Solvent concentration at 50% protein unfolding may reform enzyme stability ranking and process window identification

As water miscible organic co-solvents are often required for enzyme reactions to improve e.g., the solubility of the substrate in the aqueous medium, an enzyme is required which displays high stability in the presence of this co-solvent. Consequently, it is of utmost importance to identify the most suitable enzyme or the appropriate reaction conditions. Until now, the melting temperature is used in general as a measure for stability of enzymes. The experiments here show, that the melting temperature does not correlate to the activity observed in the presence of the solvent. As an alternative parameter, the concentration of the co-solvent at the point of 50% protein unfolding at a specific temperature T in short $$ is introduced. Analyzing a set of ene reductases, $$ is shown to indicate the concentration of the co-solvent where also the activity of the enzyme drops fastest. Comparing possible rankings of enzymes according to melting temperature and $$ reveals a clearly diverging outcome also depending on the specific solvent used. Additionally, plots of $$ versus temperature enable a fast identification of possible reaction windows to deduce tolerated solvent concentrations and temperature.