Visualizing cellulase adsorption and quantitatively determining cellulose accessibility with an updated fungal cellulose-binding module-based fluorescent probe protein

Background: Cellulose accessibility to cellulases (CAC) is a direct factor determining the enzymatic digestibility of lignocellulosic cellulose. Improving CAC by pretreatment is a prerequisite step for the efficient release of fermentable sugars from biomass cell wall. However, conventional methods to study the porosimetry of solid materials showed some limitations to be used for investigating CAC. In this work, an updated novel fusion protein comprising a fungal cellulose-binding module (CBM) from Cel7A cellobiohydrolase I (CBH I) of Trichoderma reesei QM6 and a di-green fluorescent protein - (GFP(2)) was constructed for quantitative determination of CAC. Results: The obtained probe protein had similar molecular size (e.g., weight) with that of Cel7A and could give detectable signal for quantitative analysis. Several construction strategies were compared with regard to the site of His-tag and order of CBM and - GFP(2) modules in the protein sequence, in order to achieve good expression quantity and usability of the probe protein. His6-CBM-GFP(2) has been identified as the best probe protein for investigating the effects of structural features of cellulosic substrates on cellulose accessibility. Substrate samples with different contents of xylan, lignin, and degree of substitution of cellulose -OH by formyl group were obtained, respectively, by mild - H2SO4 pre-hydrolysis, - NaClO2 selective delignification, and treatment of filter paper cellulose with concentrated formic acid. The determined CAC was in a wide range of 0.6-20.4 m(2)/g depending on the contents of hemicelluloses, lignin, and formyl group as well as cellulose degree of crystallization. Conclusions: The obtained fusion probe protein could be used as a versatile tool to quantitatively investigate the impacts of biomass structural features on CAC and hydrolyzability of cellulose substrates, as well as nonproductive adsorption of cellulase enzymes on lignin.