title: Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution.
abstract: 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 degrees C but not at 61 degrees C and above. Phenotypically thermostabilized strains that can grow at 61 degrees C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 degrees C and mutants that showed a leucine-autotrophic growth at 70 degrees 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 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.
authors.affiliation: Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Japan. akanuma@ls.toyaku.ac.jp
authors.forename: S
authors.initials: S
authors.lastname: Akanuma
authors.forename: A
authors.initials: A
authors.lastname: Yamagishi
authors.forename: N
authors.initials: N
authors.lastname: Tanaka
authors.forename: T
authors.initials: T
authors.lastname: Oshima
mesh_terms: D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology
chemical_list: D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase