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

- Tailor-made oriented immobilization of the haem-catalase KatA from Bordetella pertussis fused to the binding module Zbasic2.
- Highly effective one-step purification and immobilization of Zbasic2_KatA fusion on anionic solid support.
- O2 production from H2O2 by the immobilized Zbasic2_KatA directly measured within solid support.
- Superoxygenation of the solid material due to O2 formation by immobilized Zbasic2_KatA.
- Potential application for intensification of O2 dependent biotransformations confined to solid support.

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.

By tailored immobilization of B. pertussis catalase an efficient biocatalytic system for H2O2 conversion in porous solid support was developed. Potential use of the immobilized catalase for superoxygenation of solid supports from externally added H2O2 was demonstrated.208