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タイトル: Effects of the mutational combinations on the activity and stability of thermolysin.
著者: Kusano, Masayuki
Yasukawa, Kiyoshi  kyouindb  KAKEN_id
Inouye, Kuniyo
著者名の別形: 井上, 國世
キーワード: Activity–stability relationship
Mutational combination
Site-directed mutagenesis
Thermal stability
Thermolysin
発行日: 3-May-2010
出版者: Elsevier B.V.
誌名: Journal of biotechnology
巻: 147
号: 1
開始ページ: 7
終了ページ: 16
抄録: We have previously indicated that three single mutations (Leu144-->Ser, Asp150-->Glu, and Ile168-->Ala) in the site-directed mutagenesis of thermolysin increase the activity and two single (Ser53-->Asp and Leu155-->Ala) and one triple (Gly8-->Cys/Asn60-->Cys/Ser65-->Pro) mutations increase the stability. In the present study, aiming to generate highly active and stable thermolysin variants, we combined these mutations and analyzed the effect of combinations on the activity and stability of thermolysin. The combination of the mutations of Leu144-->Ser and Asp150-->Glu yielded the most significant increase in the hydrolytic activities for N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide (FAGLA) and N-carbobenzoxy-L-Asp-L-Phe methyl ester (ZDFM), while that of Leu144-->Ser and Ile168-->Ala abolished the activity. The combination of Ser53-->Asp and Leu155-->Ala yielded the greatest increase in the thermal stability, while that of Ser53-->Asp and Gly8-->Cys/Asn60-->Cys/Ser65-->Pro increased the stability as high as the individual mutations do. The combination of three mutations of Leu144-->Ser, Asp150-->Glu, and Ser53-->Asp yielded a variant L144S/D150E/S53D with improved activity and stability. Its k(cat)/K(m) values in the hydrolysis of FAGLA and ZDFM were 8.6 and 10.2 times higher than those of wild-type thermolysin (WT), respectively, and its rate constant for thermal inactivation at 80 degrees C was 60% of that of WT.
著作権等: © 2010 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/2433/128776
DOI(出版社版): 10.1016/j.jbiotec.2010.02.024
PubMed ID: 20214932
出現コレクション:学術雑誌掲載論文等

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