Biochemical characterization of an alkaline metallopeptidase secreted by a Pseudomonas fluorescens isolated from soil.
The extracellular endopeptidase synthesized by soil bacterium Pseudomonas fluorescens was purified to homogeneity in a four-step procedure. The enzyme was purified 45-fold, with a 20% recovery. The endopeptidase appeared to be a monomer with a molecular mass of approx. 50 kDa. The enzyme was active in the pH range of 7 to 10. The optimal activity was detected at pH 9.0 and at 42 degrees C. Enzyme activity was inhibited by EDTA, EGTA and 1,10 phenanthroline, typical metalloprotease inhibitors. Ca(2+) activated the enzyme while Zn(2+), Co(2+), Cd(2+)(in high concentration) strongly inhibited it. The presence of calcium ions strongly stabilized the enzyme with regard to thermal resistance. The amino acid sequence of fragments of the enzymatic protein determined by MS analysis revealed a high similarity to the sequences of other alkaline metalloendopeptidases of bacteria belonging to the genus Pseudomonas.
Jankiewicz
Urszula
U
Warsaw University of Life Sciences - SGGW, Department of Biochemistry, Warsaw, Poland. urszula_jankiewicz@sggw.pl
Szawłowska
Urszula
U
Sobańska
Monika
M
D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D003067:Coenzymes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013696:Temperature
D002413:Cations, Divalent; D002614:Chelating Agents; D003067:Coenzymes; D004791:Enzyme Inhibitors; D008670:Metals; D045726:Metalloproteases