## 3 Results and Discussion
### Cytotoxicity of the PsIsG\({}_{h}\)
Figure 5: \(\mathrm{L}\)92 cell viability after 24 h (**a**) and 48 h (**b**) incubation with the recombinant \(\mathrm{PslG_{h}}\). To explain the enzymes' release mechanisms from the BC and Ar_BC, pharmacokinetic models were used, including zero-order, first-order, Higuchi, and Korsmeyer-Peppas models. The fitting results are summarized in Table 3. For the PsI\({}_{\text{G}}\)h loaded on the nonmodified BC, the release of the protein was fitted to the Higuchi and Korsmeyer-Peppas models for both types of BC, which indicated that the release of PsI\({}_{\text{G}}\)h is controlled by diffusion. The n exponents of the Korsmayer-Peppas model were around 0.25 and 0.22 for the LPArP-modified and native BC, respectively. This demonstrated that PsI\({}_{\text{G}}\)h was released through Fickan diffusion. In the case of PeI\({}_{\text{A}}\)h, it was observed that the release profile for the Ar_BC fit well with the first order, with a correlation coefficient of R\({}^{2}\) equal to 0.98, which indicates that the PeI\({}_{\text{A}}\)h release is dependent on the differential concentration [54]. Moreover, the PeI\({}_{\text{A}}\)h release mechanism also fit the Higuchi and Korsmayer-Peppas models, with a correlation coefficient of R\({}^{2}\)\(>\) 0.9. The value of exponent n for the PeI\({}_{\text{A}}\)h release was \(>\)0.5 and followed non-Fickan diffusion [54, 55]. Figure 6: Morphology of L92 cells of 24 h (**a**) and 48 h controls (**c**) and exposed to the recombinant PsI\({}_{\text{G}}\)h (250 \(\upmu\)g/mL) for 24 h (**b**) and 48 h (**d**). Figure 8: Experimental in vitro PeAl\({}_{\text{h}}\) (**a**) and PslG\({}_{\text{h}}\) (**b**) release profiles from BC and Ar_BC (in 50 mM phosphate buffer at pH 7.4. Figure 7: The 3D crystal structure models of PeAl\({}_{\text{h}}\) (PDB:5 TCB) and PslG\({}_{\text{h}}\) (PDB:5 BX9) proteins with highlighted elements of secondary structure and distribution of the electrostatic potential of their molecular surfaces. The electrostatic potential of the protein molecules’ molecular surfaces at pH 7.4 was computed according to predictions of the pKa value by the PROPKA server and the APBS solver via the PDB2 PQR server. The final analysis was performed with UCSF Chimera 1.17.1 rc package [52; 53]. Influence of the Immobilization of PelA\({}_{h}\) and PsI\({}_{\rm G}\)\({}_{h}\) on P. aeruginosa PAO-1 Biofilm Eradication  
The effect of PelA\({}_{h}\) and PsI\({}_{\rm G}\)\({}_{h}\) in soluble and immobilized forms on the ability of _P. aeruginosa_ PAO-1 to form biofilm was measured in a microplate model using crystal violet staining. The results for the biofilm inhibition assay are presented in Figure 9. For the soluble form of PsI\({}_{\rm G}\)\({}_{h}\), around 70% of the biofilm inhibition was observed when 2.5 and 1.25 \(\upmu\)g of enzyme were applied, while for 0.62 \(\upmu\)g, the inhibition was slightly lower, amounting to around 50% (Figure 9a). When PelA\({}_{h}\) in the same concentration was added to the culture medium, the biofilm inhibition rate was lower compared to PsI\({}_{\rm G}\)\({}_{h}\) and was around 50% for 2.5 and 1.25 \(\upmu\)g of enzyme and 30% for 0.62 \(\upmu\)g (Figure 9d). The composition of the _P. aeruginosa_ biofilm matrix is strain dependent. The tested strain of _P. aeruginosa_ PAO-1 produces mainly PsI as a dominant exopolysaccharideacharide of the biofilm matrix, which may be the reason for the more effective inhibition of biofilm formation after the use of the PsI\({}_{\rm G}\)\({}_{h}\) enzyme [56; 57]. In the case of the immobilized enzymes on native BC, it was observed that the biofilm inhibition rate was slightly lower compared to the soluble forms of PelA\({}_{\rm h}\) and PsI\({}_{\rm G}\)\({}_{h}\) due to lowering release rate of the proteins into the culture medium (Figure 9b,e). The biofilm inhibition rate for the PelA\({}_{\rm h}\) released from the Ar_BC (Figure 9f) was similar to the native enzyme released from the BC.