besides merely destroying H₂O₂, an important use of the catalase reaction, H₂O₂ → 1/2 O₂ + H₂O, is to supply O₂ to oxygenation reactions. Due to convenient spatiotemporal control over O₂ release, oxygenation from H₂O₂ is useful in particular for enzymatic reactions confined to solid supports. Because commercial catalases are difficult to immobilize, we have developed a one-step procedure of purification and immobilization of Bordetella pertussis catalase, recombinantly produced in Escherichia coli. Fusion of the catalase to a positively charged binding module enabled effective immobilization of the chimeric enzyme on anionic support (Relisorb SP 400), giving a controllable activity loading of between 5000 and 100,000 units/g support. Use of the immobilized catalase in combination with H₂O₂ feeding provided O₂ to the reaction of glucose oxidase in solution for a range of volumetric conversion rates (0.2–1.5 mM/min). Using optical sensing to measure the O₂ concentration in the liquid but also in the solid phase, we showed that internal superoxygenation of the support was made possible under these conditions, resulting in an inverted (that is, negative) O₂ concentration gradient between the bulk and the particle and allowing the internal O₂ concentration to exceed by up to 4-fold the limit of atmospheric-pressure air saturation in solution. By tailored immobilization of B. pertussis catalase, therefore, an efficient biocatalytic system for hydrogen peroxide conversion in porous solid support was developed. This could find application for bubble-free oxygenation of O₂-dependent enzymes co-immobilized with the catalase whereby enhanced internal availability of O₂ would contribute to biocatalytic reaction intensification.