초록
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- (IT<SUB>50</SUB> 50 <SUP>o</SUP>C = 199 +/- 0.1 h) and solvent stable (IS<SUB>50</SUB> > 4%) thiolase derived from the thermophilic bacterium Meiothermus ruber. The observed catalytic constants were K<SUB>m</SUB> = 0.07 +/- 0.01 mM and k<SUB>cat</SUB> = 0.80 +/- 0.01 s<SUP>-1</SUP>. In analogy to other thiolases, the enzyme was inhibited by NAD<SUP>+</SUP> (K<SUB>i</SUB> = 38.7 +/- 5.8 mM) and CoA (K<SUB>i</SUB> = 105.1 +/- 6.6 μM) but not NADH. The enzyme was stable under harsh process conditions (T = 50 <SUP>o</SUP>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.