The Pseudomonas aeruginosa san-ai strain was isolated from water-soluble cutting oil used for cooling and lubrication during industrial metal-working processes. This strain, which is grown in a high alkaline (pH 10) mixture of surfactants and mineral oil, produces an extracellular proteolytic enzyme. We have purified and characterized this 18 kDa protease. The P aeruginosa san-ai protease functions optimally at pH 9.0 and 60degreesC. Additionally, it is a Zn-containing metalloenzyme, and its monomeric structure contains at least one disulfide bond. Because the enzyme is stable in the presence of organic solvents, it is suitable for peptide synthesis. Furthermore, the P aeruginosa san-ai protease could be used in an intelligent drug delivery system (DDS) designed for applications in the metal industry for prevention of putrefaction of cutting oil.