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Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds
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Methods for identifying lipoxygenase producing microorganisms on agar plates.

Antti Nyyssölä1, Ruud Heshof, Thomas Haarmann

  • 1Wageningen University, Laboratory of Systems and Synthetic Biology, Fungal Systems Biology, Dreijenplein 10, 6703 HB Wageningen, The Netherlands. leo.degraaff@wur.nl.

AMB Express
|March 28, 2012
PubMed
Summary

New plate assays effectively detect lipoxygenase activity in microorganisms. The potassium iodide-starch and indamine dye methods show promise for screening microbial lipoxygenase production and metagenomic libraries.

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Aseptic Laboratory Techniques: Plating Methods
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Aseptic Laboratory Techniques: Plating Methods

Published on: May 11, 2012

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Aseptic Laboratory Techniques: Plating Methods
18:00

Aseptic Laboratory Techniques: Plating Methods

Published on: May 11, 2012

Area of Science:

  • Biochemistry
  • Microbiology
  • Molecular Biology

Background:

  • Lipoxygenases are enzymes involved in various biological processes.
  • Detecting lipoxygenase activity in microorganisms is crucial for research and biotechnology.
  • Existing methods for detecting lipoxygenase activity can be limited in scope or sensitivity.

Purpose of the Study:

  • To develop and evaluate simple plate assay methods for detecting lipoxygenase-producing microorganisms.
  • To compare the effectiveness of different detection methods, including potassium iodide-starch, indamine dye, and β-carotene bleaching.
  • To assess the utility of these assays for identifying lipoxygenase production in genetically modified organisms and for screening metagenomic libraries.

Main Methods:

  • Development of agar plate assays utilizing potassium iodide-starch and indamine dye formation.
  • Application of the β-carotene bleaching method for comparative analysis.
  • Testing the developed assays on various microbial systems, including Trichoderma reesei, Pichia pastoris, Aspergillus nidulans, and Escherichia coli.
  • Inclusion of soybean lipoxygenase and fungal lipoxygenase gene expression in transformants.

Main Results:

  • Both potassium iodide-starch and indamine dye formation methods successfully detected soybean lipoxygenase activity on agar plates.
  • The β-carotene bleaching method also detected activity but with lower sensitivity.
  • Lipoxygenase production was identified in Trichoderma reesei and Pichia pastoris transformants expressing the Gaeumannomyces graminis lipoxygenase gene.
  • The potassium iodide-starch method was effective for detecting G. graminis lipoxygenase in Aspergillus nidulans.
  • The indamine dye formation method clearly detected lipoxygenase activity when applied to Escherichia coli cultures.

Conclusions:

  • Potassium iodide-starch and indamine dye formation are effective and sensitive methods for detecting microbial lipoxygenase activity on agar plates.
  • These assays are suitable for identifying lipoxygenase production in engineered microorganisms.
  • The indamine dye formation method shows potential for screening metagenomic libraries in E. coli for novel lipoxygenase activities.