DECREASING THE STABILITY AND CHANGING THE SUBSTRATE-SPECIFICITY OF THE BACILLUS-STEAROTHERMOPHILUS ALCOHOL-DEHYDROGENASE BY SINGLE AMINO-ACID REPLACEMENTS
G. Fiorentino et al., DECREASING THE STABILITY AND CHANGING THE SUBSTRATE-SPECIFICITY OF THE BACILLUS-STEAROTHERMOPHILUS ALCOHOL-DEHYDROGENASE BY SINGLE AMINO-ACID REPLACEMENTS, Protein engineering (Print), 11(10), 1998, pp. 925-930
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.