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 residues at positions of 45 and 115, which react with sulfhydryl reagents resulting in a significant decrease in the enzymatic activity. To understand the role of the cysteine residues in catalytic regions of the esterase, the residues were replaced with serine or alanine by site-directed mutagenesis to construct four single-mutated enzymes (C45A, C45S, C115A, C115S) and two double-mutated ones (C45/115A and C45/115S). Wild-type and mutant enzymes were produced in Escherichia coli cells and purified to homogeneity to examine their chemical and kinetic properties. These mutant enzymes had esterase activity, which suggested that none of the cysteines were required for its activity. Moreover, replacement of both two-cysteine residues made the enzyme insensitive to p-chloromercuribenzoic acid and extensively stabilized it at high temperatures of around 70 degrees C. These results demonstrate that replacement of free cysteine residues by site-directed mutagenesis can improve the thermostability of thermophilic enzymes.
Amaki
Y
Y
Department of Applied Biological Sciences, School of Agriculture, Nagoya University, Japan.
Nakano
H
H
Yamane
T
T
D001483:Base Sequence; D001709:Biotechnology; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D017931:DNA Primers; D004269:DNA, Bacterial; D004950:Esterases; D003545:Cysteine