Development of a new algicidal bioremediator for preventing the occurrence of red tides

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The marine bacterium Pseudoalteromonas sp. strain A28 is able to kill the diatom Skeletoned costatum NIES-324. The culture supernatant of strain A28 showed 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 toultrafiltration with a 10,000-Mw-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatium cells. 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 … More potent algicidal activity, designated AspI. The purified AspI had a molecular mass for 50 kDa. 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 tetra-ethylenepentamine. The determined N-terminal amino acid sequence of purified AspI was identical and internal amino acid sequences showed high similarity with AprI, which is an extracellualr serine protease of marine bacterium Altermonas sp. strain 0-7. Molecular cloning of the serine protease gene, aspI, was performed using the mature AprI encoding DNA fragment as a probe of southern hybridization. The sequencing of cloned aspI gene revealed an open reading frame of 2,073 bp with the capacity, to encode a polypeptide of 691 amino acids and with a molecular size of 71,007. Less