Functional identification of carbohydrate-binding module 13 and its application to quantification of hemicellulose in...

Gramineous biomass is a type of lignocellulose commonly used as a renewable energy source. Accurate quantification of hemicellulose within this biomass is crucial for its efficient conversion. Carbohydrate-binding modules (CBMs) help catalytic domains anchor substrates, representing the potential to be developed as fluorescence probes for hemicellulose content determination. In this study, we discovered a CBM named EmCBM13, derived from the bifunctional enzyme Xyn10A/Fae1A within the bacterial consortium EMSD5. This CBM displays a remarkable ability to bind soluble and insoluble hemicellulose components of gramineous plants. Through molecular docking and mutational analysis, we pinpointed two essential tyrosine residues mediating ligand interaction of EmCBM13. Leveraging the binding characteristics of EmCBM13, we created a fluorescence probe called EmCBM13-GFP by fusing a GFP with EmCBM13. We observed a positive correlation between the fluorescence contents of EmCBM13-GFP bound to hemicellulose and the hemicellulose contents in various gramineous biomasses. Utilizing this correlation, we rapidly determined the hemicellulose content in different gramineous plants with 90-110 % accuracy. This probe shows promise in quickly evaluating the characteristics of gramineous biomass feedstock for research and production purposes.