Enhancement of the thermostability and the catalytic efficiency of an alkaline protease from pseudomonas sp. KFCC10818 by site-directed mutagenesis

Abstract

To obtain increasement of industrial value by enhancement of thermostability, site-directed mutagenesis in an alkaline protease, AprP from Pseudomonas sp. KFCC 10818 was carried out. By sequence alignments with aqualysin I of Thermus aquaticus YT-1, sites for cysteine substitutions to form a disulfide bond in this protease, AprP, were chosen. Gly199 and Phe236 were replaced by cysteine residues in the enzyme by site-directed mutagenesis. The G199C/F236C mutant enzyme appeared to form a disulfide bond spontaneously in its expression and showed improved kinetic parameters compared to that of the wild-type enzyme for hydrolysis of an artificial synthetic substrate at pH 8.5 and pH 10.5. The half-life of the G199C/F236C mutant was found to be 2 - 4.8 times longer than that of the wild-type enzyme depending on experimental conditions. There was not any significant differences in the half-life of both enzymes when examined under reducing condition. These results suggest that the introduction of a disulfide bond by site-directed mutagenesis enhanced both of the thermostability and the catalytic efficiency of the enzyme.