In situ characterization of amine-forming enzymes shows altered oligomeric state

Enzyme stability can be measured in a number of ways, including melting temperature, activity retention, and size analysis. However, these measurements are often conducted in an idealized storage buffer and not in the relevant enzymatic reaction media. Particularly for reactions that occur in alkaline, volatile, and high ionic strength media, typical analyses using differential scanning calorimetry, light scattering, and sodium dodecyl-sulfate polyacrylamide gel electrophoresis are not satisfactory to track the stability of these enzymes. In this work, we monitor the stability of engineered and native dehydrogenases that require a high amount of ammonia for their reaction to occur. We demonstrate the benefits of analyzing these enzymes in their reaction buffer, uncovering trends that were not observable in the typical phosphate storage buffer. This work provides a framework for analyzing the stability of many other enzymes whose reaction media is not suitable for traditional techniques. We introduce several strategies for measuring the melting temperature, oligomeric state, and activity of these enzymes in their reaction media. Further, we have identified opportunities for integration of computational tools into this workflow to engineer enzymes more effectively for solvent tolerance and improved stability.