The impact of the carbohydrate-binding module on how a lytic polysaccharide monooxygenase modifies cellulose fibers

Fredrik G. Støpamo, Irina Sulaeva, David Budischowsky, Jenni Rahikainen, Kaisa Marjamaa, Kristiina Kruus, Antje Potthast, Vincent G.H. Eijsink, Anikó Várnai*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Background: In recent years, lytic polysaccharide monooxygenases (LPMOs) that oxidatively cleave cellulose have gained increasing attention in cellulose fiber modification. LPMOs are relatively small copper-dependent redox enzymes that occur as single domain proteins but may also contain an appended carbohydrate-binding module (CBM). Previous studies have indicated that the CBM “immobilizes” the LPMO on the substrate and thus leads to more localized oxidation of the fiber surface. Still, our understanding of how LPMOs and their CBMs modify cellulose fibers remains limited. 

Results: Here, we studied the impact of the CBM on the fiber-modifying properties of NcAA9C, a two-domain family AA9 LPMO from Neurospora crassa, using both biochemical methods as well as newly developed multistep fiber dissolution methods that allow mapping LPMO action across the fiber, from the fiber surface to the fiber core. The presence of the CBM in NcAA9C improved binding towards amorphous (PASC), natural (Cell I), and alkali-treated (Cell II) cellulose, and the CBM was essential for significant binding of the non-reduced LPMO to Cell I and Cell II. Substrate binding of the catalytic domain was promoted by reduction, allowing the truncated CBM-free NcAA9C to degrade Cell I and Cell II, albeit less efficiently and with more autocatalytic enzyme degradation compared to the full-length enzyme. The sequential dissolution analyses showed that cuts by the CBM-free enzyme are more evenly spread through the fiber compared to the CBM-containing full-length enzyme and showed that the truncated enzyme can penetrate deeper into the fiber, thus giving relatively more oxidation and cleavage in the fiber core. 

Conclusions: These results demonstrate the capability of LPMOs to modify cellulose fibers from surface to core and reveal how variation in enzyme modularity can be used to generate varying cellulose-based materials. While the implications of these findings for LPMO-based cellulose fiber engineering remain to be explored, it is clear that the presence of a CBM is an important determinant of the three-dimensional distribution of oxidation sites in the fiber.

Original languageEnglish
Article number118
Number of pages16
JournalBiotechnology for Biofuels and Bioproducts
Volume17
Issue number1
DOIs
Publication statusPublished - 24 Aug 2024
MoE publication typeA1 Journal article-refereed

Keywords

  • AA9 LPMOs
  • Carbonyl detection
  • CBM
  • Cellulose
  • Enzymatic fiber engineering
  • Fluorescence
  • Functional variation
  • Oxidation
  • Size exclusion chromatography

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