Biochemical and structural characterization of a keratin-degrading M32 carboxypeptidase from Fervidobacterium islandicum AW-1

Lee, Yong Jik; Dhanasingh, Immanuel; Ahn, Jin-Soo; Jin, Hyeon Su; Choi, Jin Kyeong; Lee, Sung Haeng; Lee, Dong Woo

doi:10.1016/j.bbrc.2015.11.058

Cited by 23 publications

(23 citation statements)

References 24 publications

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“…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.…”

Section: Discussionsupporting

confidence: 91%

“…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.…”

Section: Dynamic Expression Profiling Of Protease-encoding Genes Idenmentioning

confidence: 99%

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

Kang

Jin

et al. 2019

Microbial Biotechnology

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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.

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Section: Discussionsupporting

confidence: 91%

Section: Dynamic Expression Profiling Of Protease-encoding Genes Idenmentioning

confidence: 99%

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

Kang

Jin

et al. 2019

Microbial Biotechnology

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“…The results show that amino acid production was 2-fold higher in FisCP-supplemented whole cell extractions than in whole cell extractions alone. This feature supports the notion that FisCP together with numerous other highly up-regulated proteases on keratin are mainly involved in the degradation of native chicken feathers ( Lee et al, 2015a ).…”

Section: Classification Of Enzymes Involved In Keratin Degradationsupporting

confidence: 87%

“…These enzymes catalyze hydrolysis of the peptide bond at the C-terminus of peptides and have been widely studied ( Lee et al, 1994 ). The protease AMW32563 (FisCP) plays a pivotal role in the decomposition of keratin when added to reaction mixtures containing whole cell extractions derived from F. islandicum AW-1 cells ( Lee et al, 2015a ). The results show that amino acid production was 2-fold higher in FisCP-supplemented whole cell extractions than in whole cell extractions alone.…”

Section: Classification Of Enzymes Involved In Keratin Degradationmentioning

confidence: 99%

Microbial enzymes catalyzing keratin degradation: Classification, structure, function

Qiu

Wilkens

Barrett

et al. 2020

Biotechnology Advances

143

103

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Keratin is an insoluble and protein-rich epidermal material found in e.g. feather, wool, hair. It is produced in substantial amounts as co-product from poultry processing plants and pig slaughterhouses. Keratin is packed by disulfide bonds and hydrogen bonds. Based on the secondary structure, keratin can be classified into α-keratin and β-keratin. Keratinases (EC 3.4.-.- peptide hydrolases) have major potential to degrade keratin for sustainable recycling of the protein and amino acids. Currently, the known keratinolytic enzymes belong to at least 14 different protease families: S1, S8, S9, S10, S16, M3, M4, M14, M16, M28, M32, M36, M38, M55 (MEROPS database). The various keratinolytic enzymes act via endo -attack (proteases in families S1, S8, S16, M4, M16, M36), exo -attack (proteases in families S9, S10, M14, M28, M38, M55) or by action only on oligopeptides (proteases in families M3, M32), respectively. Other enzymes, particularly disulfide reductases, also play a key role in keratin degradation as they catalyze the breakage of disulfide bonds for better keratinase catalysis. This review aims to contribute an overview of keratin biomass as an enzyme substrate and a systematic analysis of currently sequenced keratinolytic enzymes and their classification and reaction mechanisms. We also summarize and discuss keratinase assays, available keratinase structures and finally examine the available data on uses of keratinases in practical biorefinery protein upcycling applications.

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“…For instance, a novel type of M32 protease from F . islandicum AW-1 enhanced native feather degradation synergistically with crude extracts [ 24 ]. Nevertheless, there remain barriers to investigating the substrate specificity of keratinases and determining their kinetic parameters using insoluble feather keratins and keratin-like derivatives such as azo-keratin [ 25 ], keratin azure [ 26 ], and ball-milled feather powder [ 27 ].…”

Section: Introductionmentioning

confidence: 99%

Development of a keratinase activity assay using recombinant chicken feather keratin substrates

et al. 2017

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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.

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Biochemical and structural characterization of a keratin-degrading M32 carboxypeptidase from Fervidobacterium islandicum AW-1

Cited by 23 publications

References 24 publications

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

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

Microbial enzymes catalyzing keratin degradation: Classification, structure, function

Development of a keratinase activity assay using recombinant chicken feather keratin substrates

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