## 3 Results and Discussion
### Antimicrobial Activity of AMP-ELR and AMP-SELP Films  
The antimicrobial activity of Hep-A200, Syn-A200, Hep-SELP, Syn-SELP, CM4-SELP and BMAP18-SELP films (produced from aqueous or formic acid solutions) was evaluated against _S. aureus_ ATCC6538 (Gram-positive) and _E. coli_ HB101 (Gram-negative) by direct contact after 120 min. Analysis of antibacterial assays reveals that antimicrobial performance is highly dependent on the structural (ELR or SELP) and functional (AMP) modules and less dependent on the solvent used for film production, although films produced with formic acid display slightly higher average % kill values. Overall, the AMP-A200 films demonstrated better antimicrobial performance than the AMP-SELP films, independently of the solvent used (Figure 3). Syn-SELP and Hep-SELP films produced using formic acid as solvent show average lethality percentage (% kill) values for _E. coli_ near 17%, whereas Syn-A200 and Hep-A200 films produced using the same solvent demonstrated average values of approximately 96% and 88%, respectively (Figure 3a, Table 2). This same trend is observed for CM4-SELP and BMAP18-SELP compared to reference values obtained in previous works [8,12] (Table 2). The SELP-based films produced from formic acid present values of % kill around 35% and 60% for CM4-SELP and BMAP18-SELP, respectively, whereas this value increases to 86% and 100% using A200 as a structural backbone (Table 2). The distinct antimicrobial performance observed for AMP-A200 and AMP-SELP films is possibly due to the different conformations adopted by the structural modules (SELP and ELR) in the protein polymers. ELRs adopt a highly hydrated structure with conformational \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Sample**} & \multicolumn{2}{c}{**Water**} & \multicolumn{2}{c}{**Formic Acid**} \\ \cline{2-5}  & **Untreated** & **MetOH-Treated** & **Untreated** & **MetOH-Treated** \\ \hline Hep-SELP & 45.62\% & 54.91\% & 44.90\% & 48.48\% \\ Syn-SELP & 38.40\% & 54.56\% & 48.70\% & 51.86\% \\ CM4-SELP & 40.86\% & 49.75\% & 44.42\% & 55.68\% \\ BMAP18-SELP & 37.62\% & 55.02\% & 45.95\% & 52.7\% \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Beta-structure content of AMP-SELP films, produced from water and formic acid solutions, after and before methanol (MetOH) treatment. Results presented as fractional distribution of total \(\beta\)-structure content determined by second derivative and Gaussian curve fitting. Figure 3: Antimicrobial activity of AMP-SELP and AMP-A200 hybrid materials assessed by direct contact with the surface of the films against **(a)**_Escherichia coli_ and **(b)**_Staphylococcus aureus_. Cell suspensions inoculated in empty wells were used as reference for 100% survival. Error bars represent means \(\pm\) SD (ns, non-significant; \({}^{\star}\)\(p\)\(\leq\) 0.05; \({}^{\star\star}\)\(p\)\(\leq\) 0.01; \({}^{\star\star}\)\(p\)\(\leq\) 0.001; \({}^{\star\star\star}\)\(p\)\(\leq\) 0.0001). Experiments were performed in triplicate in independent days with three replicates each (see Tables S4 and S5 for CFU enumeration). No statistical significance was found between films prepared using water or formic acid as solvents. flexibility [68, 69]. This flexibility favors the ability of the AMPs to interact with bacteria. In contrast, the methanol-treated SELPs adopt a more rigid crystalline structure, characterized by antiparallel hydrogen-bonded \(\beta\)-sheet structures (silk II-like) [5, 70, 71]. As the AMPs are fused to the N-terminal of the SELP, that is, to the silk blocks, the conformational flexibility of the AMPs is therefore restricted, limiting the ability of AMP moieties to contact with bacteria. Comparing the mean values of lethality percentage (% kill) against the tested bacteria, best results were generally obtained against _E. coli_ than against _S. aureus_ (Figure 3). The antibacterial assays against _S. aureus_ demonstrated high standard deviation (SD) values, which are a consequence of the ability of _S.