An Optimized Microplate Assay System for Quantitative Evaluation of Plant Cell Wall-Degrading Enzyme Activity of Fungal Culture Extracts
An Optimized Microplate Assay System for Quantitative Evaluation of Plant Cell Wall-Degrading Enzyme Activity of Fungal Culture Extracts
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DOI：
10.1002/bit.22151
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发表时间：
2009-03-01
影响因子：
3.8
通讯作者：
Gibson, Donna M.
中科院分区：
文献类型：
--
作者：
King, Brian C.;Donnelly, Marie K.;Gibson, Donna M.
开发用于纤维素生物质水解的酶混合物来补充当前的纤维素酶系统是经济上可行的生物燃料生产所需的关键步骤。最近的基因组分析表明，声波植物病原真菌可能是一种很大程度上尚未开发的资源，可以在其中寻找用于生物质转化的新型水解酶。为了开发用于酶生物勘探的高通量筛选测定，开发了一种标准化的微孔板测定，用于快速分析真菌提取物（掺入生物质底物）的多糖水解。真菌在含有纤维素或柳枝稷的培养基上生长 10 天，以产生用于分析的酶提取物。使用基于 3,5-二硝基水杨酸的小型比色测定法对滤纸、Avicel、玉米秆、柳枝稷、羧甲基纤维素和阿拉伯木聚糖释放的还原糖进行定量。真菌种类、生长培养基成分、测定底物和温度之间存在显着的相互作用。在少量植物病原真菌样本中，一些提取物的粗活性与里氏木霉相当或更高，特别是在较低温度和生物质基质上进行测定时。该微孔板检测系统应可用于高通量生物勘探，寻找用于生物燃料生产的新型酶的新来源。
Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that sonic plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose- or switchgrass-containing media to produce enzyme extracts for analysis. Reducing Sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethyl-cellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new Sources of novel enzymes for biofuel production.