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

## TL;DR

This study identifies and characterizes a fungal protein that enhances cellulose breakdown, offering insights into how anaerobic fungi digest plant material.

## Contribution

The first functional characterization of a fungal cellulosomal expansin-like protein with dockerin domains.

## Key findings

- NcaEXLX1 binds more strongly to cellulose than its truncated version.
- Both NcaEXLX1 and its truncated form enhance the activity of an endoglucanase.
- Enhanced cellulase activity is not directly linked to the protein's binding strength.

## Abstract

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.

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.

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.

The online version contains supplementary material available at 10.1186/s13068-026-02744-8.