Title: Engineering heterogeneous biocatalysis
Description: In heterogeneous catalysis, the phase of a catalytic agent, which is responsible for reducing the activation energy of a reaction, is different from the phase of its reactants or substrates. Often, soluble catalysts are tightly associated with an inert carrier in order to artificially alter their phase. Applying this concept to biocatalysis yields a system in which enzyme molecules are immobilized on a solid support. This often serves to stabilize the enzyme, as well as enhance the recyclability of the enzyme since it is no longer soluble. In this dissertation, two methods of enzyme immobilization are evaluated: adsorption to a solid surface and whole-cell biocatalysis. The latter is then engineered for improved kinetics and functional activity using principles of synthetic biology. Adsorption of a protein to a solid surface is driven by the same thermodynamic factors that are responsible for the folding of a protein. Hydrophobic interactions, ionic interactions, covalent bonding, and weak forces all contribute to minimizing the free energy of a protein, which defines its secondary, tertiary, and quaternary structures.

Upon introduction to a surface, these different forces rearrange across the surface of the substrate to minimize the free energy of the system. Many factors influence this behavior, including particle curvature, material properties of the surface, and the stability of the protein. In the preexisting body of work, much of the research performed regarding the effects of thermal stability on adsorption were performed using mutant proteins whose structures were intentionally altered for a range of stabilities. In Chapter 2, we evaluate the effects of thermal stability on adsorption behavior using naturally evolved enzymes from the AKR superfamily, namely AdhD and hAR. These enzymes were selected for their structural homology, but vastly different thermal stabilities. Using these proteins, we demonstrate that the previously held theories of thermostable protein adsorption behavior are not entirely applicable to naturally evolved proteins that are not artificially stabilized. We also propose a modification to the classic 4-state adsorption/desorption model by introducing new pathways and protein states based on our experiments.

In addition to physisorption, whole-cell biocatalysis was explored as an enzyme immobilization platform. In general, this can be accomplished by cytosolic expression, periplasmic expression, or surface display. After weighing these options, we chose periplasmic expression in E. coli for our biocatalysts. As for the catalytic component, we selected carbonic anhydrase (CA), a class of Zn+2-binding metalloenzymes that are capable of catalyzing the reversible hydration of CO2. This enzyme was selected for the breadth of applications it can be used for, as well as its ubiquity in nature and extremely fast kinetics. Two isoforms were selected (Cab and Cam) for their respective benefits and were periplasmically expressed using 2 different leader peptides, which we discuss in Chapter 3. The enzyme loading in the periplasm, kinetics, thermal stability, and functional activity are all reported for the resulting whole-cell biocatalysts. We also describe a new method for the measurement of the operational stability of CA-based biocatalysts.

Modifications to the whole-cell biocatalysts are described in Chapter 4 and Chapter 5. In Chapter 4, we demonstrate that expression of a viral envelope protein enhances the permeability of the outer membranes of E. coli cells. We characterize this improvement by measuring small-molecule permeance, whole-cell kinetics, and functional activity of the modified biocatalysts. We also quantify this enhancement by applying concepts of porous chemical catalysts to our whole-cells. In doing so, we show improvements in parameters such as the effectiveness factor, Thiele modulus, diffusivity, and permeability. Finally, in Chapter 5 we show enhancement of the functional activity of the whole-cell biocatalysts by displaying small peptides on the outer surfaces of the cells. The modified cells are shown to enhance precipitation of calcium carbonate, a common end product of carbon mineralization. Improved solid formation rates are also reported and possible explanations for these effects are discussed.

Overall, this dissertation explores immobilization of enzymes to create heterogeneous biocatalysts. First, the effects of immobilization on enzyme structure, stability, and activity are shown for two different immobilization techniques: adsorption to a solid surface and periplasmic expression in E. coli cells. After characterization, engineering of the whole-cell biocatalysts for improved properties is presented. Finally, future directions for this work are discussed which would advance our understanding of heterogeneous biocatalysts, as well as enhance their utility.