Enhanced thermostability of the single-Cys mutant subtilisin E under oxidizing conditions.
We obtained enhanced thermostability by replacing Ser161 with Cys in subtilisin E from Bacillus subtilis, a cysteine-free alkaline serine protease. The Ser161Cys mutant subtilisin E was purified from the culture supernatant of the recombinant B. subtilis in an oxidizing environment. SDS-polyacrylamide gel electrophoresis and mass spectrometry under oxidizing conditions indicated that the mutant enzyme in part formed an oligomeric protein, which may contain an intermolecular disulfide bond between two surface Cys residues at position 161. Further, no free sulfhydryl groups were detected in the mutant enzyme, suggesting the sulfhydryl modification in a monomeric form under oxidizing conditions. The Ser161Cys mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme. The half-life of thermal inactivation of the mutant was found to be 2-4 times longer than that of the wild-type enzyme. The optimum temperature of the mutant was 55 degrees C, which was 5 degrees C higher than that of the wild-type enzyme. Under reducing conditions, however, the characteristics of the mutant enzyme reverted to those of the wild-type enzyme. Similar results were obtained for another Cys mutant as to position 194 (wild-type, Ser), which is the same surface residue as Ser161. Possible reasons for the enhanced thermostability of the single-Cys mutant subtilisins E under oxidizing conditions are discussed in terms of two different mechanisms.
Takagi
H
H
Department of Bioscience, Fukui Prefectural University, Kenjojima, Matsuoka-cho, Fukui 910-1195, Japan. hiro@fpu.ac.jp
Hirai
K
K
Wada
M
M
Nakamori
S
S
D019943:Amino Acid Substitution / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D003198:Computer Simulation; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006207:Half-Life; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D016877:Oxidants / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D007527:Isoenzymes; D016877:Oxidants; D013381:Subtilisins; D003545:Cysteine