A fast and sensitive activity assay for lytic polysaccharide monooxygenase

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Abstract

Background

Lytic polysaccharide monooxygenases (LPMO) release a spectrum of cleavage products from their polymeric substrates cellulose, hemicellulose, or chitin. The correct identification and quantitation of these released products is the basis of MS/HPLC-based detection methods for LPMO activity. The duration, effort, and intricate analysis allow only specialized laboratories to measure LPMO activity in day-to-day work. A spectrophotometric assay will simplify the screening for LPMO in culture supernatants, help monitor recombinant LPMO expression and purification, and support enzyme characterization.

Results

Based on a newly discovered peroxidase activity of LPMO, we propose a fast, robust, and sensitive spectrophotometric activity assay using 2,6-dimethoxyphenol (2,6-DMP) and H ₂O ₂. The fast enzymatic assay (300 s) consists of 1 mM 2,6-DMP as chromogenic substrate, 100 µM H ₂O ₂ as cosubstrate, and an adequate activity of LPMO in a suitable buffer. The high molar absorption coefficient of the formed product coerulignone ( ε ₄₆₉ = 53,200 M ⁻¹ cm ⁻¹) makes the assay sensitive and allows reliable activity measurements of LPMO in concentrations of approx. 0.5–50 mg L ⁻¹.

Conclusions

The activity assay based on 2,6-DMP detects a novel peroxidase activity of LPMO. This activity can be accurately measured and used for enzyme screening, production, and purification, and can also be applied to study binding constants or thermal stability. However, the assay has to be used with care in crude extracts, because other enzymes such as laccase or peroxidase will interfere with the assay. We also want to stress that the peroxidase activity is a homogeneous reaction with soluble substrates and should not be correlated to heterogeneous LPMO activity on polymeric substrates.

Electronic supplementary material

The online version of this article (10.1186/s13068-018-1063-6) contains supplementary material, which is available to authorized users.

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Most cited references 33

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Limit of blank, limit of detection and limit of quantitation.

David Armbruster, Terry Pry (2008)

* Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) are terms used to describe the smallest concentration of a measurand that can be reliably measured by an analytical procedure. * LoB is the highest apparent analyte concentration expected to be found when replicates of a blank sample containing no analyte are tested. LoB = mean(blank) + 1.645(SD(blank)). * LoD is the lowest analyte concentration likely to be reliably distinguished from the LoB and at which detection is feasible. LoD is determined by utilising both the measured LoB and test replicates of a sample known to contain a low concentration of analyte. * LoD = LoB + 1.645(SD (low concentration sample)). * LoQ is the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met. The LoQ may be equivalent to the LoD or it could be at a much higher concentration.

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Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes

Anthony Levasseur, Elodie Drula, Vincent Lombard … (2013)

Background Since its inception, the carbohydrate-active enzymes database (CAZy; http://www.cazy.org) has described the families of enzymes that cleave or build complex carbohydrates, namely the glycoside hydrolases (GH), the polysaccharide lyases (PL), the carbohydrate esterases (CE), the glycosyltransferases (GT) and their appended non-catalytic carbohydrate-binding modules (CBM). The recent discovery that members of families CBM33 and family GH61 are in fact lytic polysaccharide monooxygenases (LPMO), demands a reclassification of these families into a suitable category. Results Because lignin is invariably found together with polysaccharides in the plant cell wall and because lignin fragments are likely to act in concert with (LPMO), we have decided to join the families of lignin degradation enzymes to the LPMO families and launch a new CAZy class that we name “Auxiliary Activities” in order to accommodate a range of enzyme mechanisms and substrates related to lignocellulose conversion. Comparative analyses of these auxiliary activities in 41 fungal genomes reveal a pertinent division of several fungal groups and subgroups combining their phylogenetic origin and their nutritional mode (white vs. brown rot). Conclusions The new class introduced in the CAZy database extends the traditional CAZy families, and provides a better coverage of the full extent of the lignocellulose breakdown machinery.

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An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides.

