Abstract
Keratinases are proteases that can catalyze the degradation of insoluble keratinous biomass. Keratinases in protease family M36 (MEROPS database) are endo-acting proteases. In total, 687 proteases are classified in family M36. In the present study, new keratinolytic enzymes were identified in protease family M36 using the bioinformatics tool Conserved Unique Peptide Patterns (CUPP). Via CUPP, M36 family members were classified into 11 groups, with CUPP group 1 containing the three currently known and sequenced family M36 keratinases (derived from the fungi Fusarium oxysporum, Microsporum canis and Onygena corvina) as well as an additional 71 uncharacterized M36 proteases. In order to assess the relevance of CUPP group 1 categorization to keratinolytic function, four uncharacterized M36 proteases and the known keratinase from F. oxysporum (in CUPP group 1) were selected for recombinant expression and keratinolytic activity assessment. The four hitherto unknown M36 proteases were from Phaeosphaeria nodorum, Aspergillus clavatus, Pseudogymnoascus pannorum and Nectria haematococca, and represent four different fungal taxonomical classes. The genes encoding the selected M36 proteases were individually expressed in Pichia pastoris and all proteases displayed keratinase activity on keratin azure. Additionally, the activity on different keratinase substrates, optimal reaction conditions and thermal stability were determined for the two most active new keratinases. The results validate the applicability of CUPP for function-based discovery of non-characterized keratinases and present new robust keratinases for potential use in keratin upgrading.
Highlights
Keratin is an abundant, insoluble, fibrous protein that constitutes the structural protein of mammalian horns, wool and claws (α-keratin), and feathers, beaks, and reptile shells (β-keratin)
The members of MEROPS peptidase family M36 were classified into 11 groups based on conserved unique peptide patterns using the bioin formatics tool Conserved Unique Peptide Patterns (CUPP)
Combined analysis of CUPP grouping and phylo genetic clustering of these M36 keratinases and peptidases revealed that members in CUPP group 1 have a high possibility of being keratinolytic proteases: group 1 contained all the confirmed M36 keratinases and an additional 71 peptidases of unknown keratinolytic function
Summary
Insoluble, fibrous protein that constitutes the structural protein of mammalian horns, wool and claws (α-keratin), and feathers, beaks, and reptile shells (β-keratin). Keratinases (EC 3.4.-.-) can catalyze hydrolytic degradation of keratin to produce amino acids and/or small soluble peptides [2]. It has been recognized that microbial keratinases may be used for utilization of keratin via controlled enzymatic degradation to create peptides and amino acid products for applications in functional skin care products, animal feed, or fertilizers [3,4]. Keratin is an abundant material, e.g. the annual global chicken feather waste from poultry processing was estimated to amount to more than 4.7 million tons per year in 2019 [5]. Horns, beaks, and pig bristles are keratin-rich side products of meat production [6]. Discovery of robust microbial keratinases is an impor tant first step for development of new bioprocesses for keratin utilization
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