Enhancing Enzymatic Activity for Cellulose Degradation

Abstract

Cellulose is converted to combustible fuels via cellulolytic enzymes (CE). These enzymes contain carbohydrate-binding modules (CBM) that tune enzymatic affinity for cellulose in plant walls. Understanding the role of specific residues in binding various CBMs to cellulose can lead to recognizing site-directed point mutations in wild type CBMs that can assist engineering enzymes with desired targeting capabilities. Here, we investigated the mechanism by which direct interactions are formed between cellulose binding modules of CE and cellulose. After finding all key residues involved in the CE-cellulose complex formation, further mutagenesis studies revealed mutations that may potentially increase the efficiency of enzymatic activity by reinforcing its anchorage point, which may in turn influence the rate of fuel production. Our results indicated that most CBMs aligned their aromatic residues flat against the hydrophobic surface of cellulose. Conversely, family 3 CBM did not show such behavior and its contact surface with cellulose was relatively curved. We tried mutations that could change the curvature of the CBM surface and examined their effect on the cellulose binding affinity. Interestingly, simulations showed that robust secondary structure of CBM limited its flexibility for adapting to the cellulose surface. Furthermore, we investigated the mechanism by which CBMs were driven towards the cellulose surface mainly through entropic forces. The molecular dynamics simulation revealed distinct binding mechanisms of different CBM families, which were governed by both the type of aromatic residues that directly contributed to cellulose association as well as groups of polar residues proximal to the binding site.