A bienzymatic biocatalyst constituted by glucose oxidase and Horseradish peroxidase immobilized on ordered mesoporous silica

•A bienzymatic biocatalyst was obtained by immobilizing GOx and HRP on SBA-15.•The biocatalyst was obtained either via co-immobilization or separated immobilization.•The biocatalyst activity is high only when it is kept wet after immobilization.•The bienzymatic biocatalyst could be reused for 14 reaction cycles.•The bienzymatic biocatalyst was able to oxidize two model phenolic pollutants.

It is presently extremely challenging to realize an active immobilized multi-enzyme biocatalyst which allows to run in vitro multi-step cascade reactions. This work deals with the obtainment of a bienzymatic immobilized biocatalyst constituted by Glucose Oxidase (GOx) and Horseradish Peroxidase (HRP) immobilized onto SBA-15 mesoporous silica. The effect of co-immobilization (GOx/HRP@SBA-15) versus the separated immobilization (GOx@SBA-15/HRP@SBA-15), and the effect of covalent versus physical immobilization, on protein loading and enzymatic activity were investigated. Regardless the different immobilization strategy used, it was found that the catalytic activity could be retained only if the immobilized bienzymatic biocatalyst was kept wet. The obtained wet GOx/HRP@SBA-15 biocatalyst could be recycled 14 times keeping a good activity. Finally, the bienzymatic biocatalyst was tested for the oxidation of two model phenolic (caffeic acid and ferulic acid) pollutants of agricultural wastewaters, as olive mill wastewaters (OMWs). The biocatalyst was able to reach a 70% conversion within 15 min.

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