Lorenz, Michael G.
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Lorenz, Michael G.
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Lorenz, Michael G.
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Item-typ:Veröffentlichung, Heterologe Expression und biochemische Charakterisierung von extrazellulären halotoleranten bzw. halophilen Peptidasen in Hinblick auf ihre biotechnologische Nutzung(2007-03-27); ; ; 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.Dissertation298 173 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Physiologische und molekularbiologische Untersuchungen an hydrolytischen extrazellulären Enzymen aus extremophilen marinen Mikroorganismen unter besonderer Berücksichtigung von Nucleasen(2003-12-10); ; ; Screening on plate assays of halophilic and halotolerant bacteria from hypersaline environments - mainly marine salterns - on Lanzarote, Canary Islands, Spain, and near La Baule, Pays de la Loire, France, led to the isolation of a relatively high proportion of strains with extracellular hydrolytic enzyme activity (66% = a total of 862 isolates with DNases, RNases, proteases, esterases, or amylases). Growth was performed on an adapted medium with salinities from 6 to 30% and pH-values of 8.0 and 9.5, respectively. DNase-producers were less abundant than other enzyme producers. Combined hydrolytic activity was detected in 45% (Lanzarote) and 67% (La Baule) of all positive strains. Haloalkaliphilic and haloalkalitolerant isolates showed enzyme activity extraordinarily often. The comparison of physico-chemical and biological parameters revealed significant differences between the sampling sites and led to the supposition of the salterns of Lanzarote being more promising for the detection of new hydrolases with outstanding extreme properties for biotechnological applications than the sites near La Baule. 223 enzyme-positive strains from Lanzarote were identified as members of the following taxonomic groups, typical for hypersaline habitats: Halomonadaceae (69 strains), Pseudomonadaceae (50 strains), Vibrionaceae (47 strains), Bacillaceae (27 strains), Alteromonadaceae (6 strains), and Archeae (12 strains). Several of the tested strains seem to represent new species. Hierarchical screening of 118 DNase producers from Lanzarote led to the detection of a borderline-halophilic bacterium (97.7% 16S-rRNA-partialsequence-identity to Halomonas halmophila) with an extracellular, salt-dependent 31 kDa endo-DNase. Further properties of the enzyme, like high tolerance for detergents and a broad pH- and temperature spectrum, are promising aspects for applying this enzyme in an improved laboratory RNA-isolation-kit.Dissertation253 307