## 4 Discussion
A phylogenetic tree was constructed based on the C-terminal extended sequence of DgeKer and those of other keratinases. As shown in Figure S1, the C-terminal extension of DgeKer was homologous with those of keratinases from _Deinococcus_. Obviously, the C-terminal extension was different from PPC (bacterial pre-peptidase C-terminal domain) and PKD (polycystic kidney disease). The modeled structure of C-terminal extension of DgeKer was also built (Figure S2) based on the structure of a keratinase (ProN-TK-SP) from _Thermococcus kodakaranensis_ (3D structure data in Protein Data Bank, PDB ID: 3 AFG) [33] which exhibited about 33% sequence identity with C-terminal extension of DgeKer in the PDB database. The special structure of C-terminal domain of DgeKer was characterized as \(\beta\)-jelly roll with six \(\beta\)-strands and loops (Figure S2). According to previous studies, PPC and PKD domains may inhibit keratinase activity [34], enhance heat resistance [10,33], and bind insoluble substrates [35]. However, there are also studies that have the opposite outcome [15]. In general, at present, the mechanism of C-terminal extension is still unclear [36]. Therefore, we constructed the C-terminal extension deletion mutant protein DgeKer-C to explore the effect of C-terminal extension on DgeKer thermal stability and pH stability. As shown in Figure 4, DgeKer and DgeKer-C exhibited maximal activity at 70 \({}^{\circ}\)C, which was higher than most of the other keratinases [18,32], such as KerA-derived (50 \({}^{\circ}\)C) [30] and MtaKer (65 \({}^{\circ}\)C) [11]. In terms of thermostability, DgeKer-C performed better than DgeKer (Figure 4). As incubation time extended, the residual enzyme activity of DgeKer-C was higher than DgeKer. DgeKer-C was stable with half-life (t\({}_{1/2}\)) of 169.10 min, which was 6.4-fold more than KerA. In general, DgeKer and DgeKer-C have high optimal temperature and thermal stability. Feather degradation fermentation is a continuous process and the temperature stability of keratinase is very important for feather degradation. These properties give DgeKer and DgeKer-C greater industrial application potential. The optimal pH of DgeKer and DgeKer-C was 9-10, which was higher than that of most keratinases, such as KerA (pH 8). In the pH range of 3-7, DgeKer and DgeKer-C were greatly affected by pH, and in the pH range of 7-11, neither of them was significantly affected by pH, indicating that DgeKer and DgeKer-C were stable in the pH range of 7-Studies have shown that the feather degradation process involves sulfitolysis and deamination [1, 37], which leads to an elevated pH in the fermentation environment [38, 39, 40]. The pH of the feather degradation system increased to 8.8 [41]. Therefore, reaction pH and pH stability are important factors in industrial applications. The activity and stability of DgeKer under alkaline conditions give this keratinase a certain potential for industrial applications. DgeKer and DgeKer-C retained high activity over several metal ions, surfactants, reducing agents and organic solvents. In generally, keratinase is stimulated by some metal ions (Ca\({}^{2+}\) and Mg\({}^{2+}\)) [3, 42, 43] which might maintain the active enzyme conformation stabilize the enzyme-substrate complex [38]. Mg\({}^{2+}\) increased the enzymatic activity of DgeKer, which is consistent with previous studies. Ca\({}^{2+}\) had no positive effect on the enzymatic activity of DgeKer and DgeKer-C, although two hypothetical Ca\({}^{2+}\) ion binding sites were predicted in their sequence by NCBI. In general, both DgeKer and DgeKer-C had good metal ion tolerance. At present, there are several degradation hypotheses about the mechanism of microbial degradation of keratin, but the key step is the degradation of disulfide bonds, so that keratinase can better hydrolyze keratin. Reducing agent DTT can improve the activity of some keratinases [30]. It might be the reducing agent reacting with the substrate rather than keratinases [38].