Improvement of thermal stability of alkaline serine protease from Pseudomonas sp. by modification of autoproteolytic sites

Abstract

The alkaline serine protease, AprP, from Pseudomonas sp. KFCC 10818 has optimum activity at 70°C. However, the thermal stability of the protease is relatively low at high temperature due to autoproteolytic degradation. Two cleavage sites have been previously identified at the amino terminus of Ser307 and Ser331 through N-terminal sequence analysis of the self-digested AprP fragments. These sites appeared to be located on the exposed surface loop based on sequence fitting with Proteinase K. Serine residues of these sites were replaced by other amino acids by site-directed mutagenesis in order to enhance the thermal stability of AprP. Among 17 mutant proteins obtained, 6 mutant enzymes, S331E, S331Y, S331R, S331H, S307A, and S307G showing the similar halo sizes of the wild-type AprP in the skim milk plates were purified and analyzed the thermal stability. Five mutant AprPs, i.e. S331E, S331Y, S331R, S331H, and S307A showed longer half-life than the wild-type AprP. The half-life of the S331E and S331Y mutants was 4 and 3.3 times longer at 55°C, respectively and 2.8 and 3 times longer at 60°C, respectively than that of the wild-type AprP in the presence of 1 mM $CaCl_2$. The half-life of the S331R, S331H, and S307A mutants was 26 min in the presence of 1 mM $CaCl_2$. The half-life was increased 2 times longer than that of the wild-type AprP at 55°C. However, there was no significant increase of the half-life in the S307G mutant. Western blot analysis showed the S331E, S331Y, and S331R mutants were not cleaved at the cleavage site, Ser331. These observations suggest that the thermal stability of the mutant AprPs was increased by the protection of the cleavage site. The catalytic parameters of these mutant proteases were also determined. When analyzed at pH 10.5, the S307A mutant was shown increased Kcat/Km value and the S331E, S331H and S331R mutants maintained similar Kcat/Km values of the wild type AprP.