BACKGROUND: Keratin, the main component of chicken feather, is the third most abundant material after cellulose and chitin. Keratin can be converted into high-value compounds and is considered a potential high-quality protein supplement; However, its recalcitrance makes its breakdown a challenge, and the mechanisms of action of keratinolytic proteases-mediated keratinous substrates degradation are not yet fully elucidated. Bacillus sp. CN2, having many protease-coding genes, is a dominant species in keratin-rich materials environments. To explore the degradation patterns of feather keratin, in this study, we investigated the characteristics of feather degradation by strain CN2 based on the functional-degradomics technology.
RESULTS: Bacillus sp. CN2 showed strong feather keratin degradation activities, which could degrade native feathers efficiently resulting in 86.70% weight loss in 24 h, along with the production of 195.05 ± 6.65 U/mL keratinases at 48 h, and the release of 0.40 mg/mL soluble proteins at 60 h. The extracellular protease consortium had wide substrate specificity and exhibited excellent biodegradability toward soluble and insoluble proteins. Importantly, analysis of the extracellular proteome revealed the presence of a highly-efficient keratin degradation system. Firstly, T3 γ-glutamyltransferase provides a reductive force to break the dense disulfide bond structure of keratin. Then S8B serine endopeptidases first hydrolyze keratin to expose more cleavage sites. Finally, keratin is degraded into small peptides under the synergistic action of proteases such as M4, S8C, and S8A. Consistent with this, high-performance liquid chromatography (HPLC) and amino acid analysis showed that the feather keratin hydrolysate contained a large number of soluble peptides and essential amino acids.
CONCLUSIONS: The specific expression of γ-glutamyltransferase and co-secretion of endopeptidase and exopeptidase by the Bacillus sp. CN2 play an important role in feather keratin degradation. This insight increases our understanding of the keratinous substrate degradation and may inspire the design of the optimal enzyme cocktails for more efficient exploration of protein resources in industrial applications.

The study aimed to propose an efficient and eco-friendly strategy to improve the utilization of feather waste and converting it into high-valued antimicrobial products. Under the synergistic effect of instant catapult steam explosion (ICSE) (1.5 MPa-120 s), over 90% of chicken feather powder (CFP) was degraded into soluble peptides via keratinolysis within 3 h, about 90% of which were smaller than 3 kDa, indicating an overwhelming advantage than general proteolysis. Importantly, the keratinolysis hydrolysate of CFP was able to inhibit E. coli growth, among which the fraction < 3 kDa exhibited highest antimicrobial activity with a minimal inhibitory concentration of 30 mg/mL. Compared to other fractions, the fraction < 3 kDa contained higher content of hydrophobic amino acids (364.11 mg/g), in which about 79% of peptides had more than 60% hydrophobic ratio, potentially contributing to its antimicrobial activity. ICSE-keratinolysis process holds potential in reducing both protein resource waste and environmental pollution by valorizing feathers into antimicrobial product.

Four new Keratinophyton species (Ascomycota, Pezizomycotina, Onygenales), K. gollerae, K. lemmensii, K. straussii, and K. wagneri, isolated from soil samples originating from Europe (Austria, Italy, and Slovakia) are described and illustrated. The new taxa are well supported by phylogenetic analysis of the internal transcribed spacer region (ITS) region, the combined data analysis of ITS and the nuclear large subunit (LSU) rDNA, and their phenotype. Based on ITS phylogeny, within the Keratinophyton clade, K. lemmensii is clustered with K. durum, K. hubeiense, K. submersum, and K. siglerae, while K. gollerae, K. straussii and K. wagneri are resolved in a separate terminal cluster. All four new species can be well distinguished from other species in the genus based on phenotype characteristics alone. Ten new combinations are proposed for Chrysosporium species which are resolved in the monophyletic Keratinophyton clade. A new key to the recognized species is provided herein.

Cysteine dioxygenase (CDO) is involved in regulation of intracellular cysteine levels by catabolising the cysteine to sulphite and sulphate. In keratinolytic fungi, sulphite is actively excreted to reduce disulphide bridges in keratin before its enzymatic degradation. The pathogenicity role of CDO was confirmed in cysteine-hypersensitive and growth-defective ΔCdo mutant of Arthroderma benhamiae on hair and nails. We analysed the CDO expression regulation in T. mentagrophytes (anamorph of A. benhamiae) mycelia by determining the Cdo mRNA and CDO protein levels and by analysing the proportion of two molecular forms of CDO in response to l-cystine exposure. Cdo mRNA levels in mycelia lysates were detected by reverse-transcription real-time polymerase chain reaction and CDO protein by western blot using mouse CDO-specific hyperimmune serum. The Cdo mRNA level increased gradually 2.5-4.5 h after exposure of the mycelium to l-cystine. The CDO protein, detected as two bands of different mobility, appeared earlier in comparison to mRNA (1 h) and culminated after 24 h. More mobile form prevailed after 4.5 h. The comparison of the dynamics in the Cdo mRNA and CDO protein levels indicates that T. mentagrophytes responds to l-cystine by increased transcription and apparently decreased degradation of the CDO and by changing towards higher mobility molecular form, similar to previous reports describing mammalian analogue.