DECREASING THE STABILITY AND CHANGING THE SUBSTRATE-SPECIFICITY OF THE BACILLUS-STEAROTHERMOPHILUS ALCOHOL-DEHYDROGENASE BY SINGLE AMINO-ACID REPLACEMENTS

The gene encoding the alcohol dehydrogenase (adh-hT) from the thermoph ilic bacterium Bacillus stearothermophilus LLD-R strain has been overe xpressed in Escherichia coli and the corresponding recombinant protein purified to homogeneity, Two putative structural determinants contrib uting to the higher stability of ADH-hT had been identified by compari son with the less thermostable ADH (ADH-T) from the less thermophilic B,stearothermophilus NCA 1503, In order to ascertain their role, mutat ions were designed to eliminate in ADH-hT a salt bridge at the N-termi nus and a proline residue in the coenzyme binding domain replacing the amino acids located at the same positions in ADH-T. Three mutants-Glu 11Lys, Pro242Ala, and Glu11Lys/Pro242Ala-were expressed at high level and the proteins purified and characterized. In general, the mutations had little effect on the activity, indicating that they were not disr uptive. The thermal resistance was changed displaying quite additive e ffects.