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United States Department of Agriculture

Agricultural Research Service

Title: An Optimized Microplate Assay System for Quantitative Evaluation of Plant Cell Wall Degrading Enzyme Activity of Fungal Culture Extracts

Authors
item King, B. - CORNELL UNIVERSITY
item Donnelly, M. - CORNELL UNIVERSITY
item Bergstrom, G. - CORNELL UNIVERSITY
item Walker, L. - CORNELL UNIVERSITY
item Gibson, Donna

Submitted to: Biotechnology and Bioengineering
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 14, 2008
Publication Date: March 1, 2009
Citation: King, B.C., Donnelly, M.K., Bergstrom, G.C., Walker, L.P., Gibson, D.M. 2009. An Optimized Microplate Assay System for Quantitative Evaluation of Plant Cell Wall Degrading Enzyme Activity of Fungal Culture Extracts. Biotechnology and Bioengineering. 102:1033-1044.

Interpretive Summary: Breakdown of plant cell walls to fermentable sugars is a critical step in the conversion process to bioethanol. The most effective means of breakdown uses a cocktail of different cellulose-degrading enzymes, but conversion is still not optimal so there is a need to identify enzymes to enhance the breakdown process. During this study, we designed a high throughput platform for assessing complex carbohydrate breakdown using standard substrates as well as biomass substrates. We chose to investigate a limited number of plant pathogenic fungi in comparison to the industrial standard. This screening system should prove useful for high throughput bioprospecting for new sources of novel enzymes for biofuel production.

Technical Abstract: 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.

Last Modified: 9/3/2014
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