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Simple Summary: Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, a process for the production of biofuels. Thermostable and oxygen-stable PDC activity is highly desirable for biotechnological applications at high temperatures. The enzymes from the thermoacidophiles Saccharolobus (formerly Sulfolobus) solfataricus (Ss, Topt = 80 °C) and Sulfolobus acidocaldarius (Sa, Topt = 80 °C) were purified and characterized, and their biophysical and biochemical properties were determined comparatively. The purified enzymes were CoA-dependent and thermostable. There was no loss of activity in the presence of oxygen. In conclusion, both thermostable SsPDC and SaPDC catalyze the CoA-dependent production of acetaldehyde from pyruvate in the presence of oxygen. Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, and it catalyzes the decarboxylation of pyruvate to acetaldehyde and CO2. Bifunctional PORs/PDCs that also have additional pyruvate:ferredoxin oxidoreductase (POR) activity are found in hyperthermophiles, and they are mostly oxygen-sensitive and CoA-dependent. Thermostable and oxygen-stable PDC activity is highly desirable for biotechnological applications. The enzymes from the thermoacidophiles Saccharolobus (formerly Sulfolobus) solfataricus (Ss, Topt = 80 °C) and Sulfolobus acidocaldarius (Sa, Topt = 80 °C) were purified and characterized, and their biophysical and biochemical properties were determined comparatively. Both enzymes were shown to be heterodimeric, and their two subunits were determined by SDS-PAGE to be 37 ± 3 kDa and 65 ± 2 kDa, respectively. The purified enzymes from S. solfataricus and S. acidocaldarius showed both PDC and POR activities which were CoA-dependent, and they were thermostable with half-life times of 2.9 ± 1 and 1.1 ± 1 h at 80 °C, respectively. There was no loss of activity in the presence of oxygen. Optimal pH values for their PDC and POR activity were determined to be 7.9 and 8.6, respectively. In conclusion, both thermostable SsPOR/PDC and SaPOR/PDC catalyze the CoA-dependent production of acetaldehyde from pyruvate in the presence of oxygen. [ABSTRACT FROM AUTHOR]