Expression and purification of pseudomonas aeruginosa keratinase in Bacillus subtilis DB104 expression system.
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.
Lin
Hsin-Hung
HH
Department of Food Science, National Taiwan Ocean University, Keelung 20224, Taiwan.
Yin
Li-Jung
LJ
Jiang
Shann-Tzong
ST
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
D011994:Recombinant Proteins; D010447:Peptide Hydrolases; C035855:keratinase