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Colorimetric LPMO assay with direct implication for cellulolytic activity.

Søren Brander1, Stine Lausten2, Johan Ø Ipsen2

  • 1Department of Geosciences and Natural Resource Management, University of Copenhagen, 1958, Copenhagen, Denmark.

Biotechnology for Biofuels
|February 28, 2021
PubMed
Summary
This summary is machine-generated.

A new assay uses a dye to rapidly measure lytic polysaccharide monooxygenase (LPMO) activity. This method is specific for LPMOs and can be used for high-throughput screening and enzyme characterization.

Keywords:
CelluloseDehydroascorbateEnzyme assayHigh throughputLytic polysaccharide monooxygenasePhenolphthalein

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Area of Science:

  • Biochemistry
  • Enzymology
  • Industrial Biotechnology

Background:

  • Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes for degrading recalcitrant polymers like cellulose and chitin.
  • Existing LPMO activity assays are either time-consuming (HPLC) or lack specificity for high-throughput screening.
  • A need exists for a fast, specific assay to facilitate LPMO discovery and characterization.

Purpose of the Study:

  • To develop a novel, rapid, and specific assay for measuring lytic polysaccharide monooxygenase (LPMO) activity.
  • To enable high-throughput screening and accelerate the biochemical characterization of LPMOs.
  • To explore new co-substrates for LPMO activity and their implications for enzyme function.

Main Methods:

  • Developed a colorimetric assay based on the oxidation of reduced phenolphthalein (rPHP) to phenolphthalein (PHP) catalyzed by LPMOs.
  • Investigated the effect of co-substrates, notably dehydroascorbate (DHA), on rPHP oxidation.
  • Optimized the assay for sensitivity, kinetics, and specificity using the cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A).

Main Results:

  • The novel assay demonstrated high specificity for LPMOs among 12 tested metalloenzymes.
  • Addition of dehydroascorbate (DHA) significantly enhanced the color response, increasing sensitivity to 15 nM within 30 minutes.
  • The assay followed Michaelis-Menten kinetics and was used to determine kinetic parameters (kcat, KM), copper-enzyme binding, and thermal stability.

Conclusions:

  • A convenient, high-throughput microtiter plate-compatible assay for LPMO activity was successfully developed.
  • Dehydroascorbate (DHA) was identified as a highly effective and specific co-substrate for rPHP oxidation by LPMOs.
  • The assay revealed that DHA and fructose can facilitate both rPHP oxidation and cellulose degradation, expanding understanding of LPMO catalysis and industrial applications.