CHARGE OPTIMIZATION OF HYDROLASE ENZYMES AND THEIR ASSOCIATED BINDING MODULES FOR WASTE POLYMER DEGRADATION

Abstract

Lignocellulosic biomass is one of the most abundantly available renewable energy sources, but it is entirely underutilized. Composed of a matrix of cellulose and hemicellulose polysaccharides, biochemical deconstruction of this substrate can yield fermentable sugars for biofuel production. Within a typical biorefinery, enzymatic hydrolysis of these polysaccharides is relied upon by Carbohydrate Active enZymes (CAZymes) to cleave the glycosidic linkages between sugar moieties. This technology is significantly hindered though due to the inherent recalcitrance of biomass to enzymatic degradation. This recalcitrance is caused by challenges related to the structural phenolic polymer lignin which limits enzyme accessibility and non-productively binds CAZymes, as well as low overall activity of the soluble enzymes on a highly insoluble, crystalline substrate. Limited accessibility to polysaccharides is typically alleviated by thermochemical pretreatment of biomass, but this does little to overcome the challenges related to CAZyme functionality. Thus, CAZymes and their appended carbohydrate binding modules (CBMs) must be engineered for improved hydrolytic activity. Lignin and cellulose are both found to have slight negative charges on their surfaces, and exploiting these electrostatics using enzyme supercharging is one promising route for improving enzyme activity. Enzyme supercharging refers to the process of mutating several surface exposed amino acid residues to either positive (R, K) or negative (D, E) charged residues to produce high theoretical net charges. Supercharging was applied to both glycosyl hydrolase (GH) CAZymes and their appended CBMs to tune enzyme surfaces to a critical net charge where binding affinity, and ultimately activity, may be optimized. This principle is closely related to the Sabatier principle which plainly states that enzyme turnover is maximized at intermediate strength binding affinity where neither adsorption nor desorption are limiting factors. This design principle was first applied to a GH family 5 endocellulase Cel5A and its native fused family-2a CBM to generate and systematically screen a supercharged library of CBM2a-Cel5A fusions. As a result of supercharging, several supercharged mutants were identified that showed enhanced binding and catalysis with greater than 2-fold improvements in hydrolysis yields. Enzymes with optimized binding affinity additionally exhibit robust thermotolerance in the presence of substrate compared to the native enzyme. Ultimately, strong substrate dependent correlations between net charge and activity were identified, resembling charge related Sabatier optima. Further probes into these effects were performed by applying this supercharging rationale to a GH family 6 exocellulase Cel6B and its native family-2a CBM. Systematic screening identified key differences resulting from supercharging compared to the endocellulase system. Most strikingly, similar Sabatier effects were not identified, yet a key charge engineered enzyme was isolated from the library. Instead of favorable adsorptive properties, this enzyme exhibited altered unfolding pathways between both domains, revealing a 10 °C increase in melt temperature for the catalytic unit. These processive enzymes possess a more complex mechanism for cellulose degradation, and as such, simple affinity modulation was not an adequate predictor of enzyme performance. Exo-endo synergism is a hallmark of efficient biomass utilization, where endo and exo active cellulases work in conjunction to effectively process cellulose. As such, supercharged enzymes of both classes were combined in several ratios to identify how net charge impacts synergistic hydrolysis. In these cases, the enzymes with the highest individual activity were also the best synergistic partners. These findings suggested that electrostatic engineering may provide a broader strategy for modulating enzyme behavior at heterogeneous polymer interfaces beyond lignocellulosic systems alone. To investigate the transferability of these interfacial engineering principles, charge engineered CBMs were applied to poly(ethylene terephthalate) (PET) synthetic polymer hydrolase systems. Several charge engineered CBMs were fused to a Cutinase enzyme that is capable of hydrolyzing ester linkages in the PET backbone. Results found that, while positively supercharged CBMs enhanced PET binding interactions, hydrolysis improvements did not correlate directly with substrate adsorption. Instead, a slightly negative CBM fusion produced substantial improvements in PET depolymerization through enhanced thermostability and catalytic persistence, revealing that enzyme stability, rather than substrate binding, was the dominant limitation governing hydrolysis performance in this system. The synthesis of this work provides a broader framework to the application of protein supercharging as a rational design technique for increasing enzyme activity. Collectively, the findings in this dissertation demonstrate that heterogeneous biocatalysis cannot be reduced to substrate binding interactions alone. Instead, enzyme activity improvement depends on a dynamic balance between substrate engagement, catalytic turnover, enzyme stability, and catalytic persistence at insoluble polymer interfaces. These results establish electrostatic surface engineering as a powerful, but highly context-dependent strategy for modulating enzyme behavior across diverse heterogeneous polymer degradation systems.