A fast, sensitive and fluorescent LPMO activity assay

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Lytic polysaccharide monooxygenases (LPMOs) are industrially relevant enzymes that utilize a copper co-factor and an oxygen species to break down recalcitrant polysaccharides. These enzymes are secreted by microorganisms and are used in lignocellulosic refineries. As such, they are interesting from both the ecological/biological and industrial perspectives. Here we describe the development of a new fluorescence-based kinetic LPMO activity assay. The assay is based on the enzymatic production of fluorescein from its reduced counterpart. The assay can detect as little as 1 nM LPMO with optimized assay conditions. Furthermore, the reduced fluorescein substrate can also be used to identify peroxidase activity as seen by the formation of fluorescein by horseradish peroxidase. The assay was shown to work well at relatively low H2O2 and dehydroascorbate concentrations. The applicability of the assay was demonstrated.

TidsskriftFrontiers in Microbiology
StatusUdgivet - 2023

Bibliografisk note

Funding Information:
The authors thank Cristina Hernández Rollán and co-workers, The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark, for the expression of Pf CopC, Lp AA10A, Ls AA10A, and Sc AA10B. Furthermore we thank Meike Burrow for providing space at the Department of Plant and Environmental Sciences.

Funding Information:
This study was supported by the Novo Nordisk Foundation, grant nos. NNF17SA0027704 and NNF20OC0059697 to KJ.

Publisher Copyright:
Copyright © 2023 Ipsen, Johansen and Brander.

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