Figure 1.
Function of glyceraldehyde dehydrogenase (AlDH) in our synthetic reaction cascade.
Glucose is degraded enzymatically to pyruvate and glyceraldehyde. AlDH catalyzes the oxidation of d-glyceraldehyde to d-glycerate, which is then dehydrated to pyruvate. Ethanol and isobutanol are synthesized by further reaction steps. AlDH must not catalyze isobutyraldehyde or acetaldehyde oxidation since the irreversibly formed carboxylates are unwanted side products, thus lowering the overall yield. Important reactions are shown in detail (continuous arrows), while some reaction steps are summarized (dashed arrows).
Table 1.
Characteristics of thermostable AlDHs for the reaction cascade shown in Figure 1.
Figure 2.
A) solubility of Thermoplasma acidophilum AlDH (TaAlDH) and B) inclusion body purification.
After recombinant expression of TaAlDH in E. coli, soluble (S) and insoluble fraction (P) were analyzed by SDS-PAGE. TaAlDH inclusion bodies from fermentation were purified with washing buffer in 2 steps (W1, W2) from insoluble fraction (P). Protein marker (M) indicates size of TaAlDH (arrow).
Table 2.
Purification of soluble TaAlDH from 10 g of E. coli cells produced in 1 L of fed-batch fermentation.
Table 3.
TaAlDH enzyme activity after refolding purified TaAlDH inclusion bodies by dilution under different renaturing conditions.
Table 4.
Refolding of TaAlDH from 10 g of E. coli cells produced in 1 L of fed-batch fermentation.
Figure 3.
Size exclusion chromatography of TaAlDH.
Samples contained soluble TaAlDH (blue) and refolded TaAlDH (green).
Figure 4.
A) Far-UV CD spectrum of soluble TaAlDH (blue) and refolded TaAlDH (green) with standard deviation (black). B) Near-UV CD of soluble TaAlDH (blue), refolded TaAlDH (green) and unfolded TaAlDH (red).
Figure 5.
Michaelis-Menten kinetics of refolded TaAlDH with cofactors A) NAD+ and B) NADP+.
Reaction rates were determined in 100 mM HEPES pH 6.2 at 50°C with 5 mM d-glyceraldehyde and various concentrations of NAD+ or NADP+, respectively.
Table 5.
Kinetic parameters of refolded TaAlDH with different cofactors NAD+ and NADP+ at 50°C, pH 6.2.
Table 6.
Substrate specificity of refolded TaAlDH at 50°C, pH 7.0.
Figure 6.
TaAlDH activity in the presence of different organic solvents.
Refolded TaAlDH was incubated at 50°C for 30 min in 100 mM HEPES pH 7 containing various concentrations of ethanol (green), isobutanol (blue) or n-butanol (red). Remaining enzyme activity was tested at 50°C in respective incubation buffers.
Figure 7.
Time course of TaAlDH deactivation by organic solvent and its reactivation.
Deactivation of refolded TaAlDH was measured after incubation for indicated time at 50°C in 3% v/v isobutanol (red) or 0.3% v/v isobutanol (blue). Furthermore, samples incubated in 3% v/v isobutanol were also measured after 10-fold dilution to 0.3% v/v isobutanol (green) to test reactivation within 2 min.
Figure 8.
Fluorescence analysis of unfolding and refolding of TaAlDH in the presence of A) GdmCl (25°C) and B) isobutanol (40°C).
The fluorescence emissions of TaAlDH at λ = 330 nm were monitored upon excitation at λmax = 280 nm. Data were collected for protein unfolding (red symbols) and refolding (green symbols) at indicated concentrations of GdmCl or isobutanol. The transition curve for protein unfolding is presented as the best fit using nonlinear regression (black curve).