A novel approach to analyze the impact of lytic polysaccharide monooxygenases (LPMOs) on cellulosic fibres

Irina Sulaeva; David Budischowsky; Jenni Rahikainen; Kaisa Marjamaa; Fredrik Gjerstad Støpamo; Hajar Khaliliyan; Ivan Melikhov; Thomas Rosenau; Kristiina Kruus; Anikó Várnai; Vincent G.H. Eijsink; Antje Potthast

doi:10.1016/j.carbpol.2023.121696

A novel approach to analyze the impact of lytic polysaccharide monooxygenases (LPMOs) on cellulosic fibres

Irina Sulaeva, David Budischowsky, Jenni Rahikainen, Kaisa Marjamaa, Fredrik Gjerstad Støpamo, Hajar Khaliliyan, Ivan Melikhov, Thomas Rosenau, Kristiina Kruus, Anikó Várnai, Vincent G.H. Eijsink, Antje Potthast^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Scientific › peer-review

9 Citations (Scopus)

Abstract

Enzymatic treatment of cellulosic fibres is a green alternative to classical chemical modification. For many applications, mild procedures for cellulose alteration are sufficient, in which the fibre structure and, therefore, the mechanical performance of cellulosic fibres are preserved. Lytic polysaccharide monooxygenases (LPMOs) bear a great potential to become a green reagent for such targeted cellulose modifications. An obstacle for wide implementation of LPMOs in tailored cellulose chemistry is the lack of suitable techniques to precisely monitor the LPMO impact on the polymer. Soluble oxidized cello-oligomers can be quantified using chromatographic and mass-spectrometric techniques. A considerable portion of the oxidized sites, however, remain on the insoluble cellulose fibres, and their quantification is difficult. Here, we describe a method for the simultaneous quantification of oxidized sites on cellulose fibres and changes in their molar mass distribution after treatment with LPMOs. The method is based on quantitative, heterogeneous, carbonyl-selective labelling with a fluorescent label (CCOA) followed by cellulose dissolution and size-exclusion chromatography (SEC). Application of the method to reactions of seven different LPMOs with pure cellulose fibres revealed pronounced functional differences between the enzymes, showing that this CCOA/SEC/MALS method is a promising tool to better understand the catalytic action of LPMOs.

Original language	English
Article number	121696
Journal	Carbohydrate Polymers
Volume	328
DOIs	https://doi.org/10.1016/j.carbpol.2023.121696
Publication status	Published - 15 Mar 2024
MoE publication type	A1 Journal article-refereed

Funding

The financial support of ERA-NET Cofund Action “ForestValue” which includes the Academy of Finland (grant number 326359 ), the Research Council of Norway (grant agreement no. 297907 ) and the Austrian Federal Ministry of Agriculture, Forestry, Environment and Water Management (BMLFUW, Project 101379 ) is gratefully acknowledged. ForestValue has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement N° 773324. The support of the Austrian Biorefinery Center Tulln (ABCT-II) is gratefully acknowledged. The authors would like to thank Dr. Sonja Schiehser for support with SEC measurements. VTT expresses gratitude to support by the FinnCERES Materials Bioeconomy Ecosystem and thanks Riitta Alander for technical assistance. NMBU also acknowledges financial support from the Research Council of Norway through the infrastructure grant 270038 . Novozymes and Kasper Bay Tingsted and Pedro Loureiro are thanked for provision of enzymes and guidance for their use.

Keywords

Carbonyl
Cellulose
Oxidation
Size exclusion chromatography
Solid fraction analysis

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10.1016/j.carbpol.2023.121696Licence: CC BY

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Sulaeva, I., Budischowsky, D., Rahikainen, J., Marjamaa, K., Støpamo, F. G., Khaliliyan, H., Melikhov, I., Rosenau, T., Kruus, K., Várnai, A., Eijsink, V. G. H., & Potthast, A. (2024). A novel approach to analyze the impact of lytic polysaccharide monooxygenases (LPMOs) on cellulosic fibres. Carbohydrate Polymers, 328, Article 121696. https://doi.org/10.1016/j.carbpol.2023.121696

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AU - Marjamaa, Kaisa

AU - Støpamo, Fredrik Gjerstad

AU - Khaliliyan, Hajar

AU - Melikhov, Ivan

AU - Rosenau, Thomas

AU - Kruus, Kristiina

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AU - Eijsink, Vincent G.H.

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AB - Enzymatic treatment of cellulosic fibres is a green alternative to classical chemical modification. For many applications, mild procedures for cellulose alteration are sufficient, in which the fibre structure and, therefore, the mechanical performance of cellulosic fibres are preserved. Lytic polysaccharide monooxygenases (LPMOs) bear a great potential to become a green reagent for such targeted cellulose modifications. An obstacle for wide implementation of LPMOs in tailored cellulose chemistry is the lack of suitable techniques to precisely monitor the LPMO impact on the polymer. Soluble oxidized cello-oligomers can be quantified using chromatographic and mass-spectrometric techniques. A considerable portion of the oxidized sites, however, remain on the insoluble cellulose fibres, and their quantification is difficult. Here, we describe a method for the simultaneous quantification of oxidized sites on cellulose fibres and changes in their molar mass distribution after treatment with LPMOs. The method is based on quantitative, heterogeneous, carbonyl-selective labelling with a fluorescent label (CCOA) followed by cellulose dissolution and size-exclusion chromatography (SEC). Application of the method to reactions of seven different LPMOs with pure cellulose fibres revealed pronounced functional differences between the enzymes, showing that this CCOA/SEC/MALS method is a promising tool to better understand the catalytic action of LPMOs.

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A novel approach to analyze the impact of lytic polysaccharide monooxygenases (LPMOs) on cellulosic fibres

Abstract

Funding

Keywords

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