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.

Identification of keratinases from Fervidobacterium islandicum AW‐1 using dynamic gene expression profiling

“…When added to reaction mixtures containing WCEs derived from F. islandicum AW-1 cells, these enzymes accelerated and/or enhanced the degradation of native chicken feathers, resulting in the release of free amino acids from feather keratin (Fig. 4), which was in accordance with the previous results (Lee et al, 2015a;Jin et al, 2017). It could be speculated that peptide fragments produced from feathers by activated cytoplasmic endo-and exoproteases might act as transcription triggering factors or signalling molecules via peptide transporters to promote feather decomposition and utilization.…”

“…In the light of this, the extremely thermophilic bacterium F. islandicum AW‐1, which can degrade chicken feathers completely within 48 h at 70ºC (Nam et al , ), would be an excellent model system to investigate the mechanism of keratinolysis under anaerobic conditions, because this bacterium belongs to the order of Thermotogales, which is the most ancient form of bacteria. Intriguingly, our previous LC‐MS/MS analysis revealed that unlike other general serine proteases such as trypsin and proteinase K, F. islandicum AW‐1 proteases exhibited a distinct substrate specificity towards feather keratin (Jin et al , ). Unlike aerobic superficial dermatophytes such as M. canis and T. rubrum that secrete the highly conserved set of S3 and M3 endoproteases (Monod, ; Sriranganadane et al , ), anaerobic F. islandicum AW‐1 appears to degrade native feathers via direct cellular adhesion to the substrate by expressing membrane‐bound proteases (Nam et al , ).…”

“…C. Venn diagram of the genes differentially expressed in bacteria grown on feathers compared with those in bacteria grown on glucose. in vitro feather degradation activity described previously (Lee et al, 2015a;Jin et al, 2017), purified recombinant enzymes were added to crude F. islandicum AW-1 extract (AWCE) from glucose-grown cells containing basal levels of proteases, which was then incubated with 0.2% (w/v) native feathers in the presence of 10 mM DTT under anaerobic conditions at 72°C ( Fig. 4 and Fig.…”

“…Unlike aerobic superficial dermatophytes such as M. canis and T. rubrum that secrete the highly conserved set of S3 and M3 endoproteases (Monod, ; Sriranganadane et al , ), anaerobic F. islandicum AW‐1 appears to degrade native feathers via direct cellular adhesion to the substrate by expressing membrane‐bound proteases (Nam et al , ). Indeed, this anaerobe did not have any significant extracellular proteases, but whole‐cell extracts including membrane compartment exhibited strong keratinolytic activity (Jin et al , ), implying that F. islandicum AW‐1 might use an evolutionarily primordial and a different version of keratinolytic machineries under anaerobic conditions. Moreover, despite its minimal genome (Lee et al , ), it has been known as the fastest keratin degrader among the microbial strains reported so far (Gupta and Ramnani, ; Brandelli et al , ).…”

Feather degradation by keratinolytic bacteria and biofertilizing potential for sustainable agricultural production

“…[45] and Streptomyces pactum [49]. The initial step in keratin degradation catalyze by disulfide reductase has also been reported by other researchers [50][51][52][53]. Reduction of disulfide bonds changes the conformation of amino acids in β-sheet of keratin rendering different hydrolytic sites for proteolytic attack by keratinase [16,54].…”