Involvement of an extracellular protease in algicidal activity of the marine bacterium Pseudoalteromonas sp strain A28
Involvement of an extracellular protease in algicidal activity of the marine bacterium Pseudoalteromonas sp strain A28
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DOI：
10.1128/aem.66.10.4334-4339.2000
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发表时间：
2000-10-01
影响因子：
4.4
通讯作者：
Ohtake, H
中科院分区：
文献类型：
--
作者：
Lee, SO;Kato, J;Ohtake, H
海洋细菌假交替单胞菌（Pseudoalteromonassp.）A28菌株对硅藻中肋骨条藻NIES-324菌株具有较强的杀藻活性。中肋骨NIES 324.通过用10，000-M-w-截留膜对A28培养上清液进行超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。中肋骨细胞然后发现浓缩的上清液具有蛋白酶和DNA酶活性。经N-甲基-N ′-亚硝基胍诱变后，获得两株无杀藻活性的假交替单胞菌，命名为NH 1和NH 2。NH 1和NH 2的培养上清的蛋白酶活性低于亲本菌株A28的15%。纸片法测定表明，纯化的蛋白酶具有较强的杀藻活性。纯化的蛋白酶的分子量为50 kDa，N-末端氨基酸序列测定为Ala-Thr-Pro-Asn-Asp-Pro。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 ℃。苯甲基磺酰氟、氟磷酸二异丙酯、抗痛剂、凝乳酶抑制剂和亮抑酶素对蛋白酶活性有强烈的抑制作用，EDTA、EGTA、菲咯啉和四亚乙基五胺对蛋白酶活性无明显抑制作用。这些结果表明，假交替单胞菌菌株A28产生的胞外丝氨酸蛋白酶负责该海洋细菌的杀藻活性。
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 NIES324. 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 degrees 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.