ROLE OF CYSTEINE RESIDUES IN ESTERASE FROM BACILLUS-STEAROTHERMOPHILUS AND INCREASING ITS THERMOSTABILITY BY THE REPLACEMENT OF CYSTEINES

Bacillus stearothermophilus esterase contains two free cysteine residu es at positions of 45 and 115, which react with sulfhydryl reagents re sulting in a significant decrease in the enzymatic activity. To unders tand the role of the cysteine residues in catalytic regions of the est erase, the residues were replaced with serine or alanine by site-direc ted mutagenesis to construct four single-mutated enzymes (C45A, C45S, C115A, C115S) and two double-mutated ones (C45/115A and C45/115S). Wil d-type and mutant enzymes were produced in Escherichia coli cells and purified to homogeneity to examine their chemical and kinetic properti es. These mutant enzymes had esterase activity, which suggested that n one of the cysteines were required for its activity. Moreover, replace ment of both two-cysteine residues made the enzyme insensitive to p-ch loromercuribenzoic acid and extensively stabilized it at high temperat ures of around 70 degrees C. These results demonstrate that replacemen t of free cysteine residues by site-directed mutagenesis can improve t he thermostability of thermophilic enzymes.