title: Refolding of a thermostable glyceraldehyde dehydrogenase for application in synthetic cascade biomanufacturing.
abstract: 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 Thermoplasma acidophilum, which catalyzes the highly substrate specific oxidation of d-glyceraldehyde to d-glycerate. In this work the enzyme was recombinantly expressed in Escherichia coli. 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.
authors.affiliation: Straubing Center of Science, Technische Universität München, Straubing, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Steffler
authors.forename: Volker
authors.initials: V
authors.lastname: Sieber
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005985:Glyceraldehyde / Q000378:metabolism*; D013696:Temperature
chemical_list: D005985:Glyceraldehyde; D000444:Aldehyde Dehydrogenase
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