Involvement of an Extracellular Protease in Algicidal Activity of the Marine Bacterium Pseudoalteromonas sp. Strain A28
| Autor: | Tsukasa Ikeda, Hisao Ohtake, Atsushi Mitsutani, Junichi Kato, Sun-Og Lee, Akio Kuroda, Noboru Takiguchi |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2000 |
| Předmět: |
medicine.medical_treatment
Applied Microbiology and Biotechnology Microbiology chemistry.chemical_compound Pseudoalteromonas Cellulase Endopeptidases medicine Protease Inhibitors Seawater Pest Control Biological Polyacrylamide gel electrophoresis Serine protease Diatoms Protease Ecology biology Strain (chemistry) Leupeptin biology.organism_classification Physiology and Biotechnology Chromatography Ion Exchange Kinetics chemistry Biochemistry Amylases biology.protein Diisopropyl fluorophosphate Antipain Electrophoresis Polyacrylamide Gel Gammaproteobacteria Food Science Biotechnology medicine.drug |
| Popis: | The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000- M w -cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N -methyl- N ′-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30°C, respectively, by using succinyl-Ala-Ala-Pro-Phe- p -nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium. |
| Databáze: | OpenAIRE |
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