Improvement of thermal stability of subtilisin J by changing the primary autolysis site

The thermostability of subtilisin J, an extracellular serine protease secreted from Bacillus stearothermophilus, has been improved by changing the primary autolysis site of the Asp-49 mutant protein. Previously we have shown that the Asp-49 mutant protein has proteolytic activity, but so unstable that it was primarily autolyzed in Tyr-58-Gln-59 peptide bond during cultivation (Jang et al. Biochim. Biophys. Acta. 1162, 233-235 1993). In the present study, to mitigate the autolytic degradation and increase the thermostability, we deleted the Tyr-58 residue using the Asp-49 mutant as a template. This mutant (Asp-49/delta Tyr-58 mutant) protein showed an improved resistance to heat treatment without changing the catalytic efficiency of the enzyme. These results show that change of primary autolysis site can stabilize the subtilisin.