Ser-49 in subtilisin J, an extracellular serine protease from B. stearothermophilus, is highly conserved residue among subtilisins from Bacillus sp. Moreover, Ala-48 - Ser-49 peptide bond was known as a primary autolysis site in subtilisin BPN`` recently. Previously constructed Asp-49 mutant protein, corresponding to that a more thermostable subtilisin variants, shows proteolytic activity, but so unstable that it was degraded during cultivation in Tyr-58 - Gln-59 peptide bond. The enzymatic activity of the Asp-49 mutant protein is higher than that of wild-type in early growth phase. To elucidate the role of Ser-49 and increase the heat-stability, first, the Ser-49 was replaced with Thr,Asn and Glu. From results of substitution of Ser-49, we concluded that hydroxyl of Ser-49, as H-bond donor and acceptor, is important in prevention of autolysis. Second, in consideration of substrate specificity of subtilisin, The Tyr-58 or Gln-59 weae replaced with various amino acid using Asp-49 mutant as a template. Among the mutants, Asp-49 -$\Delta$Tyr-58 mutant showed a considerably improved resistance to heat. These results demonstrate the powerful probability of protein-engineering in enhancement of heat stability of various enzymes.