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

Abstract

Summary Keratin degradation is of great interest for converting agro‐industrial waste into bioactive peptides and is directly relevant for understanding the pathogenesis of superficial infections caused by dermatophytes. However, the mechanism of this process remains unclear. Here, we obtained the complete genome sequence of a feather‐degrading, extremely thermophilic bacterium, Fervidobacterium islandicum AW‐1 and performed bioinformatics‐based functional annotation. Reverse transcription PCR revealed that 57 putative protease‐encoding genes were differentially expressed in substrate‐dependent manners. Consequently, 16 candidate genes were highly expressed under starvation conditions, when keratin degradation begun. Subsequently, the dynamic expression profiles of these 16 selected genes in response to feathers, as determined via quantitative real‐time PCR, suggested that they included four metalloproteases and two peptidases including an ATP‐dependent serine protease, all of which might act as key players in feather decomposition. Furthermore, in vitro keratinolytic assays supported the notion that recombinant enzymes enhanced the decomposition of feathers in the presence of cell extracts. Therefore, our genome‐based systematic and dynamic expression profiling demonstrated that these identified metalloproteases together with two additional peptidases might be primarily associated with the decomposition of native feathers, suggesting that keratin degradation can be achieved via non‐canonical catalysis of several membrane‐associated metalloproteases in cooperation with cytosolic proteases.

Microbial enzymes catalyzing keratin degradation: Classification, structure, function

“…Investigations of the keratinases of the keratinolytic non-pathogenic fungus O. corvina suggest that a blend of fungal keratinases – namely an endoprotease (S8), exoprotease (M28), and an oligopeptidase (M3) – act synergistically to break down pig bristle keratin ( Huang et al, 2015 ). Moreover, the enzymology of feather degradation by F. islandicum AW-1, which has recently been reported in detail ( Kang et al, 2020 ), points out that synergies among different types of proteases help the degradation. Hence, after sulfitolysis of feathers, the CPBP family (M48 protease family) intramembrane metalloprotease and five additional membrane proteases (of family S41, S54, S8, M24, A24, respectively) act as key keratinases to catalyze the feather degradation.…”

“…In addition to an S16 keratinolytic protease (see Section 4.1.3 ), the F. islandicum produce one M16 protease (accession number: AMW32060 ). Addition of this enzyme on top of a F. islandicum extract also enhanced the feather degradation rate in vitro (more than 1.5 fold) compared with the crude F. islandicum extract only ( Kang et al, 2020 ) but the keratinolytic activity of this protease alone is unknown. Furthermore, it is worth noting that not all family M16 proteases act by endo -attack, for instance, subfamily M16A contains oligopeptidases denoted as insulysin and nardilysin ( Rawlings and Barrett, 2013b ).…”

“…Unlike the peptidases in the S1 and S8 family, the active site of members in family S16 is a Ser-Lys catalytic dyad ( Rawlings and Barrett, 2013a ). One protease in the S16 family (derived from Fervidobacterium islandicum , accession number: AMW33508 ) has recently been confirmed to be a keratinolytic protease ( Kang et al, 2020 ). The enzymatic rates free amino acids release from chicken feather with addition of purified S16 protease is around 1.3-fold higher than those of a crude F. islandicum extract.…”

“…The M38 and M55 keratinolytic proteases, with accession number AMW33776 and AMW33601 , respectively, are also derived from F. islandicum . Both of the purified M38 and M55 proteases improved the free amino acid release rate by comparison of the rate of F. islandicum extract only ( Kang et al, 2020 ). During keratin degradation by F. islandicum , the M38 protease is presumably responsible for regulating proteolysis, while M55 protease might be activated during starvation conditions and involved in controlling the cell's flow of peptides as degradation products ( Kang et al, 2020 ).…”

“…the redox half reaction) ( Burmester et al, 2011 ; Descamps et al, 2002 ). Regarding the process of keratin degradation by keratinolytic organisms, the reducing agent sulfite or disulfide reductase may be secreted from the organism and may be involved in cleavage of disulfide bonds in the keratinous biomass ( Grumbt et al, 2013 ; Kang et al, 2020 ). Some organisms cannot secrete disulfide reductase or sulfite (e.g.…”