King Brian C, Donnelly Marie K, Bergstrom Gary C, Walker Larry P, Gibson Donna M
Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, New York 14853, USA.
Biotechnol Bioeng. 2009 Mar 1;102(4):1033-44. doi: 10.1002/bit.22151.
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 some 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, carboxymethylcellulose, 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.
开发与当前纤维素酶系统互补的用于纤维素生物质水解的酶混合物，是经济可行的生物燃料生产所需的关键一步。最近的基因组分析表明，一些植物病原真菌可能是一个很大程度上未被开发的资源，可从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法，开发了一种标准化的微孔板测定法，用于快速分析真菌提取物对多糖的水解作用，并结合生物质底物。将真菌在含纤维素或柳枝稷的培养基上培养10天，以制备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从滤纸、微晶纤维素、玉米秸秆、柳枝稷、羧甲基纤维素和阿拉伯木聚糖中释放的还原糖进行定量。在真菌种类、生长培养基组成、测定底物和温度之间发现了显著的相互作用。在一小部分植物病原真菌中，一些提取物的粗酶活性与里氏木霉相当或更高，特别是在较低温度下和在生物质底物上进行测定时。这种微孔板测定系统对于高通量生物勘探新的生物燃料生产新型酶来源应该是有用的。
