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

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 ( Nca EXLX1) was recombinantly expressed with and without ( Nca EXLX1tr) the dockerin domains. The adsorption characteristics of Nca EXLX1 and Nca EXLX1tr, and impact on cellulase (Cel7B) activity, were then investigated using quartz crystal microbalance with dissipation (QCM-D). Nca EXLX1 exhibited higher binding to cellulose nanofibrils (CNF) compared to Nca EXLX1tr. Despite the lower binding of Nca EXLX1tr to CNF, both Nca EXLX1 and Nca EXLX1tr 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 Nca EXLX1 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 Nca EXLX1 or Nca EXLX1tr was not correlated to extent of substrate binding.