Heterologe Expression und biochemische Charakterisierung von extrazellulären halotoleranten bzw. halophilen Peptidasen in Hinblick auf ihre biotechnologische Nutzung

Abstract

Extracellular peptidases from halotolerant resp. halophilic bacteria, exhibiting proteolytic activity at highly chaotropic conditions, offer a potential application for nucleic acid extraction procedures. In this study, peptidases secreted by 9 isolates from solar salterns showed a high tolerance towards guanidinium salts and detergents, including sodium dodecyl sulfate (SDS). All 9 isolates belonged to the genus Salinivibrio. Two different types of extracellular peptidases (Peptidase A and B) could be separated by hydrophobic interaction chromatography in the cell-free culture supernatants. Peptidase A was characterised as a moderate alkaliphilic and thermoactive enzyme (opt. activity: pH 8.5, 40-50Ã °C) with a molecular mass of about 35 kDa (SDS-PAGE). This enzyme was strongly inhibited by 1,10-phenantroline and EDTA, classifying as metallopeptidase. Peptidase B was shown to be a moderate alkaliphilic and thermoactive enzyme (opt. activity: pH 9-9.5; 50-55Ã °C) with a molecular mass of about 50 kDa (SDS-PAGE). Both peptidases exhibited optimal enzymatic activity at low salt concentrations (0-200 mM NaCl) and residual activity of under 20% at 2 M NaCl. The proAS gene encoding Peptidase B was cloned, sequenced and expressed in E. coli. The deduced amino acid sequence of proAS displayed homology with peptidases from the subtilisin family.