Butanol is an important renewable building block for the chemical, textile, polymer and biofuels industry due to its increased energy density. Current biotechnological butanol production is a Clostridial based anaerobic fermentation process. Thiolase (EC 2.3.1.9/EC 2.3.1.16) is a key enzyme in this biosynthetic conversion of glucose to butanol. It catalyzes the condensation of two acetyl-CoA molecules, forming acetoacetyl-CoA, which is the first committed step in butanol biosynthesis. The well characterized clostridial thiolases are neither solvent nor thermo stable, which limits butanol yields. We have isolated and characterized a new thermo- (IT50 50 °C = 199 ± 0.1 h) and solvent stable (IS50 > 4%) thiolase derived from the thermophilic bacterium Meiothermus ruber. The observed catalytic constants were Km = 0.07 ± 0.01 mM and kcat = 0.80 ± 0.01 s(-1). In analogy to other thiolases, the enzyme was inhibited by NAD(+) (Ki = 38.7 ± 5.8 mM) and CoA (Ki = 105.1 ± 6.6 μM) but not NADH. The enzyme was stable under harsh process conditions (T = 50 °C, Butanol = 4% v/v) for prolonged time periods (τ = 7 h). The new enzyme provides for targeted in-vivo and in-vitro butanol biosynthesis under industrially relevant process conditions.
Keywords: Biocatalysis; Butanol production; Meiothermus ruber; Thermophilic enzymes; Thiolase.
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