## 4 Discussion  
We proposed the modification of L-asparaginases with polycations as a promising method to improve their biopharmaceutical properties. From the results obtained, we can see that polyamines affect the activity, structure, and stability of the enzyme. With HPLC, we were able to see that RrA is present in the solution in two different forms, dimeric and monomeric (Figure 2). It was previously known that L-asparaginases of the mesophilic bacteria _Escherichia coli_ and _Erwinia carotorova_ could exist in dimeric and tetrameric forms [20]. In this case, the active site of L-asparaginase is located at the junction of two subunits, so the monomer has no catalytic activity. Figure 2 shows that the covalent modification of RrA can stabilize the tetrameric form. Spermic probably covalently binds the protein oligomers together and allows for a fully tetrameric form of RrA, which presumably exhibits greater activity than the dimer. Indeed, RrA-Spm has greater activity than native RrA. The PEI-PEG also shifts the equilibrium toward tetrameric, but to a lesser extent, because of the longer, branched chain of PEG-PEI. RrA-PEI-PEG activity also increases, which is probably due to the formation of a tetramer. In the case of the interaction with the dimer and tetramer of RrA, the most likely and energetically advantageous binding sites of spermine were identified using molecular docking. It is known that oligoamines, including spermine, can stabilize and increase the activity of enzymes when forming noncovalent complexes. For example, it was shown on chymotrypsin that polyelectrolyte complexes, with oligoamines, increase the activity of the enzyme and protect it from aggregation in the water-organic phase [18]. At the same time, the effect of the increased enzyme activity was observed only at high molar concentrations of the oligoamines, exceeding the enzyme concentration by \(\sim\)100 times. We also did not observe any particular effect of increased activity in the formation of the protein-polyelectrolyte complexes of spermine and spermidine with RrA at concentrations below 0.25 mM (data not shown). In addition, the large number of multipoint interactions with the amino groups can corrupt the native conformation and destabilize the enzyme. Spermine can also increase the activity and thermal stability of lysozyme [21]. The binding of the lysoszymes and alpha-globulin with spermine can be related to the interaction of oligoamine with asparagine and glutamine amino acid residues. In another study, the effect of spermine on the properties of myoglobin was evaluated [22], where it was also shown that spermine binds to the protein molecule and increases its stability. Using molecular docking, it was shown that the amino groups of spermine form hydrogen bonds with the carboxyl groups of the amino acid residues, Asp and Glu. In addition, spermine influenced the secondary structure of the protein by decreasing the percentage of alpha-helices and increasing the content of the beta structures. Electrostatic bonds, however, are highly dependent on the solvent and its ionic strength and may be unstable when the drug is administered intravenously. Therefore, obtaining the most active stable tetrameric form will provide a potential drug for use in therapy. This increased activity is also influenced by the local pH of the solution where the enzyme is located. The increase in the activity of RrA-PEI-PEG at a pH of 7.5 is related to the polycationic nature of the copolymer. PEI-PEG shifted the pH optimum of the enzyme to a more acidic region by changing the pH near the active center of the enzyme. For chitosan-PEG and other chitosan copolymers, the same results were previously shown. Spermine also increased the RrA activity by approximately 17% at a pH of 7.5-8.0. This is probably due to the stabilization of the tetrameric form of the enzyme, the ratio of which can change with changes in the pH of the solution.