Analysis of autodegradation sites of thermolysin and enhancement of its thermostability by modifying Leu155 at an autodegradation site.
Analysis of autodegradation sites of thermolysin and enhancement of its thermostability by modifying Leu155 at an autodegradation site.
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DOI：
10.1093/jb/mvh067
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发表时间：
2004-04
影响因子：
2.7
通讯作者：
Y. Matsumiya;K. Nishikawa;H. Aoshima;K. Inouye;M. Kubo
中科院分区：
文献类型：
--
作者：
Y. Matsumiya;K. Nishikawa;H. Aoshima;K. Inouye;M. Kubo
研究了嗜热菌蛋白酶（TLN）的自降解与热稳定性的关系。在Ca（2+）存在下，TLN有4个自降解位点。其中一个位点被鉴定为Gly（154）-Leu（155），并且Leu（155）通过定点突变被各种氨基酸（X = Ala、Ser、Phe和Gly）取代。80 ℃热稳定性随氨基酸取代的顺序为Ala>Phe>Ser>Gly>Leu（WT TLN）。对于检查的所有突变型TLN，均出现了WT TLN未观察到的额外自降解片段。自降解位点从Gly（154）-Leu（155）键转移到X（155）-Ile（156）键，Leu（155）发生突变。此外，Ile（164）-Asp（165）键被新识别为突变型TLNs中产生AF 3 '的自降解位点。
The relationship between the autodegradation and thermostability of thermolysin (TLN) was studied. Four autodegradation sites in TLN were identified in the presence of Ca(2+). One of the sites was identified as Gly(154)-Leu(155), and Leu(155) was substituted with various amino acids, X = Ala, Ser, Phe, and Gly, by site-directed mutagenesis. The thermostability at 80 degrees C increased with the amino acid substitutions in the order of Ala>Phe>Ser>Gly>Leu (WT TLN). An additional autodegradation fragment that was not observed with WT TLN appeared for all mutant TLNs examined. The autodegradation site shifted from the Gly(154)-Leu(155) bond to the X(155)-Ile(156) one with the mutation at Leu(155). Furthermore, the Ile(164)-Asp(165) bond was recognized newly as an autodegradation site in the mutant TLNs for the production of AF3'.