Further stabilization of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation method

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&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-1723Val substitution in all of the mutants. The thermal stability of the thermophile IPMDH was improved by intro-ducing the amino acid substitution. Improvement of the thermal stability of proteins is one of the major concerns in protein engineering. Several principles have been proposed to increase the thermal stability of pro