Combined Cross-Linked Enzyme Aggregates of Monoamine Oxidase and Putrescine Oxidase as a Bifunctional Biocatalyst for Determination of Biogenic Amines in Foods

In order to determine total biogenic amines in fermented foods, the combined cross-linked enzyme aggregates of a monoamine oxidase and a putrescine oxidase (combi-CLEAs) and the cross-linked enzyme aggregates (CLEAs) of the fused enzyme of two amine oxidases (MonoAmine Putrescien Oxidase, MAPO) were prepared. The effects of various parameters were examined to optimize the CLEAs formation. Biochemical characterization and stability of free and the CLEAs enzymes were performed. Through optimization of the CLEAs formation condition, the combi-CLEAs and the CLEAs-MAPO were prepared with 82% and 78% of residual activities relative to the activities of the subjected enzymes were in a preparative scale. The optimal pH for tyramine-activities of the CLEAs enzymes were shifted to relatively basic pH, leading to synchronization of the optimal performances of combi-CLEAs over pH for tyramine and putrescine. In addition, thermostability of the CLEAs enzymes were improved with almost double half-lives at 65 °C in comparison to the free enzymes. The catalytic efficiencies of combi-CLEAs for tyramine, histamine and putrescine were reduced by 41%, 56%, and 31%, respectively, and the inhibition potency by the substrate was reduced by two-fold in comparison of the mixed free enzymes. In conclusion, combi-CLEAs are a promising catalyst with the improved stability and the same optimum pH for dual activities in enzymatic determination of biogenic amines in foods.