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

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 56°C 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 70°C 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 acidreplacements 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.