# Molecular Identification of Keratinase DogkerA from Deinococcus gobiensis for Feather Degradation
## 4 Discussion  
The extremophiles are an important source for the industrial enzymes with excellent properties [24]. In this study, the keratinase DgoKerA was identified by comparative genomic analysis from _D. gobiensis_ I-0 from the Gobi desert. The phylogenetic tree showed that DgoKerA is consistent with the evolutionary origin of _Deinococcus_ keratinase and is closely  
Figure 6: Comparison of intact feather degradation capacities of DgoKerA and mutants. Tris-HCl is a control without keratinase. The reaction systems were incubated at 60 \({}^{\circ}\)C with shaking. related to the thermotolerant bacteria _Calidithermus_ and _Thermus_ keratinase, suggesting that DgoKerA may have better thermal stability. Sequence alignment shows that DgoKerA, like the classical keratinase BliKerA, contains a signal peptide, an N-terminal propeptide and a mature region. The N-propeptide of keratinase functions as an intramolecular chaperone to facilitate the folding of keratinase, and is cleaved off by mature keratinase in order to function [9, 25, 26]. Using the _E. coli_ expression system, DgoKerA was successfully expressed and purified. the molecular weight of the purified DgoKerA was consistent with the predicted size of the protein after cleavage of the N-propeptide, indicating that DgoKerA was correctly folded and present as a mature enzyme in _E. coli_, as with other exogenously expressed keratinases [27, 28]. The thermal stability of keratinase is important for feather degradation [29]. DgoKerA showed optimal enzymatic activity at 60 \({}^{\circ}\)C, and the higher temperature adaptation of this enzyme may be related to the high temperature environment of the Gobi where it originated from. It was shown that the half-life (t1/2) of DgoKerA at 60 \({}^{\circ}\)C is 1.76 folds higher than that of BliKerA (46.52 min vs. 26.46 min). The sequence analysis showed that the DgoKerA mature region contains four cysteine residues. The homology modeling results using MtaKerA (PDB ID: 5 wsl.1) as a template showed that two intramolecular disulfide bonds, Cys200-Cys232 and Cys297-Cys330, were formed in the structure of DgoKerA, which enhanced the keratinase thermal stability. An amount of 5 mmol/L K\({}^{+}\) and Li\({}^{+}\) increased the enzymatic activity of DgoKerA, while 5 mmol/L Mg\({}^{2+}\) and Co\({}^{2+}\) also promoted the degradation of soluble keratin by DgoKerA. In general, certain metal ions can stabilize the conformation of the enzyme-substrate complex, thereby enhancing keratinase activity [30, 31, 32, 33]. At present, most reported keratinases are alkaline and neutral proteases with an optimum pH value of 7.5-9.0. DgoKerA showed stable activity at pH 5-9. The residual activity of DgoKerA was 88% with incubated at pH 6 for 60 min and was 65% when incubated at pH 9 for 60 min. Extracellular enzymes encoded by microbes need to be evolved to survive in the environment [7]. Compared with BliKerA isolated from a poultry waste digester, DgoKerA, which was cloned from a high-temperature and arid environment, was more sensitive to pH changes. The results of the study also showed that DgoKerA had a good tolerance to chemical reagents and the residual enzyme activity of DgoKerA remained above 90% after Tween 80, Tween 20 and DMSO treatment for 1 h. Non-specific binding between the ionic surfactant SDS and the protein inhibits the activity of DgoKerA, probably due to complex formation and protein defolding [34]. Reducing agents protect the free sulfhydryl groups on proteins from oxidation, thus avoiding aggregation or denaturation of proteins [35]. However, the reductant \(\beta\)-mercaptoethanol-ional significantly inhibited the activity of DgoKerA on soluble keratin, with only 6.73% residual enzyme activity. To test the ability of the keratinase DgoKerA to degrade feathers, a final concentration of 5 mmol/L DTT was added to the enzyme-feather mixture (0.03 mg purified enzyme, 1.5 mL) in the present work.