title: Random mutagenesis of the weak calcium binding site in subtilisin Carlsberg and screening for thermostability by temperature-gradient gel electrophoresis.
abstract: 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.
authors.affiliation: Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Sättler
authors.forename: S
authors.initials: S
authors.lastname: Kanka
authors.forename: W
authors.initials: W
authors.lastname: Schrörs
authors.forename: D
authors.initials: D
authors.lastname: Riesner
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004795:Enzyme Stability; D005820:Genetic Testing; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D015202:Protein Engineering; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics*; D013696:Temperature
chemical_list: D013381:Subtilisins; D002118:Calcium