A Novel Industrially Promising Keratinase Isolated from Aspergillus aureolatus Changes its Substrate Specificity after pH-Mediated Autolysis

Microbial proteolytic enzymes are green catalysts with many potential applications. Keratinolytic enzymes (or keratinases) are crucial for efficient keratin degradation, making them essential in waste management, biomedicine, cosmetics, and various industrial applications. This study describes the production and characterization of a protease with keratinolytic activity from Aspergillus aureolatus. The protease was isolated from the culture fluid on the 5^th day of cultivation with keratin hydrolysate through precipitation, dialysis, and isoelectric focusing. The enzyme exhibited limited self-cleavage at a moderately acidic pH range of 4.0-4.5. The molecular weight of the ancestral protease was identified as 27 kDa, while the self-cleaved form was 20 kDa. The self-cleaved protease showed increased specificity to keratin and maintained activity in acidic pH buffers, distinguishing it from its ancestral form. These properties suggest the enzyme's potential applications in biomedicine, biotechnology, and various industrial processes, where efficient keratin degradation is highly beneficial.