Protective role of hematin in alcohol oxidase-mediated glycerol oxidation: A kinetic modeling approach

Mathematical modeling of complex multi-catalytic reaction pathways is a powerful tool not only for determining optimal operational conditions but also for gaining insights into the kinetic behavior of efficient yet product-sensitive enzymes. This approach was applied to the biooxidation of glycerol using oxidases, aided by hematin, a natural mimic of heme peroxidases and catalases, which acts as a scavenger for enzymatically produced H₂O₂. The bi-catalytic reaction system was coupled with the formation of a colored imine product from phenol and 4-aminoantipyrine, enabling spectrophotometric monitoring of reaction kinetics. Alcohol oxidase (AOX) from Komagataella pastoris (formerly Pichia pastoris) was submitted to this strategy to overcome some kinetic drawbacks previously observed with galactose oxidase (GAO) from Dactylium dendroides. Although both enzymes exhibited comparable oxidation yields, computer-aided kinetic analyses revealed significant differences in efficiency and sensitivity to H₂O₂. AOX demonstrated two orders of magnitude greater efficiency and a similarly higher affinity for glycerol. However, AOX showed a stronger tendency to bind H₂O₂ compared to hematin or GAO, as suggested by the ratio of the inhibition constant (k_inh) to the activation constant (k₁) for hematin. The kinetic model was also used to simulate optimal conditions for enhancing the stability of AOX and hematin. Reducing glycerol concentration or increasing hematin concentration improved AOX stability, while the addition of phenol helped preserve hematin. The predicted stabilities turn the AOX/hematin/phenol system promising for biosensing or analytical applications.