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首页> 外文期刊>Applied Microbiology >Use of Label-Free Quantitative Proteomics To Distinguish the Secreted Cellulolytic Systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis
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Use of Label-Free Quantitative Proteomics To Distinguish the Secreted Cellulolytic Systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis

机译：使用无标记的定量蛋白质组学来区分产钙梭菌贝西和产黑曜石的纤维素分解系统

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The extremely thermophilic, Gram-positive bacteria Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis efficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses from C. bescii and C. obsidiansis , label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative growth on crystalline cellulose. Both organisms' secretomes consisted of more than 400 proteins, of which the most abundant were multidomain glycosidases, extracellular solute-binding proteins, flagellin, putative pectate lyases, and uncharacterized proteins with predicted secretion signals. Among the identified proteins, 53 to 57 significantly changed in abundance during cellulose fermentation in favor of glycosidases and extracellular binding proteins. Mass spectrometric characterizations, together with cellulase activity measurements, revealed a substantial abundance increase of a few bifunctional multidomain glycosidases composed of glycosidase (GH) domain family 5, 9, 10, 44, or 48 and family 3 carbohydrate binding (CBM3) modules. In addition to their orthologous cellulases, the organisms expressed unique glycosidases with different domain organizations: C. obsidiansis expressed the COB47_1671 protein with GH10/5 domains, while C. bescii expressed the Athe_1857 (GH10/48) and Athe_1859 (GH5/44) proteins. Glycosidases containing CBM3 domains were selectively enriched via binding to amorphous cellulose. Preparations from both bacteria contained highly thermostable enzymes with optimal cellulase activities at 85°C and pH 5. The C. obsidiansis preparation, however, had higher cellulase specific activity and greater thermostability. The C. bescii culture produced more extracellular protein and additional SDS-PAGE bands that demonstrated glycosidase activity.
机译：嗜热革兰氏阳性细菌Caldicellulosiruptor bescii和Caldicelluloiruptor obsidiansis有效地降解了纤维素和半纤维素，这使其成为木质纤维素生物质解构以生产可持续生物燃料的相关模型。为了鉴定来自C. bescii和Cobisdiansis的分泌型纤维素分解设备的共有和独特特征，使用无标签定量蛋白质组学分析了在结晶纤维素上发酵生长过程中的蛋白质丰度。两种生物的分泌组均由400多种蛋白质组成，其中最丰富的是多域糖苷酶，细胞外溶质结合蛋白，鞭毛蛋白，假定的果胶裂合酶和具有预测分泌信号的未表征的蛋白质。在鉴定出的蛋白质中，有53至57种蛋白质在纤维素发酵过程中的丰度发生了显着变化，有利于糖苷酶和细胞外结合蛋白。质谱表征以及纤维素酶活性测量表明，由糖苷酶（GH）域家族5、9、10、44或48和家族3碳水化合物结合（CBM3）模块组成的一些双功能多域糖苷酶的丰度大大提高。除直系纤维素酶外，这些生物还表达具有不同域结构的独特糖苷酶：黑曜石病菌表达具有GH10 / 5域的COB47_1671蛋白，而贝氏梭菌则表达Athe_1857（GH10 / 48）和Athe_1859（GH5 / 44）蛋白。 。含有CBM3结构域的糖苷酶通过与无定形纤维素结合而选择性富集。两种细菌的​​制剂均含有高度热稳定的酶，在85°C和pH 5时具有最佳的纤维素酶活性。然而，黑曜石芽孢杆菌制剂具有较高的纤维素酶比活性和较高的热稳定性。贝氏梭状芽胞杆菌培养物产生更多的胞外蛋白和另外的显示糖苷酶活性的SDS-PAGE带。

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