Bae K H, Jang J S, Park K S, Lee S H, Byun S M
Department of Life Science, Korea Advanced Institute of Science and Technology, Taejon.
Biochem Biophys Res Commun. 1995 Feb 6;207(1):20-4. doi: 10.1006/bbrc.1995.1147.
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.
嗜热脂肪芽孢杆菌分泌的胞外丝氨酸蛋白酶枯草杆菌蛋白酶J的热稳定性，通过改变Asp-49突变蛋白的主要自溶位点得到了提高。此前我们已经表明，Asp-49突变蛋白具有蛋白水解活性，但非常不稳定，以至于在培养过程中主要在Tyr-58-Gln-59肽键处发生自溶（Jang等人，《生物化学与生物物理学报》，1162卷，233 - 235页，1993年）。在本研究中，为了减轻自溶降解并提高热稳定性，我们以Asp-49突变体为模板删除了Tyr-58残基。这种突变体（Asp-49/ΔTyr-58突变体）蛋白在不改变酶催化效率的情况下，对热处理的抗性有所提高。这些结果表明，主要自溶位点的改变可以使枯草杆菌蛋白酶稳定。
