## 4 Discussion  
Extremophile microorganisms are a source of enzymes with good tolerances to extreme conditions [19]. Many keratinases from extremophiles have attractive properties, such as _Nocardiopsis_[20] and _Salicola Manasensis_[21], etc. _M. tuunensis_ WR-220 is a thermophilic microorganism with a growth temperature of 55-65 \({}^{\circ}\)C, and the keratinase in this bacterium also has thermal stability [11]. Many studies have demonstrated the presence of peptidase in the extreme _Deinococcus_ microorganisms [22], such as serine peptidase from _D. geothermalis_[23] and _Deinococcus_ sp. D7000 [24]. The degrading feather temperature of _Deinococcus fucus_[25] (feather-degrading bacterium) and _D. radiodurans_ R1 [22] was 30 \({}^{\circ}\)C, which is limited for industrial applications. In this study, DgeKer gene was found  
Figure 5: Visual observation of disintegration of intact feathers by the DgeKer and DgeKer-C. Tris-HCl is a control without keratinase. DTT is a reducing chemical reagent dithiothreitol, which was added to feather degradation system to reduce feather disulfide bonds. (**a**), The feather degradation in the absence of DTT. \({}^{\prime\prime}-\) DTT\({}^{\prime\prime}\) represents the system without DTT. (**b**), The feather degradation in the presence of DTT. \({}^{\prime\prime}+\)DTT\({}^{\prime\prime}\) represents the system with 5 mmol/L DTT. The reaction systems were incubated at 70 \({}^{\circ}\)C with shaking. in \(D\). _geothermalis_, which grow at 50 \({}^{\circ}\)C [14]. According to Figure 2, DgeKer has high homology with peptidases from _Calidithermus_ and _Thermus_. These bacteria were isolated from extreme environments such as hot springs and are known for their thermal stability. _Thermus_ and _Calidithermus_ bacteria generally grow at medium and high temperature, and their keratinocytes might have high temperature range and temperature stability, such as MtaKer [11] and aqulysin I [26]. The amylosucrase from _Calidithermus timidus_ has high thermostability with a half-life of 1.09 h at 70 \({}^{\circ}\)C [27]. As mentioned above, DgeKer has a long C-terminal extension, as with aqulysin-I [26] and VPR from _Vibrio_ sp. PA44 [28]. As [PERSON], etc., reported, C-terminal extension only exists in some keratinocytes from _Thermus_, _Thermococcus_, _Acinetobacter_, _Vibrio_ and _Deinococcus_, which are generally mesophilic and thermophilic bacteria or extreme [11] bacteria. Therefore, it is valuable to find high temperature-resistant keratinase from \(D\). _geothermalis_. For industrial needs, it is necessary to overexpress keratinase in _E. coli_ and other cells [7,29]. In general, keratinase consists of signal peptide, N-terminal pro-peptide and catalytic domains, and some also carry an extra C-terminal extension [11,30]. The folding of keratinase requires the N-pro-peptide (intramolecular chaperones) to facilitate correct folding and regulate the activation of catalytic domains [31]. Many studies indicate that the N-pro-peptide function as an inhibitor of subtilisin to prevent premature protease activity. The peptide bonds between the intramolecular chaperones and the protease domain are automatically hydrolyzed after the protein folding is complete. However, intramolecular chaperones still bind to proteases in the form of inhibitory complexes [31]. As shown in Figure 3, DgeKer and DgeKer-C could autonomously cleave the N-terminal propeptide to become mature keratinase in _E. coli_ which was consistent with previous [11,31] reports, and the minor bands below the main brand might result from processing or autohydrolysis of the protease. However, the N-terminal cleavage process is currently unclear. Studies have shown that the cleavage process is related to the amino acid preference of the cleavage site [32]. The C-terminal extension is usually located at the C-terminal of some secreted bacterial peptidase, some proteins from metalloproteinase families M4, M9 and M28 and serine protease family S8 were found to contain the C-terminal extension.