Further stabilization of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation method

被引:57
作者
Kotsuka, T
Akanuma, S
Tomuro, M
Yamagishi, A
Oshima, T
机构
[1] TOKYO PHARM & LIFE SCI,DEPT MOLEC BIOL,HACHIOJI,TOKYO 19203,JAPAN
[2] TOKYO INST TECHNOL,DEPT LIFE SCI,MIDORI KU,YOKOHAMA,KANAGAWA 226,JAPAN
关键词
D O I
10.1128/jb.178.3.723-727.1996
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
We succeeded in further improvement of the stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, by a suppressor mutation method. We previously constructed a chimeric IPMDH consisting of portions of thermophile and mesophile enzymes. The chimeric enzyme is less thermostable than the thermophile enzyme. The gene encoding the chimeric enzyme was subjected to random mutagenesis and integrated into the genome of a leuB-deficient mutant of T. thermophilus. The transformants were screened at 76 degrees C in minimum medium, and three independent stabilized mutants were obtained. The leuB genes from these three mutants were cloned and analyzed. The sequence analyses revealed Ala-172-->Val substitution in all of the mutants. The thermal stability of the thermophile IPMDH was improved by introducing the amino acid substitution.
引用
收藏
页码:723 / 727
页数:5
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