(27ah) In Situ Characterization of Ammonia-Dependent Enzymes

Process stability of enzymes is heavily influenced by the microenvironment, including the concentration of ions, the temperature, and the pH. As the medium interacts with the protein, it can begin to unfold and aggregate irreversibly, which can deactivate the enzyme. Many techniques exist for the characterization of protein structures in dilute solutions of ions, such as size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, and differential scanning calorimetry. For more ideal cases, such as dilute buffers used to store proteins in the refrigerator, these techniques can give a comprehensive overview of the storage stability of proteins. However, reaction media and the operating temperature and pH of a biocatalytic process is often vastly different from these storage conditions. These differences in microenvironment drive the formation of product molecules, but also can rapidly unfold and deactivate the enzyme of interest. Particularly for enzymes with high ion concentrations needed for their reaction, the aforementioned techniques fall short. One such class of enzymes is amine dehydrogenases, which require high concentrations of ammonia and high pH to drive the formation of their product molecule. This necessitates a more thorough and diverse set of tools for in situ characterization of these enzymes.

Using engineered and native amine dehydrogenases, an analysis of their storage stability in reaction media was investigated. A combination of techniques suited for higher salt concentrations, such as differential scanning fluorimetry, analytical ultracentrifugation, and activity assays were performed. These measurements gave the melting point, oligomeric state, and reactivity of the enzyme, allowing a comprehensive comparison of different amine dehydrogenases for operational stability. Structural analysis was then performed in silico to corroborate and interpret any differences in the surface properties that may predict the deactivation behavior of these enzymes. A correlation between initial distribution of oligomeric state and stability was identified for several enzymes, suggesting those with a more defined oligomeric state may be less susceptible to deactivation. This paradigm for in situ analysis of enzyme structure and reactivity gives a more complete picture of the total turnover for an enzymatic process, helps compare the output of several related enzyme, and gives a set of tools that can be used for higher salt or ion concentrations than typical storage buffers.