¹ Institute of Biomedical Chemistry, Moscow, Russia;

² Joint Institute for High Temperatures of the Russian Academy of Sciences, Moscow, Russia

³ Foundation of Perspective Technologies and Novations, Moscow, Russia;

⁴ Moscow State University, Faculty of Computational Mathematics and Cybernetics, Moscow, Russia

⁵ Moscow State Academy of Veterinary Medicine and Biotechnology Named after Skryabin, Moscow, Russia

Abstract: Glycerol represents a useful functional component of heat-transfer fluids. Glycerol is known to be triboelectrically active. We report how the flow of glycerol, stopped in a cylindrical coiled pipe, influences horseradish peroxidase (HRP) enzyme. Namely, glycerol flow was pumped through the coil and then stopped. After that, we incubated the 0.1 μM solution of HRP near the side of the coil, and then adsorbed HRP onto mica. This operation markedly increased the HRP aggregation on mica in comparison with the control sample. We explain this phenomenon by the influence of triboelectrically induced electromagnetic fields. Our results can be useful in the development of flow-based systems for biosensors and bioreactors.

Keywords: horseradish peroxidase; glycerol; atomic force microscopy; enzyme aggregation

Enzymes have found numerous applications in biotechnology [1] and biomedical science [2]. In nature, enzymes catalyze reactions in living cells [3], and can be employed as catalysts in a wide range of commercially important processes [1]. The list of biotechnological applications of enzymes includes, for instance, food processing, synthesis of pharmaceuticals, paper fabrication, etc. [1,4]. In biomedical science, applications of enzymes in biosensors [2] and in diagnostic test kits [5,6] should be mentioned.

Enzyme-based catalysis requires proper selection and careful maintaining of optimal process conditions, since enzymes quickly lose their functional activity at extreme temperatures [7], pH values, ion concentrations, and pressures [8]. This is why biosensors and bioreactors intended for operation with enzymes are often equipped with thermal stabilization systems in order to maintain stable temperature conditions [9,10]. In these thermal stabilization systems, cylindrically wound pipes (or simply coils) with circulating heat-transfer fluid are often employed [11–13]. The use of glycerol as a component of heat-transfer fluids was shown to be promising [14,15]. At that, it should be emphasized that the flow of glycerol in a pipe induces electromagnetic fields due to the so-called triboelectric effect [16,17]. Electromagnetic fields generated upon flow of glycerol can be quite strong [16], thus representing an important factor influencing the activity of enzymes [18]. The effect of a triboelectrically induced field on an enzyme can take place even after stopping the flow of heat transfer fluid [18].

One of possible effects of electromagnetic fields on an enzyme is exhibited in a form of a change in its aggregation state on upon adsorption onto a solid substrate surface [18–20]. This is an important point, since surface-adsorbed enzymes are widely employed in biotechnology [1]. Aggregation of proteins, including enzyme proteins, is generally attributed to misfolding or partial unfolding of their polypeptide chains [21,22]. With regard to enzymes, their aggregation can also be related to a change in their hydration [18,19,23–25]. In general, aggregation is considered to cause a decrease in the functional activity of enzymes [26]. Colombie’ et al. demonstrated that inactivation of lysozyme in a bioreactor is accompanied by its aggregation [27]. On the other hand, Gentile et al. emphasized that aggregation of an enzyme can occur in the course of its functioning and does not inevitably imply the activity loss [28]. Accordingly, enzyme aggregation and external factors, influencing this process, require further thorough investigation. In this respect, ultrasensitive methods such as atomic force microscopy (AFM) are of use [18,19,29–31], since they allow the researchers to reveal even subtle effects on the enzyme aggregation [31].

Horseradish peroxidase (HRP) is a ~44 kDa enzyme glycoprotein [32,33]. It is widely employed in diagnostic kits as a component of enzyme-linked immunosorbent assay (ELISA) kits [34], and in biosensors as a reporter enzyme [35]. Furthermore, HRP has found many industrial applications in food technology [36], wastewater purification [37] and biofuel cells [38–40]. At that, aggregation state of HRP was shown to be influenced by external magnetic [29,30] and electromagnetic fields [18–20]. Electromagnetic fields are ubiquitous in industry [41]. As mentioned above, the aggregation state of enzymes can influence their functional activity. This explains the importance of further in-deep investigation of the influence of electromagnetic fields on the aggregation state of HRP.

Our present study reveals a considerable 40-minute aftereffect (the so-called long-term effect [19]) of the glycerol flow in a cylindrically coiled heat exchanger on the aggregation state of HRP after incubation of its solution near the outer side of the coiled section. In other words, the glycerol flow has been stopped prior to the incubation of the enzyme. The enzymatic activity of HRP has been found unaffected — as opposed to the previously reported case with the incubation of the enzyme near the inlet section of the heat exchanger [19]. Nevertheless, a considerably increased content of aggregated enzyme has been revealed by AFM on the surface of mica substrates after the incubation of the enzyme in our experimental setup. The phenomenon observed can be explained by the influence of triboelectrically induced electromagnetic fields [20].

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doi: 10.20944/preprints202403.0304.v1