Meiothermus ruber thiolase - a new process stable enzyme for improved butanol synthesis.
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.
Fachgebiet Industrielle Biokatalyse, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany. Electronic address: steven.reisse@tum.de.
Steven
S
Reiße
Fachgebiet Industrielle Biokatalyse, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany. Electronic address: daniel.garbe@tum.de.
Daniel
D
Garbe
Fachgebiet Industrielle Biokatalyse, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany. Electronic address: brueck@tum.de.
Thomas
T
Brück
D000101:Acetyl-CoA C-Acetyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D000440:Butanols / Q000378:metabolism*; D003001:Cloning, Molecular; D034301:Deinococcus / Q000201:enzymology* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D012997:Solvents / Q000494:pharmacology; D013696:Temperature
D000440:Butanols; D004791:Enzyme Inhibitors; D012997:Solvents; D000101:Acetyl-CoA C-Acetyltransferase
Biocatalysis; Butanol production; Meiothermus ruber; Thermophilic enzymes; Thiolase