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Background <p>Fungal AA9 lytic polysaccharide monooxygenases (LPMOs) play a central role in oxidative cellulose degradation and are key contributors to biomass conversion. While widely distributed in fungal genomes, many LPMOs remain uncharacterized, limiting our understanding of their functional diversity and biotechnological potential. <i>Fusarium oxysporum</i>, a genetically rich but underexplored species, contains several AA9 LPMOs. This study focuses on the characterization of one such enzyme named <i>Fo</i>LPMO9A, demonstrating its role in lignocellulose degradation and its potential for cellulose-based material functionalization.</p> Results <p>The gene encoding <i>Fo</i>LPMO9A was heterologously expressed in <i>Pichia pastoris</i>, and the produced recombinant protein was evaluated for its biochemical and functional properties, revealing C1/C4 regioselectivity on cellulosic substrates and higher H₂O₂ production compared to other AA9 fungal LPMOs. Its activity was assessed through the release of oxidized cello-oligosaccharides from various substrates and its synergistic action with cellulases. The stronger synergistic effect on lignocellulosic biomass likely reflects the enzyme’s ability to target complex, less accessible structures, where oxidative cleavage complements cellulase activity. Attention was given to how <i>Fo</i>LPMO9A responds to phenolic compounds and lignin-derived fractions, both in their native form and after enzymatic oxidation by redox enzymes such as laccases and polyphenol oxidases. In the case of lignin-derived fractions, the goal was to assess how these modifications influence their ability to drive <i>Fo</i>LPMO9A activity as redox-active electron donors. Building on its characterized activity, <i>Fo</i>LPMO9A was applied in two biotechnological contexts. First, it was integrated into a three-step enzymatic process for isolating micro-fibrillated cellulose from OxiOrganosolv-pretreated wheat straw. This treatment led to enhanced fibre disruption and finer fibrils, confirmed by determination of fibre diameter distribution, while fluorescence labelling with Rhodamine 110 verified C1-specific oxidation. Second, <i>Fo</i>LPMO9A was applied to bacterial nanocellulose produced by <i>Komagataeibacter medellinensis</i>, where successful oxidation and introduction of carboxyl groups were confirmed, demonstrating its potential to modify material properties.</p> Conclusions <p>This study underscores the versatile catalytic role of AA9 LPMOs in synergistic enzyme systems, emphasizing their impact on cellulose degradation and modification. The findings highlight the need for further exploration of LPMO mechanisms in biomaterial development and industrial applications.</p>

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Characterization of an AA9 LPMO from Fusarium oxysporum and its role in cellulose hydrolysis and functionalization

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月28日

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  • Koar Chorozian,
  • Maria Dimarogona,
  • Marijana Ponjavic,
  • Jasmina Nikodinovic-Runic,
  • Evangelos Topakas,
  • Anthi Karnaouri

摘要

Background

Fungal AA9 lytic polysaccharide monooxygenases (LPMOs) play a central role in oxidative cellulose degradation and are key contributors to biomass conversion. While widely distributed in fungal genomes, many LPMOs remain uncharacterized, limiting our understanding of their functional diversity and biotechnological potential. Fusarium oxysporum, a genetically rich but underexplored species, contains several AA9 LPMOs. This study focuses on the characterization of one such enzyme named FoLPMO9A, demonstrating its role in lignocellulose degradation and its potential for cellulose-based material functionalization.

Results

The gene encoding FoLPMO9A was heterologously expressed in Pichia pastoris, and the produced recombinant protein was evaluated for its biochemical and functional properties, revealing C1/C4 regioselectivity on cellulosic substrates and higher H₂O₂ production compared to other AA9 fungal LPMOs. Its activity was assessed through the release of oxidized cello-oligosaccharides from various substrates and its synergistic action with cellulases. The stronger synergistic effect on lignocellulosic biomass likely reflects the enzyme’s ability to target complex, less accessible structures, where oxidative cleavage complements cellulase activity. Attention was given to how FoLPMO9A responds to phenolic compounds and lignin-derived fractions, both in their native form and after enzymatic oxidation by redox enzymes such as laccases and polyphenol oxidases. In the case of lignin-derived fractions, the goal was to assess how these modifications influence their ability to drive FoLPMO9A activity as redox-active electron donors. Building on its characterized activity, FoLPMO9A was applied in two biotechnological contexts. First, it was integrated into a three-step enzymatic process for isolating micro-fibrillated cellulose from OxiOrganosolv-pretreated wheat straw. This treatment led to enhanced fibre disruption and finer fibrils, confirmed by determination of fibre diameter distribution, while fluorescence labelling with Rhodamine 110 verified C1-specific oxidation. Second, FoLPMO9A was applied to bacterial nanocellulose produced by Komagataeibacter medellinensis, where successful oxidation and introduction of carboxyl groups were confirmed, demonstrating its potential to modify material properties.

Conclusions

This study underscores the versatile catalytic role of AA9 LPMOs in synergistic enzyme systems, emphasizing their impact on cellulose degradation and modification. The findings highlight the need for further exploration of LPMO mechanisms in biomaterial development and industrial applications.