Expression and Purification of Pseudomonas aeruginosa Keratinase in Bacillus subtilis DB104 Expression System
2009
Lin, Hsin-Hung | Yin, Li-Jung | Jiang, Shann-Tzong
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 °C, respectively, and was stable at pH 6.0-9.0 and 10-50 °C. It was strongly inhibited by Cu2+, Fe2+, Hg2+, Fe3+, ethylene glycol tetraacetic acid, and ethylene diamine tetraacetic acid but activated by Ca2+, Mg2+, Zn2+, dithiothreitol, glutathione, and β-mercaptoethanol. According to substrate specificity, the rK was considered to be a metalloprotease.
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