Molecular cloning of subtilisin J gene from bacillus stearothermophilus and its protein engineering = Bacillus stearothermophilus로부터 서브틸리신 J유전자의 클로닝 및 단백질공학에 의한 변형

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dc.contributor.advisorByun, Si-Myung-
dc.contributor.advisor변시명-
dc.contributor.authorJang, Jeong-Su-
dc.contributor.author장정수-
dc.date.accessioned2011-12-12T07:50:32Z-
dc.date.available2011-12-12T07:50:32Z-
dc.date.issued1993-
dc.identifier.urihttp://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=60611&flag=dissertation-
dc.identifier.urihttp://hdl.handle.net/10203/27332-
dc.description학위논문(박사) - 한국과학기술원 : 생명과학과, 1993.2, [ xiii, 142 p. ]-
dc.description.abstractThe 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...eng
dc.languageeng-
dc.publisher한국과학기술원-
dc.subject단백질 분리.-
dc.titleMolecular cloning of subtilisin J gene from bacillus stearothermophilus and its protein engineering = Bacillus stearothermophilus로부터 서브틸리신 J유전자의 클로닝 및 단백질공학에 의한 변형-
dc.typeThesis(Ph.D)-
dc.identifier.CNRN60611/325007-
dc.description.department한국과학기술원 : 생명과학과, -
dc.identifier.uid000875366-
dc.contributor.localauthorByun, Si-Myung-
dc.contributor.localauthor변시명-
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