(9f) Rational Engineering of Cellulase Enzymes for Improved Biomass Conversion Via Reduced Non-Productive Binding | AIChE

(9f) Rational Engineering of Cellulase Enzymes for Improved Biomass Conversion Via Reduced Non-Productive Binding


Nemmaru, B. - Presenter, Rutgers University
Yarbrough, J., National Renewable Energy Lab
Johnson, M., Vanderbilt University
Lang, M. J., Vanderbilt University
Chundawat, S., Rutgers University
Dissociation of non-productively bound cellulolytic enzymes from lignin and cellulose is hypothesized to be a key rate-limiting factor impeding cost-effective cellulosic biomass conversion to fermentable sugars. However, the molecular mechanisms of cellulase binding and particularly role of carbohydrate-binding modules (CBMs) in enabling non-productive enzyme binding to cellulose and lignin is not well understood. To firstly understand the molecular mechanism of non-productive binding to cellulose, we monitored the single-molecule processive motility of full-length exocellulases and developed a single-molecule CBM-cellulose rupture assay employing optical tweezers to characterize the binding of a well-studied Type-A CBM and its mutant to cellulose allomorphs. Next, we examined the subtle interplay of CBM binding and cellulose hydrolysis activity for three model Type-A CBMs (families 1, 3a, and 64) tethered to a cellulase catalytic domain (CD) on two distinct cellulose allomorphs (i.e., cellulose I and III). We finally generated a small-library of mutant CBMs with varying cellulose affinity followed by monitoring cellulose hydrolysis activity of multiple CD-CBM fusion constructs to identify novel mutants with enhanced activity towards cellulose I. Kinetic binding assays using quartz crystal microbalance with dissipation (QCM-D) were then employed to measure mutant CBM adsorption and desorption rate constants and , respectively, towards nanocrystalline cellulose. However, development of efficient enzymes for biomass conversion needs to account for non-productive binding to lignin as well. To this end, we deployed a novel protein engineering strategy called supercharging and generated a library of endocellulase mutants with varying net charges. Overall, this presentation highlights our efforts to understand non-productive binding using a suite of analytical techniques and showcases enzymes with up to 80% improved activity on cellulose.