Characterization of a highly thermostable ß-hydroxybutyryl CoA dehydrogenase from Clostridium acetobutylicum ATCC 824

• Hbd from Clostridium acetobutylicum was structurally and biochemically characterized.
• Hbd is highly thermostable with Topt of 65 °C and a half-life of 42 h at 70 °C.
• Hbd is extremely stable against the industrial solvents ethanol, isobutanol, butanol.
• pH dependence of the reversible acetoacetyl CoA oxidation was elucidated.
• NAD+ was identified as an uncompetitive inhibitor of Hbd.

Higher energy content and hydrophobicity make bio-based n-butanol a preferred building block for chemical and biofuels manufacturing. Butanol is obtained by Clostridium sp. based ABE fermentation process. While the ABE process is well understood, the enzyme systems involved have not been elucidated in detail. The important enzyme ß-hydroxybutyryl CoA dehydrogenase from Clostridium acetobutylicum ATCC 824 (Hbd) was purified and characterized. Surprisingly, Hbd shows extremely high temperature (T > 60 °C), pH (4–11) and solvent (1-butanol, isobutanol, ethanol) stability. Hbd catalyzes acetoacetyl CoA hydration to ß-hydroxybutyryl CoA up to pH 9.5, where the reaction is reversed. Substrate (acacCoA, ß-hbCoA) and cofactor (NADH, NAD+, NADPH and NADP+) specificities were determined. We identified NAD+ as an uncompetitive inhibitor. Identification of process relevant enzymes such as Hbd is key to optimize butanol production via cellular or cell-free enzymatic systems.

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