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.
Bae
K H
KH
Department of Life Science, Korea Advanced Institute of Science and Technology, Taejon.
Jang
J S
JS
Park
K S
KS
Lee
S H
SH
Byun
S M
SM
D000595:Amino Acid Sequence; D001329:Autolysis; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine; D012697:Serine Endopeptidases / Q000737:chemistry; D013381:Subtilisins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
D011994:Recombinant Proteins; C077349:subtilisin J; D012694:Serine; D012697:Serine Endopeptidases; D013381:Subtilisins