title: Development of a keratinase activity assay using recombinant chicken feather keratin substrates.
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.
authors.affiliation: School of Applied Biosciences, Kyungpook National University, Daegu, South Korea.
authors.forename: Hyeon-Su
authors.initials: HS
authors.lastname: Jin
authors.affiliation: School of Applied Biosciences, Kyungpook National University, Daegu, South Korea.
authors.forename: Seon Yeong
authors.initials: SY
authors.lastname: Park
authors.affiliation: School of Applied Biosciences, Kyungpook National University, Daegu, South Korea.
authors.forename: Kyungmin
authors.initials: K
authors.lastname: Kim
authors.affiliation: School of Applied Biosciences, Kyungpook National University, Daegu, South Korea.
authors.forename: Yong-Jik
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: Department of Cosmetic Science & Technology, Seowon University, Cheongju, South Korea.
authors.forename: Gae-Won
authors.initials: GW
authors.lastname: Nam
authors.affiliation: School of Food Science and Biotechnology, Kyungpook National University, Daegu, South Korea.
authors.forename: Nam Joo
authors.initials: NJ
authors.lastname: Kang
authors.affiliation: School of Applied Biosciences, Kyungpook National University, Daegu, South Korea.
authors.forename: Dong-Woo
authors.initials: DW
authors.lastname: Lee
mesh_terms: D000818:Animals; D002645:Chickens; D005241:Feathers / Q000378:metabolism*; D007633:Keratins / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000032:analysis*; D053719:Tandem Mass Spectrometry
chemical_list: D007633:Keratins; D010447:Peptide Hydrolases; C035855:keratinase