Functional Expression and Characterization of Keratinase from Pseudomonas aeruginosa in Pichia pastoris

Recombinant keratinase (rK) from Pseudomonas aeruginosa was secreted by Pichia pastoris SMD1168H with a final yield of 580 mg/L (1.03 kU/mL) after 72 h of induction. The rK was purified after nickel affinity chromatography and was stable at pH 6.0-9.0 and 10-60 degrees C. It was nonglycosylated protein with a molecular mass of 33 kDa and had an optimal pH and temperature at 8.0 and 60 degrees C, respectively. Ba2+ Ca2+, Mg2+, Mn2+, Zn2+, dithiothreitol, glutathione, and beta-mercaptoethanol activated, while Cu2+, Fe2+, Hg2+, Fe3+, ethylene glycol tetraacetic acid, ethylene diamine tetraacetic acid, and p-chloromercuribenzoate inhibited its activity. rK could hydrolyze broad substrates and cleave hydrophobic and aromatic amino acids at P-1 position, behaving as those from the wild type strain and E. coli transformant.