An antifungal protein from the marine bacterium Streptomyces sp strain AP77 is specific for Pythium porphyrae, a causative agent of red rot disease in Porphyra spp.
An antifungal protein from the marine bacterium Streptomyces sp strain AP77 is specific for Pythium porphyrae, a causative agent of red rot disease in Porphyra spp.
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DOI：
10.1128/aem.68.6.2666-2675.2002
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发表时间：
2002-06-01
影响因子：
4.4
通讯作者：
Kamei, Y
中科院分区：
文献类型：
--
作者：
Woo, JH;Kitamura, E;Kamei, Y
从海洋细菌链霉菌AP 77的培养上清中发现了一种新的抗真菌蛋白（SAP），并对其进行了纯化。这种蛋白质的特点是化学，生物化学和生物学分析。通过使用凝胶过滤，SAP的分子量估计为160 kDa。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和基质辅助激光解吸电离-飞行时间质谱对SAP的结构分析表明，SAP由3个异源蛋白亚基组成，其分子量分别为41.7 kDa（SAP 1）、21.7 kDa（SAP 2）和18.7 kDa（SAP 3），摩尔比为1：1：5（或1：1：6）。N-末端氨基酸序列分析和同源性搜索显示，SAP 1，SAP 2和SAP 3表现出64.3%，68.4%和86.7%的相似性，三个天蓝色链霉菌多肽，嘌呤霉素抗性蛋白（Pur 8），一个保守的假设蛋白，和细菌铁蛋白，分别。纯化的SAP对Pythium porphyllum的MIC被确定为1.6 μ g/盘，而在高达100 μ g/盘的浓度下对大多数测试的真菌和细菌菌株没有观察到抑制作用;唯一的例外是对Pythium ultimum的相对强的抗真菌活性（MIC，6.3 μ g/盘）。体外和体内毒性试验表明，SAP对条斑紫菜细胞、人正常皮肤成纤维细胞和小鼠在剂量分别高达700 μ g/ml（持续24小时）、250 μ g/ml（持续12小时）和75 mg/kg（持续35天）时均无毒性。SAP是不稳定的，当它受到热空气干燥处理，这是一个很大的优势，在食品生产过程中。这些结果表明，链霉菌AP 77可作为安全的抗腐霉转基因紫菜育种的基因源。
A novel antifungal protein (SAP) was found in the culture supernatant of a marine bacterium, Streptomyces sp. strain AP77, and was purified. This protein was characterized by chemical, biochemical, and biological analyses. By using gel filtration, the molecular mass of SAP was estimated to be 160 kDa. Structural analysis of SAP by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry suggested that SAP is composed of three heterologous protein subunits of 41.7 kDa (SAP1), 21.7 kDa (SAP2), and 18.7 kDa (SAP3) at a molar ratio of 1:1:5 (or 1:1:6). N-terminal amino acid sequence analysis and a homology search revealed that SAP1, SAP2, and SAP3 exhibit 64.3, 68.4, and 86.7% similarity to three Streptomyces coelicolor polypeptides, puromycin resistance protein (Pur8), a conserved hypothetical protein, and bacterioferritin, respectively. The MIC of purified SAP against Pythium porphyrae was determined to be 1.6 mug/disk whereas no inhibitory effect was observed at concentrations up to 100 mug/disk against most of the fungal and bacterial strains tested; the only exception was relatively strong antifungal activity against Pythium ultimum (MIC, 6.3 mug/disk). In vitro and in vivo toxicity tests demonstrated that SAP showed no toxicity against Porphyra yezoensis cells, human normal dermal fibroblasts, and mice at doses up to 700 mug/ml (for 24 h), 250 mug/ml (for 12 h), and 75 mg/kg (for 35 days), respectively. SAP was labile when it was subjected to a heated-air drying treatment, which is a great advantage in food production procedures. These results indicated that Streptomyces sp. strain AP77 might be useful as a gene source for safe transgenic Porphyra breeding for tolerance to Pythium infection.