(459d) Using Protein Design to Evaluate the Relationship Between Protein Surface Potential and Protein-Lignin Binding for the Eventual of Low Lignin Binding Cellulases

Increasing total turnover number or recycling cellulases used in deconstruction is a key step in making microbial biofuel production from lignocellulosic feedstocks economically viable. Nonspecific binding interactions between cellulases and lignin denature cellulases and hinder cellulase recycling. Previous literature supports that this binding event is influenced by the surface potential characteristics of the cellulases themselves, namely surface hydrophobicity and electrostatic surface potential. To further investigate the relationship between surface potential and nonspecific protein-lignin binding, we used a hybrid computational/experimental approach to (i.) computationally redesign surface potentials spanning a large range of net charge, total charge density, and surface hydrophobicity on a model protein; and (ii.) experimentally characterize the resultant designs for function, stability, and protein-lignin binding. In all, we tested sixteen designs spanning the physiological ranges of the above potentials. We found that designs were, on average, better expressed and more stable that the parental protein, showing that the computational design process is robust. We characterized variant binding to AFEX pretreated, dioxane extracted cornstover lignin with two different experimental methods â?? quartz crystal microbalance with dissipation (QCM-D) and fluorescence based subtractive mass balance assay. Comparing the binding capacity of variants between characterization methods highlights that both methods isolate the same low, medium, or high lignin binders. Analysis of determined binding capacity with known surface potential properties suggests a weak relationship between protein net charge and protein:lignin binding capacity, while other parameters were not statistically significant. We then applied this method to redesign surfaces of different catalytic domains on endocellulases. Implications for improving the deconstruction step in the next generation of biorefineries will be discussed.