title: Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith.
abstract: The α-humulene synthase from Zingiber zerumbet Smith was expressed as a polyhistidine-tagged protein in an E. coli BL21(DE3) strain. Induction time and inductor (isopropyl-β-D-thiogalactopyranoside) concentration were optimized. The enzyme was successfully purified directly from cell lysate by NTA affinity column chromatography and careful selection of coordinated metal ion and imidazole elution conditions. Bioactivity assays were conducted with the natural substrate farnesyl diphosphate (FDP) in a two-phase system with in situ extraction of products. The conversion of FDP to α-humulene (~94.5%) and β-caryophyllene (~5.5%) could be monitored by gas chromatography-flame ionization detection (GC-FID). Optimal pH and temperature as well as kinetic parameters K M and k cat were determined using a discontinuous kinetic assay.
authors.affiliation: Institute of Technical Chemistry, Gottfried Wilhelm Leibniz University of Hannover, Callinstr. 5, 30167, Hannover, Germany.
authors.forename: Semra
authors.initials: S
authors.lastname: Alemdar
authors.affiliation: Institute of Technical Chemistry, Gottfried Wilhelm Leibniz University of Hannover, Callinstr. 5, 30167, Hannover, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Hartwig
authors.affiliation: Institute of Technical Chemistry, Gottfried Wilhelm Leibniz University of Hannover, Callinstr. 5, 30167, Hannover, Germany.
authors.forename: Thore
authors.initials: T
authors.lastname: Frister
authors.affiliation: Institute of Technical Chemistry, Gottfried Wilhelm Leibniz University of Hannover, Callinstr. 5, 30167, Hannover, Germany.
authors.forename: Jan Christoph
authors.initials: JC
authors.lastname: König
authors.affiliation: Institute of Technical Chemistry, Gottfried Wilhelm Leibniz University of Hannover, Callinstr. 5, 30167, Hannover, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Scheper
authors.affiliation: Institute of Technical Chemistry, Gottfried Wilhelm Leibniz University of Hannover, Callinstr. 5, 30167, Hannover, Germany. beutel@iftc.uni-hannover.de.
authors.forename: Sascha
authors.initials: S
authors.lastname: Beutel
mesh_terms: D019149:Bioreactors; D002849:Chromatography, Gas; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D000081222:Monocyclic Sesquiterpenes; D012717:Sesquiterpenes / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D030521:Zingiberaceae / Q000378:metabolism*
chemical_list: D000081222:Monocyclic Sesquiterpenes; D012717:Sesquiterpenes; C042686:humulene
keywords: Enzyme activity; Humulene; Purification; Recombinant expression; Sesquiterpene; Terpene synthase