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(PseudoAlternomonas sp.)菌株A28能够杀死硅藻骨架中肋骨杆菌NIES-324。菌株A28的培养上清液用于放置在中肋链霉菌NIES-324草坪上的纸盘上，具有杀藻活性。用10,000 mW截流膜过滤A28培养上清液得到的浓缩上清液具有杀藻活性，表明A28菌株产生的胞外物质能够杀死COSTATUM细胞。经N-甲基-N-亚硝胺诱变，筛选出2株无杀藻活性的假交替单胞菌突变体，命名为NH1和NH2。NH1和NH2的培养上清液的酶活不到亲本菌株A28的15%。通过离子交换层析和制备性凝胶电泳法从A28培养上清液中纯化得到了均一的蛋白水解酶。纸片鉴定表明，纯化后的酶具有…更强的杀藻活性，指定为ASPI。纯化的ASPI的相对分子质量为50 kDa。以琥珀酰丙氨酸-丙氨酸-苯丙氨酸-对硝基苯胺为底物，该酶的最适pH为8.8，最适温度为30℃。苯甲基磺酰氟、氟化二异丙酯、止痛药、糜抑素和亮肽素对该酶有较强的抑制作用。EDTA、EGTA、邻菲罗啉和四乙五胺对细胞无明显抑制作用。纯化的ASPI的N-末端氨基酸序列与海洋细菌Altermonas sp.的胞外丝氨酸蛋白酶APRI具有较高的相似性。菌株0-7。以编码AprI的成熟DNA片段为探针，进行了丝氨酸蛋白酶基因ASPI的分子克隆。对克隆的ASPI基因进行测序，发现其开放阅读框为2,073个碱基，编码691个氨基酸的多肽，分子大小为71,007。较少

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。