Continuous photometric activity assays for lytic polysaccharide monooxygenase—Critical assessment and practical considerations

Schwaiger, Lorenz; Zenone, Alice; Csarman, Florian; Ludwig, Roland

doi:10.1016/bs.mie.2022.08.054

Cited by 2 publications

(2 citation statements)

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“…Addition of ascorbate also enhances the enzyme activity to a similar level, but the full effect is reached at 3–7.5 μM and adding more gives a diminishing effect ( Supplementary Figure S2 ). Ascorbate is a known co-substrate for LPMO activity, but it can interfere with the LPMO assays through radical scavenging, H 2 O 2 formation and even enzyme autooxidation ( Schwaiger et al, 2023 ). DHA has not been shown to have similar interferences and was chosen as the preferred co-susbstrate.…”

Section: Resultsmentioning

confidence: 99%

A fast, sensitive and fluorescent LPMO activity assay

2023

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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.

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Section: Resultsmentioning

confidence: 99%

A fast, sensitive and fluorescent LPMO activity assay

2023

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“…To gain further insight into the properties of TrAA14A and PcoAA14A, we assessed the abilities of these proteins to consume and produce H 2 O 2 , as these are considered common features of LPMOs [49,55,65,66]. The production of hydrogen peroxide results from the oxidase activity of LPMOs, which implies that, in the absence of substrate, the reduced LPMO activates molecular oxygen.…”

Section: Production and Consumption Of H 2 Omentioning

confidence: 99%

Revisiting the AA14 family of lytic polysaccharide monooxygenases and their catalytic activity

et al. 2023

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Lytic polysaccharide monooxygenases (LPMOs) belonging to the AA14 family are believed to contribute to the enzymatic degradation of lignocellulosic biomass by specifically acting on xylan in recalcitrant cellulose‐xylan complexes. Functional characterization of an AA14 LPMO from Trichoderma reesei, TrAA14A, and a re‐evaluation of the properties of the previously described AA14 from Pycnoporus coccineus, PcoAA14A, showed that these proteins have oxidase and peroxidase activities that are common for LPMOs. However, we were not able to detect activity on cellulose‐associated xylan or any other tested polysaccharide substrate, meaning that the substrate of these enzymes remains unknown. Next to raising questions regarding the true nature of AA14 LPMOs, the present data illustrate possible pitfalls in the functional characterization of these intriguing enzymes.

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Continuous photometric activity assays for lytic polysaccharide monooxygenase—Critical assessment and practical considerations

Cited by 2 publications

References 68 publications

A fast, sensitive and fluorescent LPMO activity assay

A fast, sensitive and fluorescent LPMO activity assay

Revisiting the AA14 family of lytic polysaccharide monooxygenases and their catalytic activity

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