THERMOSTABLE VARIANTS OF SUBTILISIN SELECTED BY TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS

Region-specific random mutagenesis in the weak calcium binding site of subtilisin Carlsberg and subsequent screening for variants with enhan ced heat stability revealed two variants, which showed significantly e nhanced residual activity at 68 degrees C, 0.1 mM CaCl2, pH 8.0. Prese lected variants have been studied by temperature-gradient gel electrop horesis (TGGE) and were found to be stabilized due to different effect s. Whereas the point mutation (Ser188Pro) mainly enhanced autoproteoly tic stability of subtilisin, the double mutation (Ser188Pro; Ala194Glu ) additionally increased the apparent T-m-value of the molecule for 2- 3 degrees C under a variety of conditions. It was possible to differen tiate between the effects of autoproteolysis and structural unfolding to a certain degree by TGGE and to show the complex influence of chang ed calcium affinity on thermal stability for the double variant.