通过抑制突变法进一步稳定嗜热栖热菌（Thermus thermophilus）这种嗜热菌的3-异丙基苹果酸脱氢酶。
Further stabilization of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation method.
作者信息
Kotsuka T, Akanuma S, Tomuro M, Yamagishi A, Oshima T
机构信息
Department of Life Science, Tokyo Institute of Technology, Japan.
出版信息
J Bacteriol. 1996 Feb;178(3):723-7. doi: 10.1128/jb.178.3.723-727.1996.
Abstract
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.
摘要
我们通过抑制突变方法成功进一步提高了嗜热栖热菌（Thermus thermophilus）中3-异丙基苹果酸脱氢酶（IPMDH）的稳定性。我们之前构建了一种由嗜热菌和嗜温菌酶的部分组成的嵌合IPMDH。该嵌合酶的热稳定性低于嗜热菌酶。编码嵌合酶的基因进行了随机诱变，并整合到嗜热栖热菌leuB缺陷型突变体的基因组中。在76℃的基本培养基中筛选转化体，获得了三个独立的稳定突变体。克隆并分析了这三个突变体的leuB基因。序列分析显示所有突变体中均有Ala-172→Val取代。通过引入该氨基酸取代提高了嗜热菌IPMDH的热稳定性。
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