Refolding of a Thermostable Glyceraldehyde Dehydrogenase for Application in Synthetic Cascade Biomanufacturing

<div><p>The production of chemicals from renewable resources is gaining importance in the light of limited fossil resources. One promising alternative to widespread fermentation based methods used here is Synthetic Cascade Biomanufacturing, the application of minimized biocatalytic reaction cascades in cell free processes. One recent example is the development of the phosphorylation independent conversion of glucose to ethanol and isobutanol using only 6 and 8 enzymes, respectively. A key enzyme for this pathway is aldehyde dehydrogenase from <i>Thermoplasma acidophilum</i>, which catalyzes the highly substrate specific oxidation of d-glyceraldehyde to d-glycerate. In this work the enzyme was recombinantly expressed in <i>Escherichia coli</i>. Using matrix-assisted refolding of inclusion bodies the yield of enzyme production was enhanced 43-fold and thus for the first time the enzyme was provided in substantial amounts. Characterization of structural stability verified correct refolding of the protein. The stability of the enzyme was determined by guanidinium chloride as well as isobutanol induced denaturation to be ca. −8 kJ/mol both at 25°C and 40°C. The aldehyde dehydrogenase is active at high temperatures and in the presence of small amounts of organic solvents. In contrast to previous publications, the enzyme was found to accept NAD⁺ as cofactor making it suitable for application in the artificial glycolysis.</p></div

Fluorescence analysis of unfolding and refolding of <i>Ta</i>AlDH in the presence of A) GdmCl (25°C) and B) isobutanol (40°C).

<p>The fluorescence emissions of <i>Ta</i>AlDH at λ = 330 nm were monitored upon excitation at λ_max = 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).</p

Refolding of <i>Ta</i>AlDH from 10 g of <i>E. coli</i> cells produced in 1 L of fed-batch fermentation.

1<p>Protein concentration was determined with Bradford Assay/additionally after inclusion body washing and lyophilization by UV absorption spectroscopy.</p>2<p>Enzyme activity was analyzed using NAD⁺ as electron acceptor during purification.</p

Substrate specificity of refolded <i>Ta</i>AlDH at 50°C, pH 7.0.

<p>Substrate specificity of refolded <i>Ta</i>AlDH at 50°C, pH 7.0.</p

<i>Ta</i>AlDH activity in the presence of different organic solvents.

<p>Refolded <i>Ta</i>AlDH 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.</p

Purification of soluble <i>Ta</i>AlDH from 10 g of <i>E. coli</i> cells produced in 1 L of fed-batch fermentation.

<p>n. d.: Not determinable.</p>1<p>Protein concentration was determined with Bradford Assay/additionally after lyophilization by UV absorption spectroscopy.</p>2<p>Enzyme activity was analyzed using NAD⁺ as electron acceptor during purification.</p

A) solubility of <i>Thermoplasma acidophilum</i> AlDH (<i>Ta</i>AlDH) and B) inclusion body purification.

<p>After recombinant expression of <i>Ta</i>AlDH in <i>E. coli</i>, soluble (S) and insoluble fraction (P) were analyzed by SDS-PAGE. <i>Ta</i>AlDH inclusion bodies from fermentation were purified with washing buffer in 2 steps (W1, W2) from insoluble fraction (P). Protein marker (M) indicates size of <i>Ta</i>AlDH (arrow).</p

<i>Ta</i>AlDH enzyme activity after refolding purified <i>Ta</i>AlDH inclusion bodies by dilution under different renaturing conditions.

<p><i>Ta</i>AlDH enzyme activity after refolding purified <i>Ta</i>AlDH inclusion bodies by dilution under different renaturing conditions.</p

Kinetic parameters of refolded <i>Ta</i>AlDH with different cofactors NAD⁺ and NADP⁺ at 50°C, pH 6.2.

1<p>0.02 mg/mL <i>Ta</i>AlDH.</p

Size exclusion chromatography of <i>Ta</i>AlDH.

<p>Samples contained soluble <i>Ta</i>AlDH (blue) and refolded <i>Ta</i>AlDH (green).</p