Development of Binding Domains for Improved Cellulose Decrystallization and Hydrolysis

The cellulose-binding domain (CBD) has long been recognized as a key moiety in cellulolytic enzymes to bring the full-length enzyme to a close proximity of the cellulose substrate. As one of the most efficient exoglucanases, T. reesei Cel7A has attracted enormous interest. The cellulose-binding domain of Cel7A has been reported to be capable of decrystallizing Avicel. In this work, the top nine CBD variants were identified by Principal Component Analysis (PCA), a data-driven protein engineering method, with the concept of consensus. Both wild-type CBD and the identified mutants were expressed as GFPuv fusion proteins in bacterial and yeast expression systems. We identified the mutants with improved functionalities including both cellulose-binding affinity and cellulose decrystallization effect, opening the chance to combine the CBDs with the catalytic domain to yield variant cellulases with improved enzymatic activity on cellulose substrate. Besides, the effects of glycosylation and linker length on substrate binding capacity, reversibility of substrate binding and/or cellulose decrystallization will be discussed.