## 3 Results and Discussion
### Cytotoxicity of the PsIsG\({}_{h}\)  
To enable the use of recombinant enzymes in biomedicine, among other things, as elements of wound-dressing materials, it is important to assess their cytotoxicity against eukaryotic cells. The cytotoxicity of PeAl\({}_{\text{h}}\) was investigated in our previous research, and we concluded that there is no cytotoxicity effect of the enzyme on murine fibroblast (L929) cultures [26].Therefore, the PsIsG\({}_{\text{h}}\) enzyme obtained in this study was also tested to determine its potential cytotoxicity towards murine fibroblast (L929) cultures. Six different enzyme concentrations (5.0, 10.0, 25.0, 50.0, 100.0, and 250.0 \(\upmu\)g/mL) were used for 24 h and 48 h observations of cell viability and morphology. After 24 h, no reduction in cell viability (Figure 5a) and cell morphology (Figure 6) was observed. After 48 h, the cell morphology  
Figure 4: The thermal stability profile of soluble and freeze-dried forms of PeAl\({}_{\text{h}}\) and PsIsG\({}_{\text{h}}\) stored at 4 \({}^{\circ}\)C on day 0 (**a**,**c**) and after 30 days of storage (**b**,**d**). for the experimental cultures did not demonstrate changes in comparison to the control culture. In the case of cell viability, a \(\sim\)29% reduction was found for the 250.0 \(\mathrm{\SIUnitSymbolMicro g}\)/mL enzyme concentration (Figure 5b). These results are consistent with earlier studies on the cytotoxicity of \(\mathrm{PelA_{h}}\) and \(\mathrm{PslG_{h}}\) against eukaryotic cells from line IMR-90 of human lung fibroblasts and red blood cells [27; 28]. In Vitro Release Study of \(\mathrm{PelA_{h}}\) and \(\mathrm{PslG_{h}}\) from Pristine and LPArP-Modified BC  
The immobilization of proteins on different kinds of surfaces is used in many areas of industry, including the biomedical and pharmaceutical industries in drug delivery systems [49]. To evaluate the \(\mathrm{PelA_{h}}\) and \(\mathrm{PslG_{h}}\) from the BC and LPArP-modified BC, the release behavior was monitored in 50 mM of phosphate buffer at pH 7.4. The release profiles of \(\mathrm{PelA_{h}}\) and \(\mathrm{PslG_{h}}\) are shown in Figure 8. The release profiles of \(\mathrm{PelA_{h}}\) from the BC and Ar_BC membranes did not differ and were characterized by sustained diffusion. The same release profile was observed for the \(\mathrm{PslG_{h}}\) from nonmodified BC, while the \(\mathrm{PslG_{h}}\) released from Ar_BC was characterized by an initial burst release for 2 min. In both cases, higher amounts of enzyme were retained on the BC matrix, and the maximum releases of \(\mathrm{PslG_{h}}\) were 58% and 63% after 10 min for BC and Ar_BC, respectively. For \(\mathrm{PelA_{h}}\), the % of release from the carrier was faster compared to \(\mathrm{PslG_{h}}\), and the maximum release at the same time was around 80% for both unmodified and modified BC. The observed \(\mathrm{PelA_{h}}\) release profile did not differ between the unmodified and LPArP-modified BC, but it showed a significantly faster release of this protein compared to \(\mathrm{PslG_{h}}\). The charge distributions on the molecular surfaces of the analyzed enzymes at pH 7.4 showed the occurrence of large negatively charged patches. The calculated charges of the protein molecules did not differ significantly between \(\mathrm{PelA_{h}}\) and \(\mathrm{PslG_{h}}\) and were \(-8.24\) and \(-6.94\), respectively (Figure 7). In contrast to \(\mathrm{PelA_{h}}\) the structure of \(\mathrm{PslG_{h}}\) consist of large catalytic N- terminal domain and significantly smaller C-terminal domain recognized as possible carbohydrate binding domain (Figure 7). Occurrence of this domain in \(\mathrm{PslG}\) structure was reported as reason of higher binding to dextran and may be reason of different releasing profile. Furthermore, the differences in release profile of \(\mathrm{PslG_{h}}\) from native and LPArP-modified BC suggest that the CBM occur in \(\mathrm{PslG_{h}}\) molecular structure belongs to Type B CBMs, which has affinity to amorphous form of BC.