Role of sugars in the inactivation of horseradish peroxidase induced by cold atmospheric plasma

This study investigated the effect of different CAP exposures on the activity of horseradish peroxidase (HRP) in phosphate buffer and in model systems with different concentrations of monosaccharides (glucose, fructose) and disaccharides (sucrose, trehalose) to evaluate the potential role of sugars in enzyme inactivation. Spectroscopic analyses (fluorescence, circular dichroism, UV-Vis absorption) were conducted to highlight structural modifi-cations possibly responsible for HRP inactivation. In phosphate buffer, the inactivation of HRP by CAP resulted dependent on both the treatment time (up to 70% after 30 min) and ozone concentration in the chamber. HRP residual activity was fitted with a very good approximation by the Weibull model. Sugars reduced the CAP ef-ficacy, and this effect was concentration-dependent and much higher for disaccharides than monosaccharides. UV and fluorescence spectra showed that sugars differently preserved HRP tertiary structure, hindering heme degradation and quenching of aromatic amino acids. Likewise, sugars differently affected the loss of secondary structures. These findings only partially explained the diverse protective effects of sugars on HRP. Other factors, such as the different sugars' abilities to quench plasma reactive species, reduce the system mobility, and stabilize proteins by preserving their hydration shell, were retained to play a role in the reduction of HRP inactivation. This study deepened the knowledge of the effect of CAP on peroxidase activity and demonstrated that the presence of mono and disaccharides, naturally found in fruits and vegetables and widely employed in food and beverage industries, can mitigate the inactivation and structural alterations induced by CAP on peroxidase enzymes.