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An optimized microplate assay system for quantitative evaluation of plant cell wall–degrading enzyme activity of fungal culture extracts†

  1. Brian C. King1,
  2. Marie K. Donnelly2,
  3. Gary C. Bergstrom1,
  4. Larry P. Walker2,
  5. Donna M. Gibson1,3,*

Article first published online: 29 SEP 2008

DOI: 10.1002/bit.22151

Issue

Biotechnology and Bioengineering

Biotechnology and Bioengineering

Volume 102, Issue 4, pages 1033–1044, 1 March 2009

Additional Information

How to Cite

King, B. C., Donnelly, M. K., Bergstrom, G. C., Walker, L. P. and 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. doi: 10.1002/bit.22151

Author Information

  1. 1

    Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, New York 14853; telephone: 607-255-2359; fax: 607-255-2739

  2. 2

    Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853

  3. 3

    USDA Agricultural Research Service, Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853

*Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, New York 14853; telephone: 607-255-2359; fax: 607-255-2739.

  1. †

    Brian C. King and Marie K. Donnelly contributed equally to this work.

Publication History

  1. Issue published online: 20 JAN 2009
  2. Article first published online: 29 SEP 2008
  3. Accepted manuscript online: 29 SEP 2008 12:00AM EST
  4. Manuscript Accepted: 22 SEP 2008
  5. Manuscript Revised: 25 AUG 2008
  6. Manuscript Received: 30 JUN 2008

Funded by

  • Cornell University
  • Department of Plant Pathology and Plant-Microbe Biology
  • USDA-ARS
  • New York State Foundation for Science, Technology and Innovation (NYSTAR)

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Keywords:

  • bioethanol;
  • biomass conversion;
  • cellulase;
  • cell wall hydrolysis;
  • high throughput screening;
  • plant pathogens

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. Biotechnol. Bioeng. 2009;102: 1033–1044. © 2008 Wiley Periodicals, Inc.

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