Abstract
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.
Highlights
Keratin is an insoluble, sulfur-containing fibrous protein and the main constituent of skin, hair, nails, hooves, horns, scales, claws, and teeth
Given the absence of functional annotation of keratinases in F. islandicum AW-1, we first characterized the effect of temperature and pH on proteolytic activity using a crude extract (AWCE)
These results clearly indicated a robust proteolytic activity in AW-1 crude extract (AWCE) that was highly thermostable, optimal around neutral pH, and resilient to detergents
Summary
Sulfur-containing fibrous protein and the main constituent of skin, hair, nails, hooves, horns, scales, claws, and teeth. It is synthesized by keratinocytes and is resistant to degradation by general proteases [1]. Based on their sulfur content, keratins can be divided into soft keratin (
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