THE EFFECT OF ENGINEERING SURFACE LOOPS ON THE THERMAL-STABILITY OF BACILLUS-SUBTILIS NEUTRAL PROTEASE

Using genetic techniques the contribution of surface loops to the ther mal stability of Bacillus subtilis neutral protease (NP-sub) was studi ed. Mutations were designed to make the surface of NP-sub more similar to the surface of more thermostable neutral proteases such as thermol ysin (TLN). The mutations included the replacement of an irregular loo p by a shorter variant and the introduction of a ten-residue beta-hair pin. In general, these drastic mutations had little effect on the prod uction and activity of NP-sub, indicating the feasibility of major str uctural rearrangements at the surface of proteins. In the most stable mutant, exhibiting an increase in thermal stability of 1.1 degrees C, similar to 10% of the surface of NP-sub was modified. Several NP-sub v ariants carrying multiple mutations were constructed. Non-additive eff ects on thermal stability were observed, which were interpreted on the basis of a model for thermal inactivation, that emphasizes the import ance of local unfolding processes for thermal stability.