Poultry feathers consist mainly of the protein keratin, which is rich in β-pleated sheets and consequently resistant to proteolysis. Although many keratinases have been identified, the reasons for their substrate specificity towards β-keratin remain unclear due to difficulties in preparing a soluble feather keratin substrate for use in activity assays. In the present study, we overexpressed Gallus gallus chromosomes 2 and 27 β-keratin-encoding genes in Escherichia coli, purified denatured recombinant proteins by Ni2+ affinity chromatography, and refolded by stepwise dialysis to yield soluble keratins. To assess the keratinolytic activity, we compared the proteolytic activity of crude extracts from the feather-degrading bacterium Fervidobacterium islandicum AW-1 with proteinase K, trypsin, and papain using purified recombinant keratin and casein as substrates. All tested proteases showed strong proteolytic activities for casein, whereas only F. islandicum AW-1 crude extracts and proteinase K exhibited pronounced keratinolytic activity for the recombinant keratin. Moreover, LC-MS/MS analysis of keratin hydrolysates allowed us to predict the P1 sites of keratinolytic enzymes in the F. islandicum AW-1 extracts, thereby qualifying and quantifying the extent of keratinolysis. The soluble keratin-based assay has clear therapeutic and industrial potential for the development of a high-throughput screening system for proteases hydrolyzing disease-related protein aggregates, as well as mechanically resilient keratin-based polymers.
Bibliographical noteFunding Information:
This work was supported by the national coordinating centre for global cosmetics R&D (NCR) grant HN14C0097 to N.J. Kang, funded by the Ministry of Health and Welfare, by the National Research Foundation of Korea (NRF) grant 2014R1A2A2A01006765 to D.W. Lee, funded by the Korean government (MSIP), and by the Strategic Initiative for Microbiomes in Agriculture and Food grant 916006-2 to D.W. Lee, funded by the Ministry of Agriculture, Food and Rural Affairs. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)
- Agricultural and Biological Sciences(all)