Division of Microbiology, Indian Agricultural Research Institute, New Delhi-110012, India.
J Microbiol Biotechnol. 2013 Nov 28;23(11):1536-43. doi: 10.4014/jmb.1306.06062.
Proteases produced by Xenorhabdus are known to play a significant role in virulence leading to insect mortality. The present study was undertaken to purify and characterize protease from Xenorhabdus indica, an endosymbiont of nematode Steinernema thermophilum, and to decipher its role in insect mortality and its efficacy to control Helicoverpa armigera. A set of 10 strains of Xenorhabdus isolated from different regions of India were screened for protease activity on the basis of zone of clearing on gelatin agar plates. One potent strain of Xenorhabdus indica was selected for the production of protease, and the highest production (1,552 U/ml) was observed at 15-18 h of incubation at 28°C in soya casein digest broth. The extracellular protease was purified from culture supernatant using ammonium sulfate precipitation and ion-exchange chromatography. The enzyme was further characterized by SDS-PAGE and zymography, which confirmed the purity of the protein and its molecular mass was found to be ~52 kDa. Further MALDI-TOF/TOF analysis and effect of metal chelating agent 1,10-phenanthrolin study revealed the nature of the purified protease as a secreted alkaline metalloprotease. The bioefficacy of the purified protease was also tested against cotton bollworm (Helicoverpa armigera) and resulted in 67.9 ± 0.64% mortality within one week. This purified protease has the potential to be developed as a natural insecticidal agent against a broad range of agriculturally important insects.
产酶菌属 Xenorhabdus 产生的蛋白酶在导致昆虫死亡的致病作用中起着重要作用。本研究旨在从线虫 Steinernema thermophilum 的共生菌 Xenorhabdus indica 中纯化和鉴定蛋白酶，并解析其在昆虫死亡率中的作用及其对棉铃虫（Helicoverpa armigera）的防治效果。根据明胶琼脂平板上的透明圈，从印度不同地区分离的 10 株产酶菌属 Xenorhabdus 进行了蛋白酶活性筛选。选择了一株产酶能力较强的 Xenorhabdus indica 菌株用于蛋白酶的生产，在 28°C 下，大豆酪蛋白消化肉汤中培养 15-18 小时，酶产量最高（1552 U/ml）。采用硫酸铵沉淀和离子交换层析从培养上清液中纯化了胞外蛋白酶。通过 SDS-PAGE 和同工酶分析进一步对该酶进行了鉴定，证实了该蛋白的纯度，其分子量约为 52 kDa。进一步的 MALDI-TOF/TOF 分析和金属螯合剂 1,10-邻菲罗啉研究的结果表明，纯化的蛋白酶为分泌型碱性金属蛋白酶。还测试了纯化蛋白酶对棉铃虫（Helicoverpa armigera）的生物功效，结果表明在一周内死亡率为 67.9±0.64%。这种纯化的蛋白酶具有作为一种针对广泛农业重要昆虫的天然杀虫剂的潜力。
