## 4 Discussion
At an alkaline pH, PEI decreases the enzyme activity more than the other polyamines (up to 25%) and slightly increases the activity at a pH of 7.5 (by 13%). As mentioned earlier, this polycation can destabilize the enzyme due to its higher positive charge density. The loss of enzyme activity at elevated temperatures can be explained by two factors: dissociation of the enzyme to the monomers and denaturation of the protein sequence. When the temperature is increased, dissociation of the enzyme subunits is usually observed, which leads to the separation of the amino acids that make up the active center and the loss of activity. Therefore, it is logical that RrA-Spm loses activity approximately two-fold slower at an elevated temperature than the native enzyme since there is covalent cross-linking of the monomers to each other. As expected, PEI has a destabilizing effect on RrA, accelerating the decrease in activity. PEI is longer than spermine, and this prevents the oligomeric forms of the protein from cross-linking with each other. Moreover, the high concentration of positive charge can negatively affect the interaction of the enzyme subunits, destroying the hydrogen bonds between them. In PEI-PEG, a portion of the primary amino groups is modified by PEG, so conjugation with this copolymer avoids the negative effect of a large amount of positive charge on the quaternary structure of the protein. The obtained conjugates also showed a different resistance to the trypsin action, which is related to the immunogenicity of the enzyme preparation--the availability of the enzyme to the proteins of the complement system. In the previous work, it was shown that chitosan copolymers, due to their branched structure and shielding of the protein globule, allow us to protect the enzyme from degradation by proteases [9]. Inactivation constants for the conjugates with chit-PEG and chit-PEI with a branched structure decreased four- and nine-fold, respectively. In the current work, we also found that branched PEI-PEG increased the resistance of RrA to trypsinolysis more than the other conjugates. PEI had almost no effect on the resistance of RrA to trypsinolysis, which may also be due to the destabilization (a disordered tertiary structure) of the enzyme. In addition, the charged end chains of PEI may, on the contrary, promote binding to the proteases, reducing the resistance to the trypsin action. Earlier, it was shown that covalent modification of EwA with chitosan copolymers allows for increasing the enzyme activity, increasing its thermal stability and resistance to trypsinolysis, and cytotoxicity [7; 8; 23]. The RrA modification with these copolymers also improved the same physicochemical parameters and had almost no effect on the secondary structure of the protein [9; 24]. However, it was found that chitosan-PEG increased the RrA activity no more than 1.5-fold. For EwA, such modification turned out to be much more effective: the enzyme activity increased 3-4-fold depending on the degree of PEGylation of the chitosan. The different effects of the chitosan copolymers can be explained by the fact that EwA and RrA differ in their structure and properties. These enzymes have only about 25% peptide sequence homology and differ from each other in molecular weight, particle charge in a neutral solution, hydrolysis activity, and affinity for L-asparagine and L-glutamine [25]. It was also shown in a previous article that the formation of the polyelectrolyte complexes of RrA with PEI increases the activity of the enzyme by about 30% [9]. In the case of EwA, PEI had the opposite effect, reducing the hydrolysis activity of L-asparagine by several times. It is evident that the studied polyelectrolytes have different effects on the properties of L-asparaginaes from different sources.