Gustav Vaaje-Kolstad, Bjørge Westereng, Svein J. Horn … (2010)

Efficient enzymatic conversion of crystalline polysaccharides is crucial for an economically and environmentally sustainable bioeconomy but remains unfavorably inefficient. We describe an enzyme that acts on the surface of crystalline chitin, where it introduces chain breaks and generates oxidized chain ends, thus promoting further degradation by chitinases. This enzymatic activity was discovered and further characterized by using mass spectrometry and chromatographic separation methods to detect oxidized products generated in the absence or presence of H(2)(18)O or (18)O(2). There are strong indications that similar enzymes exist that work on cellulose. Our findings not only demonstrate the existence of a hitherto unknown enzyme activity but also provide new avenues toward more efficient enzymatic conversion of biomass.

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Author and article information

Contributors

Erik Breslmayr: erik.breslmayr@boku.ac.at

Marija Hanžek: mhanzek3@gmail.com

Aoife Hanrahan: a.hanrahan4@nuigalway.ie

Christian Leitner: christian.leitner@boku.ac.at

Roman Kittl: roman.kittl@boku.ac.at

Božidar Šantek: bsantek@pbf.hr

Chris Oostenbrink: chris.oostenbrink@boku.ac.at

Roland Ludwig:

ORCID: http://orcid.org/0000-0002-5058-5874

roland.ludwig@boku.ac.at

Journal

Journal ID (nlm-ta): Biotechnol Biofuels

Journal ID (iso-abbrev): Biotechnol Biofuels

Title: Biotechnology for Biofuels

Publisher: BioMed Central (London )

ISSN (Electronic): 1754-6834

Publication date (Electronic): 23 March 2018

Publication date PMC-release: 23 March 2018

Publication date Collection: 2018

Volume: 11

Electronic Location Identifier: 79

Affiliations

[1 ]ISNI 0000 0001 2298 5320, GRID grid.5173.0, Department of Food Science and Technology, Vienna Institute of Biotechnology, , BOKU-University of Natural Resources and Life Sciences, ; Muthgasse 18, 1190 Vienna, Austria

[2 ]ISNI 0000 0001 2298 5320, GRID grid.5173.0, Department of Material Science and Process Engineering, Institute of Molecular Modeling and Simulation, , BOKU-University of Natural Resources and Life Sciences, ; Muthgasse 18, 1190 Vienna, Austria

[3 ]ISNI 0000 0001 0657 4636, GRID grid.4808.4, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, , University of Zagreb, ; Pierottijeva 6, 10000 Zagreb, Croatia

Author information

Roland Ludwig http://orcid.org/0000-0002-5058-5874

Article

Publisher ID: 1063

DOI: 10.1186/s13068-018-1063-6

PMC ID: 5865291

PubMed ID: 29588664

SO-VID: d6b2c3c4-042a-48b3-9890-c7e8646021bf

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 19 December 2017

Date accepted : 24 February 2018

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100002428, Austrian Science Fund;

Award ID: I2385-N28

Award ID: W1224

Award Recipient : Chris Oostenbrink Roland Ludwig

Funded by: FundRef http://dx.doi.org/10.13039/501100005203, OeAD-GmbH;

Award ID: HR 12/2016

Award Recipient : Roland Ludwig

Custom metadata

ScienceOpen disciplines: Biotechnology

Keywords: activity assay,2,6-dimethoxyphenol,hydrogen peroxide,biomass degradation,lytic polysaccharide monooxygenase,peroxidase activity

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ScienceOpen disciplines: Biotechnology

Keywords: activity assay, 2,6-dimethoxyphenol, hydrogen peroxide, biomass degradation, lytic polysaccharide monooxygenase, peroxidase activity

A fast and sensitive activity assay for lytic polysaccharide monooxygenase

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Abstract

Background

Results

Conclusions

Electronic supplementary material

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Genome Engineering using CRISPR

Most cited references 33

Limit of blank, limit of detection and limit of quantitation.

Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes

An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides.

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