Protein Science, Vol 7, Issue 3 698-705, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

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

S. AKANUMA, A. YAMAGISHI, N. TANAKA and T. OSHIMA
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji 192-03, Japan Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226, Japan

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{deg}C but not at 61{deg}C and above. Phenotypically thermostabilized strains that can grow at 61{deg}C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70{deg}C and mutants that showed a leucine-autotrophic growth at 70{deg}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.
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