Catechol 1,2-Dioxygenase from Rhodococcus opacus is a type of intradiol dioxygenase enzyme that catalyzes the conversion of catechol to cis, cis muconic acid. This enzymatic conversion has the potential to be useful in a number of different applications such as treating wastewater contaminated with aromatic compounds to creating a greener method to produce cis, cis muconic acid which can be used to make a number of industrially important base chemicals. However, for enzymes to be used in industrial conditions, they must be highly stable. The experimental chapters in this thesis explore whether this enzyme can be stabilized to meet industrial requirements while minimizing any loss in catalytic activity. Through the studies described in Chapter 2, a mutant enzyme was generated through disulfide bond engineering with significantly improved thermostability. However overall catalytic activity was reduced. Toward addressing this loss of catalytic activity, in Chapter 3, attempts were made to implement state-of-the-art genetic code expansion strategies to increase catalytic activity of the enzymes. However, these attempts were unsuccessful. Finally, Chapter 4 describes how future stability engineering could be optimized using design pipelines similar to the one developed in this study. Additionally, it describes possible additional optimizations toward making the application of these enzymes cost effective in the near future.

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Activated neutrophils release a neutrophil serine proteinase (NSP) called Proteinase 3 (PR3). In granulomatosis with polyangiitis (GPA), an autoimmune vasculitis, enhanced PR3 release results in endothelial damage. Serine proteinase inhibitors (serpins) such as α-1 proteinase inhibitor (API) inhibit NSPs through the serpin’s reactive center loop (RCL). However, API is known to bind PR3 with a low specificity, compared to its main inhibitory target Human Neutrophil Elastase (HNE). The current treatment for GPA is immunosuppression, which leaves patients immunocompromised. Thus, the overall aim of this study was to engineer an API variant with a higher specificity to PR3 than HNE, which could serve as a possible novel therapeutic strategy for GPA. We created an API expression library, hypervariable at RCL residues A355-I356-P357-M358-S359, and expressed it in a T7 bacteriophage display system. This phage library was then biopanned for PR3 binding. Two conditions were used for each round of biopanning: experimental, with PR3, and the negative control, without PR3. The library was biopanned for a total of five consecutive rounds, with the product of one screen serving as the starting material for the next. A bacterial mass lysate screen was also employed to further probe the library with PR3. The phage-display and bacterial lysate screens resulted in the selection of two novel variants API-DA (D357/A358) and API-N (N359). Serpin-proteinase gel complexing assays indicated that API-N formed complex with PR3 similar to API-WT (wild-type), while API-DA was mainly cleaved as a substrate. There was no significant difference between the second order rate constants of API-N and API-WT reactions with PR3. Rate constants for API-DA binding to PR3 or for API-HNE reactions were not completed due to novel coronavirus (COVID-19) restrictions. However, this project successfully demonstrated the ability to screen a hypervariable API phage library with PR3, yielding two new novel API variants. / Thesis / Master of Science in Medical Sciences (MSMS) / When harmful substances enter our body such as bacteria or viruses, we have ways of protecting ourselves from them. One of those ways is through a cell called the neutrophil. This is an immune cell that can release “fighting tools” into our blood to combat the harm. Some of these tools are called proteins. One of those proteins is Proteinase 3. However, sometimes our neutrophils can be activated without the presence of viruses or bacteria by products made in our bodies called autoantibodies. When this happens, too many of the “fighting tool” Proteinase 3 is released leading to damage to the tubes or vessels that our blood flows through. This project aimed to find a new possible way to stop these extra fighting tools from doing harm to our body. We did this by creating a library of different proteins that can stop Proteinase 3 once it is released by the neutrophil.

The goals of this research were 1) to produce hydrogen in high yields from cellulosic materials and water by synthetic pathway biotranformation (SyPaB), and 2) to increase the hydrogen production rate to a level comparable to microbe-based methods (~ 5 mmol H2/L/h). Cell-free SyPaB is a new biocatalysis technology that integrates a number of enzymatic reactions from four different metabolic pathways, e.g. glucan phosphorylation, pentose phosphate pathway, gluconeogenesis, and hydrogenase-catalyzed hydrogen production, so as to release 12 mol hydrogen per mol glucose equivalent. To ensure the artificial enzymatic pathway would work for hydrogen production, thermodynamic analysis was firstly conducted, suggesting that the artificial enzymatic pathway would spontaneously release hydrogen from cellulosic materials. A kinetic model was constructed to assess the rate-limited step(s) through metabolic control analysis. Three phosphorylases, i.e. α-glucan phosphorylase, cellobiose phosphorylase, and cellodextrin phosphorylase, were cloned from a thermophile Clostridium thermocellum, and heterologously expressed in Escherichia coli, purified and characterized in detail. Finally, up to 93% of hydrogen was produced from cellulosic materials (11.2 mol H2/mol glucose equivalent). A nearly 20-fold enhancement in hydrogen production rates has been achieved by increasing the rate-limiting hydrogenase concentration, increasing the substrate loading, and elevating the reaction temperature slightly from 30 to 32°C. The hydrogen production rates were higher than those of photobiological systems and comparable to the rates reported in dark fermentations. Now the hydrogen production is limited by the low stabilities and low activities of various phosphorylases. Therefore, non-biologically based methods have been applied to prolong the stability of α-glucan phosphorylases. The catalytic potential of cellodextrin phosphorylase has been improved to degrade insoluble cellulose by fusion of a carbohydrate-binding module (CBM) family 9 from Thermotoga maritima Xyn10A. The inactivation halftime of C. thermocellum cellobiose phosphorylase has been enhanced by three-fold at 70°C via a combination of rational design and directed evolution. The phosphorylases with improved properties would work as building blocks for SyPaB and enabled large-scale enzymatic production of cellulosic hydrogen. / Ph. D.