Functional expression and characterization of keratinase from Pseudomonas aeruginosa in Pichia pastoris.

ABSTRACT

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. Ba(2+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol activated, while Cu(2+), Fe(2+), Hg(2+), Fe(3+), 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.