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Related Experiment Videos

A universal assay for screening expression libraries for carbohydrases.

P J Meeuwsen1, J P Vincken, G Beldman

  • 1Food Chemistry Group, Department of Food Technology and Nutritional Sciences, Wageningen Agricultural University, Bomenweg 2, NL-6703 HD Wageningen, The Netherlands.

Journal of Bioscience and Bioengineering
|October 20, 2005
PubMed
Summary

A new assay detects all carbohydrases by measuring reducing ends. This method successfully identified three xylogalacturonase-producing clones from Aspergillus tubigensis.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Existing assays for carbohydrases have limitations, failing to detect enzymes with incompatible substrates.
  • Carbohydrases universally increase reducing ends during substrate degradation, a common property exploitable for detection.

Purpose of the Study:

  • To explore the utility of the bicinchoninic acid (BCA) reducing value assay for detecting all types of carbohydrases.
  • To screen a cDNA library of Aspergillus tubigensis for xylogalacturonan-degrading enzymes using the BCA assay.

Main Methods:

  • Utilized the bicinchoninic acid (BCA) reducing value assay, a sensitive method for detecting increases in reducing ends.
  • Screened a Kluyveromyces lactis expression library of Aspergillus tubigensis using the BCA assay and a xylogalacturonan substrate.

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  • Implemented specific precautions to mitigate high background absorbances from culture medium, proteins, and substrate.

    • Main Results:

    • The BCA assay proved effective in detecting carbohydrases, including exo- and endo-acting enzymes simultaneously.
    • Successfully identified three clones from the Aspergillus tubigensis library that produce xylogalacturonase.
    • Demonstrated that background interference in the BCA assay can be managed effectively.

    Conclusions:

    • The bicinchoninic acid (BCA) reducing value assay is a versatile and sensitive tool for detecting a wide range of carbohydrases.
    • This method enables the simultaneous detection of both exo- and endo-acting carbohydrases, overcoming limitations of existing assays.
    • The successful screening of the Aspergillus tubigensis library highlights the applicability of this assay for enzyme discovery.