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タイトル: Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability.
著者: Kojima, Kenji  kyouindb  KAKEN_id
Nakata, Hiroki
Inouye, Kuniyo
著者名の別形: 井上, 國世
キーワード: C-terminal region
Enzyme expression
Enzyme stability
Hydropathy
Site-directed mutagenesis
Thermolysin
発行日: Feb-2014
出版者: Elsevier B.V.
誌名: Biochimica et biophysica acta -Proteins and Proteomics
巻: 1844
号: 2
開始ページ: 330
終了ページ: 338
抄録: Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of β-rich N-terminal (residues 1-157) and α-rich C-terminal (residues 158-316) domains. Expression of thermolysin variants truncated from the C-terminus was examined in E. coli culture. The C-terminal Lys316 residue was not significant in the expression, but Val315 was critical. Variants in which Val315 was substituted with fourteen amino acids were prepared. The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. Variants with charged (Asp, Glu, Lys, and Arg), bulky (Trp), or small (Gly) amino acids were lower in these characteristics than WT. All variants exhibited considerably high activities (50-100% of WT) in hydrolyzing protein and peptide substrates. The expression amount, helix content, and stability of variants showed good correlation with hydropathy indexes of the amino acids substituted for Val315. Crystallographic study of thermolysin has indicated that V315 is a member of the C-terminal hydrophobic cluster. The results obtained in the present study indicate that stabilization of the cluster increases thermolysin stability and that the variants with higher stability are expressed more in the culture. Although thermolysin activity was not severely affected by the variation at position 315, the stability and specificity were modified significantly, suggesting the long-range interaction between the C-terminal region and active site.
著作権等: © 2013 Elsevier B.V.
This is not the published version. Please cite only the published version. この論文は出版社版でありません。引用の際には出版社版をご確認ご利用ください。
URI: http://hdl.handle.net/2433/180111
DOI(出版社版): 10.1016/j.bbapap.2013.10.014
PubMed ID: 24192395
出現コレクション:学術雑誌掲載論文等

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