Molecular characterization of a keratinolytic enzyme from an alkaliphilic Nocardiopsis sp. TOA-1

Abstract A keratinolytic protease of an alkaliphilic Nocardiopsis sp. strain TOA-1, designated NAPase, was characterized and the gene was cloned and expressed. NAPase exhibited a much greater keratinolytic activity compared with proteinase K and subtilisin, especially at a higher alkaline pH. The maximal activity toward keratin was observed at a pH above 12.5 and at 60 °C. NAPase showed a pH-independent adsorption ability for keratin. The adsorption manner fitted the Langmuir-type adsorption isotherm. NAPase showed strong resistance to reducing agents such as dithiothreitol, β-mercaptoethanol and sodium thioglycolate. NAPase also exhibited a high proteolytic coefficient of 1287 mM −1  s −1 ( k cat / K m ) for the synthetic substrate of Suc-(Ala) 2 -Pro-Phe- p NA. The napA gene (encoding NAPase) was identified in a 6-kb DNA fragment and the entire nucleotide sequence was determined. The napA gene consists of 1152-bp and encodes 384 amino acids. The mature enzyme consists of 188 amino acids and is 19.1 kDa in molecular mass. The amino acid sequence showed homology to proteases from Streptomyces strains with 38–57% identities. The napA gene was heterologously expressed in Streptomyces lividans with keratinolytic activity.