title: Characterization of recombinantly expressed dihydroxy-acid dehydratase from Sulfobus solfataricus-A key enzyme for the conversion of carbohydrates into chemicals.
abstract: Dihydroxyacid dehydratases (DHADs) are excellent biocatalysts for the defunctionalization of biomass. Here, we report on the recombinant production of DHAD from Sulfolobus solfataricus (SsDHAD) in E. coli and its characterization with special focus on activity toward non-natural substrates, thermo-stability, thermo-inactivation kinetics and activation capabilities and its application within multi-step cascades for chemicals production. Using a simple heat treatment of cell lysate as major purification step we achieved a specific activity of 4.4 units per gram cell mass toward the substrate d-gluconate. The optimal temperature and pH value for this reaction are 77°C and pH 6.2. The inhibitory concentration (IC50, 50% residual activity) of different alcohols was determined to be 15% (v/v) for ethanol, 4.5% (v/v) for butanol and 4% (v/v) for isobutanol. Besides d-gluconate and the natural substrate 2,3-dihydroxyisovalerate (DHIV) SsDHAD is able to convert the C3-sugar-acid d-glycerate to pyruvate, a reaction, which does not occur in natural metabolic pathways, with a specific activity of 10.7±0.4mU/mg. The specific activity of the enzyme can be increased 3-fold by incubation with 2-mercaptoethanol. The activation has no impact on temperature dependence, but modulates the thermo-inactivation tolerance at 50°C. The total turnover numbers for all of the three reactions was found to be 35.5×10(3)±1.0×10(3) for the conversion of d-gluconate to 2-keto-3-deoxygluconate (KDG), 28.2×10(3)±0.8×10(3) for DHIV to 2-ketovalerate (KIV) and 943±0.28×10(2) for d-glycerate to pyruvate. With activated SsDHAD these values could be further increased 5- and 4-fold for the d-gluconate and d-glycerate conversion, respectively.
authors.affiliation: Lehrstuhl für Chemie Biogener Rohstoffe, Technische Universität München, Schulgasse 16, 94315 Straubing, Germany. Electronic address: Joerg.carsten@tum.de.
authors.forename: Jörg M
authors.initials: JM
authors.lastname: Carsten
authors.affiliation: Lehrstuhl für Chemie Biogener Rohstoffe, Technische Universität München, Schulgasse 16, 94315 Straubing, Germany. Electronic address: Anja.philipp@tum.de.
authors.forename: Anja
authors.initials: A
authors.lastname: Schmidt
authors.affiliation: Lehrstuhl für Chemie Biogener Rohstoffe, Technische Universität München, Schulgasse 16, 94315 Straubing, Germany. Electronic address: Sieber@tum.de.
authors.forename: Volker
authors.initials: V
authors.lastname: Sieber
mesh_terms: D020001:1-Butanol / Q000494:pharmacology; D055162:Biocatalysis / Q000187:drug effects; D053898:Biosynthetic Pathways / Q000187:drug effects; D000440:Butanols / Q000494:pharmacology; D002241:Carbohydrates / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology; D005942:Gluconates / Q000378:metabolism; D005988:Glyceric Acids / Q000378:metabolism; D006836:Hydro-Lyases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism*; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D013696:Temperature
chemical_list: D000440:Butanols; D002241:Carbohydrates; D005942:Gluconates; D005988:Glyceric Acids; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; C002957:2-keto-3-deoxygluconate; D000431:Ethanol; C040507:isobutyl alcohol; C042971:glyceric acid; D020001:1-Butanol; D006836:Hydro-Lyases; C020052:dihydroxyacid dehydratase; C030691:gluconic acid
keywords: 2-Keto-3-deoxy-gluconate; Dihydroxyacid dehydratase; Iron sulfur cluster; Synthetic cascade biomanufacturing; d-Glycerate