RESUMO
Region-specific random mutagenesis in the weak calcium binding site of subtilisin Carlsberg and subsequent screening for variants with enhanced heat stability revealed two variants, which showed significantly enhanced residual activity at 68 degrees C, 0.1 mM CaCl2, pH 8.0. Preselected variants have been studied by temperature-gradient gel electrophoresis (TGGE) and were found to be stabilized due to different effects. Whereas the point mutation (Ser188Pro) mainly enhanced autoproteolytic stability of subtilisin, the double mutation (Ser188Pro; Ala194Glu) additionally increased the apparent Tm-value of the molecule for 2-3 degrees C under a variety of conditions. It was possible to differentiate between the effects of autoproteolysis and structural unfolding to a certain degree by TGGE and to show the complex influence of changed calcium affinity on thermal stability for the double variant.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Ligação ao Cálcio/isolamento & purificação , Eletroforese/métodos , Subtilisinas/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Cloreto de Cálcio/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , DNA Bacteriano , Endopeptidases/metabolismo , Temperatura Alta , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Subtilisinas/química , Subtilisinas/genética , TemperaturaRESUMO
A random mutagenesis approach was directed to the weak calcium binding site of subtilisin Carlsberg in order to enhance the thermal stability of the enzyme by changing its calcium affinity. The structural motif of the binding site was altered by two strategies, the ligand strategy, which was directed to the amino acid ligands of the calcium ion and the conformation strategy, by which a part of the calcium cave was redesigned. Subtilisin mutants were expressed in Bacillus subtilis and screened for enhanced thermostability by a filter assay and by temperature-gradient gel electrophoresis (TGGE). Characterization of selected mutants and application of TGGE to investigate the thermal stability of proteases and protease-inhibitor complexes in general is described.