Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution

Satoshi Akanuma, Akihiko Yamagishi, Nobuo Tanaka, Tairo Oshima

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We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell. The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T. thermophilus. The resulting transformant showed a leucine-autotrophy at 56δC but not at 61°C and above. Phenotypically thermostabilized strains that can grow at 61°C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70°C and mutants that showed a leucine-autotrophic growth at 70δC were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine. The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme. Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme.

Original languageEnglish
Pages (from-to)698-705
Number of pages8
JournalProtein Science
Issue number3
Publication statusPublished - 1998 Mar
Externally publishedYes



  • 3-isopropylmalate dehydrogenase
  • Evolutionary molecular engineering
  • Integration vector system
  • Thermal stability
  • Thermus thermophilus

ASJC Scopus subject areas

  • Biochemistry

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