title: Expression and purification of pseudomonas aeruginosa keratinase in Bacillus subtilis DB104 expression system.
abstract: The DNA encoding Pseudomonas aeruginosa keratinase was ligated into pRPA expression vector and transformed into Bacillus subtilis DB104. Recombinant keratinase (rK), secreted by B. subtilis after 72 h of incubation, was purified to electrophoretical homogeneity by nickel affinity chromatography and found to have a molecular mass of 33 kDa. The rK had an optimal pH and temperature at 8.0 and 60 degrees C, respectively, and was stable at pH 6.0-9.0 and 10-50 degrees C. It was strongly inhibited by Cu(2+), Fe(2+), Hg(2+), Fe(3+), ethylene glycol tetraacetic acid, and ethylene diamine tetraacetic acid but activated by Ca(2+), Mg(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol. According to substrate specificity, the rK was considered to be a metalloprotease.
authors.affiliation: Department of Food Science, National Taiwan Ocean University, Keelung 20224, Taiwan.
authors.forename: Hsin-Hung
authors.initials: HH
authors.lastname: Lin
authors.forename: Li-Jung
authors.initials: LJ
authors.lastname: Yin
authors.forename: Shann-Tzong
authors.initials: ST
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D005822:Genetic Vectors; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010447:Peptide Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D010447:Peptide Hydrolases; C035855:keratinase