Enhanced thermal stability and catalytic efficiency of an alkaline protease from pseudomonas sp. KFCC10818 by changing the autolysis sites

Abstract

The extracellular alkaline protease, AprP, isolated from Pseudomonas sp. KFCC10818 was degraded by autoproteolysis at high temperature. Two sites of autoproteolysis were identified as amino terminal of Ser307 and Ser331 by isolation of early autolysis products and amino terminal sequence analysis. These sites seemed to be located in an exposed surface loop by fitting the sequence to model for subtilisin BPN``. To enhance thermal stability of AprP, we replaced serine residues of these sites with aspartate residues by site-directed mutagenesis. However, the only S331D mutant of two mutants was successfully purified and characterized. This mutant enzyme had a Km value 2.4-fold and a Kcat value 2-fold smaller than wild-type as measured by hydrolysis of s-AAPF-pna at pH 10.5. The half-life of S331D mutant showed 1.5 and 2.4 times longer than wild-type at 55℃ and 60℃, respectively. Therefore, these results suggest that the change of autolytic cleavage sites enhance the thermal stability and catalytic efficiency of the enyme AprP by preventing from cleaving.