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Background <p>Anaerobic microbes produce multienzyme complexes known as cellulosomes to enhance the degradation of cellulosic substrates. These complexes localize diverse enzymes onto a protein scaffold, where proteins are anchored by dockerin domains. Although the cellulosomes of anaerobic fungi incorporate a broad array of cellulolytic enzymes, they remain largely unexplored. Notably, some fungal cellulosomes reportedly comprise expansin-like proteins with potential to disrupt cellulose networks. While two bacterial cellulosomal expansin-like proteins have been characterized, no fungal cellulosomal expansin-like proteins have been&#xa0;functionally characterized to date.</p> Results <p>Sequence analyses of expansin-like proteins from the anaerobic fungus <i>Neocallimastix californiae</i> revealed similar N-terminal domains among proteins with or without appended dockerins. Those without dockerins, however, consistently lacked the first conserved aromatic residue that forms the substrate binding surface of the C-terminal family 63 carbohydrate binding module. One cellulosomal expansin-like protein from <i>N. californiae</i> (<i>Nca</i>EXLX1) was recombinantly expressed with and without (<i>Nca</i>EXLX1tr) the dockerin domains. The adsorption characteristics of <i>Nca</i>EXLX1 and <i>Nca</i>EXLX1tr, and impact on cellulase (Cel7B) activity, were then investigated using quartz crystal microbalance with dissipation (QCM-D). <i>Nca</i>EXLX1 exhibited higher binding to cellulose nanofibrils (CNF) compared to <i>Nca</i>EXLX1tr. Despite the lower binding of <i>Nca</i>EXLX1tr to CNF, both <i>Nca</i>EXLX1 and <i>Nca</i>EXLX1tr enhanced the action of Cel7B to similar extents.</p> Conclusions <p>This study reports the production and characterization of a&#xa0;fungal cellulosomal expansin-like protein. The corresponding <i>Nca</i>EXLX1 protein&#xa0;and truncated variant were shown to enhance the activity of an endoglucanase, similar to observations made with non-cellulosomal expansin-like proteins. Notably, the improvement in cellulase activity upon the addition of <i>Nca</i>EXLX1 or <i>Nca</i>EXLX1tr was not correlated to extent of substrate binding.</p> Graphical Abstract <p></p>

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Functional characterization of a dockerin-containing expansin-like protein from the anaerobic fungus Neocallimastix californiae

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月3日

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  • Taru Koitto,
  • Anna Pohto,
  • Elizaveta Sidorova,
  • Thu V. Vuong,
  • Merja Penttilä,
  • Emma R. Master

摘要

Background

Anaerobic microbes produce multienzyme complexes known as cellulosomes to enhance the degradation of cellulosic substrates. These complexes localize diverse enzymes onto a protein scaffold, where proteins are anchored by dockerin domains. Although the cellulosomes of anaerobic fungi incorporate a broad array of cellulolytic enzymes, they remain largely unexplored. Notably, some fungal cellulosomes reportedly comprise expansin-like proteins with potential to disrupt cellulose networks. While two bacterial cellulosomal expansin-like proteins have been characterized, no fungal cellulosomal expansin-like proteins have been functionally characterized to date.

Results

Sequence analyses of expansin-like proteins from the anaerobic fungus Neocallimastix californiae revealed similar N-terminal domains among proteins with or without appended dockerins. Those without dockerins, however, consistently lacked the first conserved aromatic residue that forms the substrate binding surface of the C-terminal family 63 carbohydrate binding module. One cellulosomal expansin-like protein from N. californiae (NcaEXLX1) was recombinantly expressed with and without (NcaEXLX1tr) the dockerin domains. The adsorption characteristics of NcaEXLX1 and NcaEXLX1tr, and impact on cellulase (Cel7B) activity, were then investigated using quartz crystal microbalance with dissipation (QCM-D). NcaEXLX1 exhibited higher binding to cellulose nanofibrils (CNF) compared to NcaEXLX1tr. Despite the lower binding of NcaEXLX1tr to CNF, both NcaEXLX1 and NcaEXLX1tr enhanced the action of Cel7B to similar extents.

Conclusions

This study reports the production and characterization of a fungal cellulosomal expansin-like protein. The corresponding NcaEXLX1 protein and truncated variant were shown to enhance the activity of an endoglucanase, similar to observations made with non-cellulosomal expansin-like proteins. Notably, the improvement in cellulase activity upon the addition of NcaEXLX1 or NcaEXLX1tr was not correlated to extent of substrate binding.

Graphical Abstract