ENHANCEMENT OF THERMOSTABILITY AND CATALYTIC EFFICIENCY OF APRP, AN ALKALINE PROTEASE FROM PSEUDOMONAS SP, BY THE INTRODUCTION OF A DISULFIDE BOND

A site-directed mutagenesis in AprP, an alkaline protease isolated fro m Pseudomonas sp. KFCC 10818 was carried out in order to obtain increa sed thermostability. Sites for cysteine substitutions to form disulfid e bond within AprP were chosen by comparing the sequences with aqualys in I, an alkaline thermostable serine protease whose disulfide bonds s eems to be important for its thermostability. Gly199 and Phe236 residu es were each replaced with cysteine by site-directed mutagenesis. The G199C/F236C mutant enzyme appeared to form a disulfide bond spontaneou sly during its expression. It also showed improved kinetic parameters for the hydrolysis of a synthetic peptide substrate at pH 8.5 and 10.5 compared to those of the wild-type enzyme. The half-life of the G199C /F236C mutant was found to be 2 to 4.8 times longer than that of the w ild-type under various experimental conditions, except when tested und er reducing condition, where no significant differences in the half-li fe of the two types were observed. Therefore, it is concluded that the introduction of the disulfide bond enhanced the thermostability and t he catalytic efficiency of the enzyme AprP. (C) 1996 Academic Press, I nc.