Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.
Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.
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DOI：
10.1038/srep43117
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发表时间：
2017-02-21
影响因子：
4.6
通讯作者：
Flitsch SL
中科院分区：
文献类型：
--
作者：
van Munster JM;Thomas B;Riese M;Davis AL;Gray CJ;Archer DB;Flitsch SL
可再生生物技术依靠酶将植物木质纤维素降解为单糖，转化为燃料或高价值产品。这种木质纤维素降解酶的鉴定和表征可以通过酶活性测量方法的可用性来快速跟踪，所述酶活性测量方法是快速的、无标记的、使用最少的资源并且允许直接鉴定所产生的产物。我们开发了这样一种方法，通过应用碳水化合物阵列结合MALDI-ToF质谱鉴定丝状真菌尼日尔的碳水化合物活性酶（CAZymes）的反应产物。我们描述了植物多糖衍生的低聚糖的生产和表征，以及它们与金靶上疏水自组装单分子膜的连接。我们使用商业酶验证了该阵列用于检测外作用和内作用糖苷水解酶活性的有效性，并展示了该平台如何适用于检测相关生物样品中的酶活性，即A.用麦草种的尼日尔。总之，这种通用方法广泛适用于筛选和表征CAZymes的活性，例如与生物技术应用如生物燃料生产、食品和动物饲料工业相关的用于植物木质纤维素降解的真菌酶。
Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.