Bacillus stearothermophilus로부터 서브틸리신 J유전자의 클로닝 및 단백질공학에 의한 변형

학위논문(박사) - 한국과학기술원 : 생명과학과, 1993.2, [ xiii, 142 p. ]The structural gene for subtilisin J from Bacillus stearothermophilus NCIMB10278 was cloned in Bacillus subtilis using pZ124 as a vector, and its nucleotide sequence was deterimined. The nucleotide sequence revealed only one large open reading frame, composed of 1,143 base pairs and 381 amino acid residues. A Shine-Dalgarno sequence was found 8 bp upstream from the translation start codon (GTG). The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein composed of 275 residues. The productivity of subtilisin J in the culture broth of the Bacillus subtilis was about 46-fold higher than that of the Bacillus stearothermophilus. The amino acid sequence deduced from the extracellular alkaline protease subtilisin J is highly homologous to that of subtilisin E and it shows 69\% homology with subtilisin Carlsberg, 89\% with subtilisin BPN`` and 70\% with subtilisin DY. Some properties of the subtilisin J with had been purified from the Bacillus subtilis were examined. The subtilisin J has alkaline pH characteristics and a molecular weight of 27,500. All enzymatic properties of the subtilisin J including thermostabilities were similar to those of subtilisin E and subtilisin Amylosacchariticus. Eight mutations were introduced to study the molecular basis of thermostability of subtilisin J and to analyze the structure-function relationship in the subtilisin J. Among eight mutant proteins, Gln-195 and Asp-49 mutant showed activity as wild-type subtilisin. Gln-195 mutant enzyme was expressed in Bacillus subtilis and was purified from the culture supernatant. When the mutant enzyme was expressed at 37$^\circ$C in the presence of 2 mM $CaCl_2$, the pattern of enzyme production was quite different from that of wild-type. The purified Gln-195 mutant enzyme was analyzed with respect to optimal temperature, optimal pH, and heat stability. The mutation was found to decrease the heat stability but not cataly...한국과학기술원 : 생명과학과

서브틸리신 유전자의 부위 특이 돌연변이

학위논문(석사) - 한국과학기술원 : 생물공학과, 1989.2, [ x, 71 p. ]Subtilisin is a alkaline protease secreted by Bacilli. It``s catalytic triad is composed of Asp 32, His 64 and Ser 221. Also, His 67 is located nearby His 64 in a-helix segment. To analyze the functional role of subtilisin, His 64 and His 67 were changed to Gly 64 and Gly 67, by oligonucleotide-directed site-specific mutagenesis. Furthermore, double mutant was obtained from Gly 64 and Gly 67 mutant by simple gene manipulation. Then, we were examined for streptokinase activity of each mutant. This experiment was based upon the fact that SK has evolved from a serine protease by gene duplication and fusion. In addition, catalytic site of SK is composed of Gly 24 ASP 66 and Ser 157. When transformed into B. subtilkis DB104, each mutant had not shown any detectable protease activity compared with wild-type and had not shown SK activity. Interstingly, the effect of double mutant for protease production was almost same as Gly 64 mutant. from these results, we suggest that His 67 is also necessary for the catalytic activity of subtilisin as well as His 64.한국과학기술원 : 생물공학과