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

开发新型杀藻生物修复剂以预防赤潮的发生

• 批准号: 11555221
• 负责人: OHTAKE Hisao
• 金额: $ 5.38万
• 依托单位: HIROSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11555221/
• 关键词: Red tide; Protease; Algicidal Bacteria; 細菌; 殺藻物質; 遺伝子

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

海洋细菌假交替单胞菌菌株A28能够杀死硅藻寄生的中肋骨NIES-324。菌株A28的培养上清在置于藻苔上的纸片上显示出杀藻活性。第324章.通过用10，000-MW截留膜对A28培养上清液进行透滤制备的浓缩上清液显示杀藻活性，表明菌株A28产生能够杀死S的胞外物质。肋细胞经N-甲基-N-亚硝基胍诱变，筛选出两株无杀藻活性的假交替单胞菌突变株，命名为NH 1和NH 2。NH 1和NH 2的培养物上清液显示出小于用亲本菌株A28检测到的蛋白酶活性的15%。通过使用离子交换层析，然后通过制备性凝胶电泳，从A28培养物上清液中纯化蛋白酶至均一。纸盘试验表明，纯化的蛋白酶具有 ...更多信息 有效的杀藻活性，命名为AspI。纯化的AspI具有50 kDa的分子量。以琥珀酰-Ala-Ala-Pro-Phe-对硝基苯胺为底物，该蛋白酶的最适pH为8.8，最适温度为30 ℃。蛋白酶活性强烈抑制苯甲基磺酰氟，二异丙基氟磷酸盐，抗痛剂，凝乳酶抑制剂，和亮抑酶素。EDTA、EGTA、菲咯啉或四亚乙基五胺均未检测到显著抑制。AspI的N-末端氨基酸序列与海洋细菌Altermonas sp. strain 0-7的胞外丝氨酸蛋白酶Apr I的氨基酸序列完全一致，内部氨基酸序列与Apr I有很高的相似性。以丝氨酸蛋白酶基因aspI的成熟片段为探针，进行Southern杂交，克隆了丝氨酸蛋白酶基因aspI。对克隆的aspI基因进行测序，发现其开放阅读框为2，073 bp，能够编码691个氨基酸的多肽，分子大小为71，007。少

项目成果

Lee, S-O: "Cloning and characterization of extracellular metalprotease gene of the algicidal marine bacterium Pseudoalteromonas sp. strain A28"Biosc. Biochem. Biotechnol.. 66. 1366-1369 (2002)

Lee，S-O：“杀藻海洋细菌假交替单胞菌菌株 A28 的胞外金属蛋白酶基因的克隆和表征”Biosc。

Lee S.: "Involvement of an extracellular protease in the algicidal activity of the marine bacterium Pseudoalteromonas sp.strain A28."Appl.Environ.Microbiol.. 66. 4334-4339 (2000)

Lee S.：“细胞外蛋白酶参与海洋细菌假交替单胞菌菌株 A28 的杀藻活性。”Appl.Environ.Microbiol.. 66. 4334-4339 (2000)

Lee, S-O: "Cloning and characterization of extracellular metalprotease gene of the algicidal marine bacterium Pseudoalteromonas sp. strain A28"Biosc. Biochem. Biotechnol. 66. 1366-1369 (2002)

Lee，S-O：“杀藻海洋细菌假交替单胞菌菌株 A28 的胞外金属蛋白酶基因的克隆和表征”Biosc。

Lee, S-O: "Cloning and characterization of extracellular metalprotease gene of the algicidal marine bacterium Pseudoalteromonas sp. strain A28."Biosc. Biochem. Biotechnol.. 66. 1366-1369 (2002)

Lee，S-O：“杀藻海洋细菌假交替单胞菌菌株 A28 的胞外金属蛋白酶基因的克隆和表征。”Biosc。

Lee, S-O.: "Cloning and characterization of extracellular metalprotease gene of the algicidal marine bacterium Pseudoalteromonas sp. strain A28"Biosc. Biochem. Biotechnol.. (in press).

Lee，S-O.：“杀藻海洋细菌假交替单胞菌菌株 A28 的胞外金属蛋白酶基因的克隆和表征”Biosc。