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

Abstract

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 $c_{U_{50}}^T$ is introduced. Analyzing a set of ene reductases, $c_{U_{50}}^T$ 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 $c_{U_{50}}^T$ reveals a clearly diverging outcome also depending on the specific solvent used. Additionally, plots of $c_{U_{50}}$ versus temperature enable a fast identification of possible reaction windows to deduce tolerated solvent concentrations and temperature.

Fig. 1. Phylogenetic tree and pairwise sequence identities of ene reductases.

a In the phylogenetic tree the 13 representative EREDs selected for this study are labeled in green while other known EREDs are labeled in black. b The percentage pairwise sequence identities of the selected EREDs cluster into two main similarity groups. The color scale ranges from 0% (dark green) to 100% (white) identity.

Fig. 2. Co-solvent concentration-dependent changes in ERED stability and activity.

a Change of melting temperature ΔT_m and b relative (Rel.) specific activity of four selected EREDs at increasing co-solvent concentration (% v/v) (Conc.) [dimethyl sulfoxide (DMSO) (red), ethanol (yellow), methanol (green), 2-propanol (blue), and n-propanol (magenta)]. The complete set of results can be found in Supplementary Figs. 3 and 4. The melting temperature was measured in 50 mM sodium phosphate buffer pH 7.4 and the mean change of melting temperature induced by the co-solvents of three replicates is given. The error bar marks the standard error of the mean using Gaussian error propagation. The specific initial activity was recorded under the same conditions using cyclohex-2-enone (10 mM) as substrate following the decrease of NAD(P)H (0.2 mM) at 340 nm and room temperature (i.e., 20–24 °C for DMSO, 2-propanol and n-propanol) and 25 °C for ethanol and methanol. Given is the mean of three replicates. The values are normalized by setting the activity measured without co-solvent (normal conditions) to 100%. Values above 100 indicate an increased activity while values below 100 indicate a decreased activity relative to the activity under normal conditions. The error bar marks the standard error of the mean using Gaussian error propagation. The relative specific activities calculated from measurements at 25 °C given here are shown again as part of Figs. 4b and 5b. Measured values are connected by lines to facilitate reading of the figure and do not represent measured values. All raw data that was used to obtain the derived values ΔT_m and relative specific activity can be found in Supplementary Tables 5 and 6.

Fig. 3. Model reaction for the determination of ERED specific initial activity by following NAD(P)H depletion at 340 nm.

EREDs (5–30 μg ml^-1) were analyzed using model substrate cyclohex-2-enone (10 mM) using the following reaction conditions: NAD(P)H (0.2 mM) in sodium phosphate buffer (50 mM, pH  $7.4$), a reaction volume of 200 μl, in the absence or presence of varied concentration of co-solvents [DMSO, methanol, ethanol, 2-propanol, and n-propanol at 0–45% (v/v)].

Fig. 4. The influence of ethanol on unfolding and comparison to specific activity.

a The unfolding of EREDs induced by the presence of increasing amounts of ethanol at different temperatures [color range from dark blue (25 °C) to dark red (80 °C)] measured by nanoDSF [given as the ratio of the fluorescence at 350 and 330 nm (F350/330)]. b The initial activity recorded at the same co-solvent and temperature conditions. A drop of activity can be found close to the concentration of unfolding. The dashed lines in (a) represent fits of the two-state model of unfolding Eq. (1) to the recorded data. The star marks the co-solvent concentration of unfolding $c_{U_{50}}^T$ at the specific temperature. The solid lines in (b) connect the measured values of each reaction temperature for visual guidance. The mean activity was calculated from three replicates and the error bar marks the standard error of the mean using Gaussian error propagation.

Fig. 5. The influence of methanol on unfolding and the comparison to activity.

a The unfolding of EREDs induced by the presence of increasing amounts of methanol at different temperatures [color range from dark blue (25 °C) to dark red (80 °C)] measured by nanoDSF [given as the ratio of the fluorescence at 350 and 330 nm (F350/330)]. b The initial activity recorded at the same co-solvent and temperature conditions. A drop of activity can be found close to the concentration of unfolding. The dashed lines in (a) show fits of the two-state model of unfolding Eq. (1) to the recorded data. The star marks the co-solvent concentration of unfolding $c_{U_{50}}^T$ at the specific temperature. The solid lines in (b) connect the measured values of each reaction temperature for visual guidance. The mean activity was calculated from three replicates and the error bar marks the standard error of the mean using Gaussian error propagation.

Fig. 6. Activity-solvent model [Eq. (3)] fitted to specific activity data of EREDs.

The initial specific activity data of four EREDs in the presence of increasing amount of ethanol and methanol fitted with the activity-solvent model Eq. (3). The fits are displayed as dashed lines, the measured initial activity is displayed as circles and are also shown in Figs. 4 and 5. The color of the curve represents the reaction temperature. The stars mark $c_{A_{50}}$, the concentration of largest loss in activity and the triangles mark $c_{A_{max}}$ the concentration of maximal activity.

Fig. 7. Identification of suitable reaction conditions for reaction temperature and solvent concentration from a plot of cU50T at the respective temperature for various EREDs.

Suitable reaction conditions for a ethanol and b methanol are those at co-solvent and temperature values below the curve for each enzyme. The solid lines connect the measured values of each reaction temperature T_reaction for visual guidance. $c_{U_{50}}^T$ is the concentration of half unfolding at a given temperature.