HYPERTHERMOSTABLE MUTANTS OF BACILLUS-LICHENIFORMIS ALPHA-AMYLASE - MULTIPLE AMINO-ACID REPLACEMENTS AND MOLECULAR MODELING

We have identified previously two critical positions for the thermosta bility of the highly thermostable alpha-amylase from Bacillus lichenif ormis, We have now introduced all 19 possible amino acid residues to t hese two positions, His133 and Ala209. The most favourable substitutio ns were to Ile and Val, respectively, which both increased the half-li fe of the enzyme at 80 degrees C by a factor of similar to 3, At both positions a stabilizing effect of hydrophobic residues was observed, a lthough only in the case of position 133 could a clear correlation be drawn between the hydrophobicity of the inserted amino acid and the ga in in protein stability, The construction of double mutants showed a c umulative effect of the most favourable and/or deleterious substitutio ns. Computer modelling was used to generate a 3-D structure of the wil d-type protein and to model substitutions at position 209, which lies in the conserved (alpha/beta)(8) barrel domain of alpha-amylase; Ala20 9 would be located at the beginning of the third helix of the barrel, in the bottom of a small cavity facing the fourth helix, The model sug gests that replacement by, for example, a valine could fill this cavit y and therefore increase intra- and interhelical compactness and hydro phobic interactions.