Enhancement of the thermostability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease.
Sites for Cys substitutions to form a disulfide bond were chosen in subtilisin E from Bacillus subtilis, a cysteine-free bacterial serine protease, based on the structure of aqualysin I of Thermus aquaticus YT-1 (a thermophilic subtilisin-type protease containing two disulfide bonds). Cys residues were introduced at positions 61 (wild-type, Gly) and 98 (Ser) in subtilisin E by site-directed mutagenesis. The Cys-61/Cys-98 mutant subtilisin appeared to form a disulfide bond spontaneously in the expression system used and showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. The thermodynamic characteristics of these enzymes were examined in terms of enzyme autolysis (t1/2) and thermal stability (Tm). The half-life of the Cys-61/Cys-98 mutant was found to be 2-3 times longer than that of the wild-type enzyme. Similar results were obtained by differential scanning calorimetry. The disulfide mutant showed a Tm of 63.0 degrees C, which was 4.5 degrees C higher than that observed for the wild-type enzyme. Under reducing conditions, however, the characteristics of the mutant enzyme were found to revert to those of the wild-type enzyme. These results strongly suggest that the introduction of a disulfide bond by site-directed mutagenesis enhanced the thermostability of subtilisin E without changing the catalytic efficiency of the enzyme.
Takagi
H
H
Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan.
Takahashi
T
T
Momose
H
H
Inouye
M
M
Maeda
Y
Y
Matsuzawa
H
H
Ohta
T
T
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D004220:Disulfides; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D011489:Protein Denaturation; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D013824:Thermus / Q000201:enzymology
D004220:Disulfides; D015345:Oligonucleotide Probes; D012697:Serine Endopeptidases; D013381:Subtilisins; C055102:aqualysin I