Purification and characterization of protease with high stability in organic solvents

A novel solvent-tolerant protease was produced by a solvent-tolerant Bacillus licheniformis YP1 strain isolated from soil. The protease was purified by precipitation with ammonium sulfate, hydrophobic interaction chromatography and cation exchange chromatography, leading to 37.2-fold purification with 20.8% recovery rate. The product showed eletrophoretic homogeneity, as identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the specific activity of the purified protease reached 1.18 × 10⁵ U/mg. SDS-PAGE analysis indicated that the relative molecular mass of the protein was about 28 kDa. The protease was stable and active in all the tested solvents and the protease activity was significantly enhanced in the presence of 50%(φ) DMSO and DMF. The protease was considered as a Zn²⁺-complexed enzyme with an optimal reaction temperature of 55°C. The enzyme was stable in the pH range of 8.0~13.0, with an optimum pH of 10.0. The Michaelis constant for caseinolytic activity was 0.048 g/L.