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

H. Takagi, T. Takahashi, H. Momose, M. Inouye, Y. Maeda, H. Matsuzawa, T. Ohta

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Abstract

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 (t( 1/2 )) and thermal stability (T(m)). 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 T(m) of 63.0°C, which was 4.5°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.

Original languageEnglish (US)
Pages (from-to)6874-6878
Number of pages5
JournalJournal of Biological Chemistry
Volume265
Issue number12
StatePublished - May 16 1990
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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