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

基本信息

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

项目摘要

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

海洋细菌假单胞菌sp。应变A28能够杀死硅藻骨架Costatum Nies-324。菌株A28的培养上清液将其应用于Costatum nies-324定律的纸盘时，显示出藻类活性。凝结的上清液是通过将A28培养物上清液触摸液和10,000毫W-切割的膜进行制备的，该膜显示出藻类活性，这表明菌株A28产生了能够杀死Costatium细胞的细胞外物质。在N-甲基N-硝基共糖苷氨酸诱变后选择了缺乏藻类活性的两个假偏角瘤突变体。 NH1和NH2的培养上清液显示出父母菌株A28检测到的蛋白酶活性的15％。通过使用离子 - 交换色谱法纯化蛋白酶从A28培养上清液中纯化为同质性，然后使用制备的凝胶电泳。纸盘测定法表明，纯化的蛋白酶具有……更多的藻类活性，称为ASPI。纯化的ASPI的分子质量为50 kDa。发现蛋白酶的最佳pH和温度分别为8.8和30-C，通过使用琥珀酰 - ala-ala-phe-phe-phe-p-p-phe-p-nitroanilide作为底物。苯基磺磺酰氟，二异丙基氟化物，抗蛋白酶，甲状腺胰蛋白酶和亮肽蛋白强烈抑制蛋白酶活性。用EDTA，EGTA，菲诺林或四乙基异戊胺检测到没有明显的抑制作用。确定的纯化ASPI的N末端氨基酸序列是相同的，并且内部氨基酸序列与APRI相似，这是海洋细菌Artermonas sp的细胞外丝氨酸蛋白。应变0-7。使用编码DNA片段的成熟APRI作为南部杂交的探针，对序列蛋白酶基因ASPI的分子克隆进行。克隆的ASPI基因的测序揭示了具有2,073 bp的开放式阅读框，其能力为691个氨基酸的多肽编码，分子大小为71,007。较少的