A fast, sensitive and fluorescent LPMO activity assay
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...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-03-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1128470/full |
| _version_ | 1797871282064195584 |
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| author | Johan Ø. Ipsen Katja S. Johansen Søren Brander |
| author_facet | Johan Ø. Ipsen Katja S. Johansen Søren Brander |
| author_sort | Johan Ø. Ipsen |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T00:40:22Z |
| format | Article |
| id | doaj.art-28a77f14e4a442bd90f061f004cf829a |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-10T00:40:22Z |
| publishDate | 2023-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-28a77f14e4a442bd90f061f004cf829a2023-03-14T05:50:16ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-03-011410.3389/fmicb.2023.11284701128470A fast, sensitive and fluorescent LPMO activity assayJohan Ø. IpsenKatja S. JohansenSøren BranderLytic 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.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1128470/fullfluorescinredox activitycopper enzymeperoxideLPMOenzyme assay |
| spellingShingle | Johan Ø. Ipsen Katja S. Johansen Søren Brander A fast, sensitive and fluorescent LPMO activity assay Frontiers in Microbiology fluorescin redox activity copper enzyme peroxide LPMO enzyme assay |
| title | A fast, sensitive and fluorescent LPMO activity assay |
| title_full | A fast, sensitive and fluorescent LPMO activity assay |
| title_fullStr | A fast, sensitive and fluorescent LPMO activity assay |
| title_full_unstemmed | A fast, sensitive and fluorescent LPMO activity assay |
| title_short | A fast, sensitive and fluorescent LPMO activity assay |
| title_sort | fast sensitive and fluorescent lpmo activity assay |
| topic | fluorescin redox activity copper enzyme peroxide LPMO enzyme assay |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1128470/full |
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