Colorimetric LPMO assay with direct implication for cellulolytic activity

Søren Brander, Stine Lausten, Johan Ipsen, Kristoffer B. Falkenberg, Andreas B. Bertelsen, Morten H.H. Nørholm, Lars H. Østergaard, Katja S. Johansen*

*Corresponding author for this work

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Background: Lytic polysaccharide monooxygenases (LPMOs) are important industrial enzymes known for their catalytic degradation of recalcitrant polymers such as cellulose or chitin. Their activity can be measured by lengthy HPLC methods, while high-throughput methods are less specific. A fast and specific LPMO assay would simplify screening for new or engineered LPMOs and accelerate biochemical characterization. Results: A novel LPMO activity assay was developed based on the production of the dye phenolphthalein (PHP) from its reduced counterpart (rPHP). The colour response of rPHP oxidisation catalysed by the cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A), was found to increase tenfold by adding dehydroascorbate (DHA) as a co-substrate. The assay using a combination of rPHP and DHA was tested on 12 different metallo-enzymes, but only the LPMOs catalysed this reaction. The assay was optimized for characterization of TaAA9A and showed a sensitivity of 15 nM after 30 min incubation. It followed apparent Michaelis–Menten kinetics with kcat = 0.09 s−1 and KM = 244 µM, and the assay was used to confirm stoichiometric copper–enzyme binding and enzyme unfolding at a temperature of approximately 60 °C. DHA, glutathione and fructose were found to enhance LPMO oxidation of rPHP and in the optimized assay conditions these co-substrates also enabled cellulose degradation. Conclusions: This novel and specific LPMO assay can be carried out in a convenient microtiter plate format ready for high-throughput screening and enzyme characterization. DHA was the best co-substrate tested for oxidation of rPHP and this preference appears to be LPMO-specific. The identified co-substrates DHA and fructose are not normally considered as LPMO co-substrates but here they are shown to facilitate both oxidation of rPHP and degradation of cellulose. This is a rare example of a finding from a high-throughput assay that directly translate into enzyme activity on an insoluble substrate. The rPHP-based assay thus expands our understanding of LPMO catalysed reactions and has the potential to characterize LPMO activity in industrial settings, where usual co-substrates such as ascorbate and oxygen are depleted.

Original languageEnglish
Article number51
JournalBiotechnology for Biofuels
Issue number1
Publication statusPublished - Dec 2021

Bibliographical note

Funding Information:
This study was supported by the Novo Nordisk Foundation, Grant No. NNF17SA0027704 to KSJ.

Funding Information:
We thank Cristina Hern?ndez Roll?n, The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark, for the expression of PfCopC. We thank professors Meike Burow and Henrik Frisenvang for the sample of MYB28.

Publisher Copyright:
© 2021, The Author(s).


  • Cellulose
  • Dehydroascorbate
  • Enzyme assay
  • High throughput
  • Lytic polysaccharide monooxygenase
  • Phenolphthalein


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