Purification and identification of an antibacterial protein from the symbiotic bacteria associated with novel entomopathogenic nematode, Rhabditis (Oscheius) sp.
Purification and identification of an antibacterial protein from the symbiotic bacteria associated with novel entomopathogenic nematode, Rhabditis (Oscheius) sp.
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DOI：
10.1007/s11274-015-1816-3
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发表时间：
2015-04-01
影响因子：
4.1
通讯作者：
Nambisan, Bala
中科院分区：
文献类型：
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作者：
Anju, K. M.;Archana, M. M.;Nambisan, Bala
昆虫病原线虫(EPN)属于斯氏线虫科和异杆线虫科，它们的共生细菌Xnowhabdus和Photorhabdus是著名的生防菌，能产生广泛的生物活性次生代谢产物。在中央块茎作物研究所(CTCRI)对昆虫病原线虫进行的研究导致鉴定出属于Rhabditidae科的新的EPN。本研究报告了从与昆虫病原线虫Rhabditis(Oscheius)种共生的一种细菌(蜡状芽孢杆菌)的培养滤液中纯化出一种高相对分子质量的抗菌蛋白。对发酵条件进行了优化，在30A℃下发酵48h后，胰酶酱油的抗菌活性最好。水提物经硫酸铵沉淀、离子交换层析和尺寸排斥层析纯化后得到抗菌蛋白。天然凝胶电泳法检测到一条相对分子质量为220 KDa的活性蛋白质，在SDS-PAGE上可分辨出一条90 kDa的主带和一条约40 kDa的小带。经MALDI-TOF-MS/MS分析，该90 kDa蛋白为蜡样芽孢杆菌TQXA(苏氨酸-谷氨酰胺二肽)结构域蛋白。该蛋白对枯草芽孢杆菌MTCC2756、金黄色葡萄球菌MTCC902和大肠杆菌MTCC 2622具有较强的抗菌活性，且具有热稳定性。
Entomopathogenic nematodes (EPN) belonging to the families steinernematidae and heterorhabditidae and their symbiotic bacteria Xenorhabdus and Photorhabdus are well-known as biological control agents and are found to produce a wide range of bioactive secondary metabolites. Studies carried out at the Central Tuber Crops Research Institute (CTCRI) on entomopathogenic nematodes resulted in the identification of novel EPN belonging to the family Rhabditidae. This study reports the purification of a high molecular weight antibacterial protein from culture filtrates of a bacterium (Bacillus cereus) symbiotically associated with a novel entomopathogenic nematode Rhabditis (Oscheius) species, maintained at CTCRI laboratory. Fermentation conditions were standardized and optimum antibacterial activity was observed in tryptic soy broth after 48 h incubation at 30 A degrees C. The aqueous extracts yielded antibacterial proteins which were purified by ammonium sulfate precipitation followed by ion exchange chromatography and size exclusion chromatography. Native gel electrophoresis indicated an active protein of molecular mass 220KDa which resolved into a major band of 90 kDa and a minor band of about 40 kDa on SDS-PAGE. The 90 kDa protein showed antibacterial activity and was further analysed by MALDI TOF-MS/MS. The protein was identified as a TQXA (Threonine-glutamine dipeptide) domain containing protein from Bacillus cereus. The protein was found to be active against Bacillus subtilis MTCC2756, Staphylococus aureus MTCC902 and Escherichia coli MTCC 2622 and was thermally stable.