Heterologe Expression und biochemische Charakterisierung von extrazellulären halotoleranten bzw. halophilen Peptidasen in Hinblick auf ihre biotechnologische Nutzung
Veröffentlichungsdatum
2007-03-27
Autoren
Betreuer
Gutachter
Zusammenfassung
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.
Schlagwörter
Halophiles
;
peptidases
;
chaotropes
;
nucleic acid extraction
;
Salinivibrio
Institution
Fachbereich
Dokumenttyp
Dissertation
Zweitveröffentlichung
Nein
Sprache
Deutsch
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