Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith

Autor: Jan C. L. König, Steffen Hartwig, Thore Frister, Semra Alemdar, Sascha Beutel, Thomas Scheper
Rok vydání: 2015
Předmět:
Sesquiterpene
0301 basic medicine
Dewey Decimal Classification::500 | Naturwissenschaften::540 | Chemie
Lysis
Dewey Decimal Classification::500 | Naturwissenschaften::570 | Biowissenschaften
Biologie
Synthases
Applied Microbiology and Biotechnology
Biochemistry
Assays
chemistry.chemical_compound
Bioreactors
Enzyme activity
Metal ions
Purification
Chromatography
Gas chromatography
biology
Humulene
Biochemical characterization
Chemistry
Temperature
General Medicine
Hydrogen-Ion Concentration
Terpene synthase
Enzymes
Metals
ddc:540
Electrophoresis
Polyacrylamide Gel
Sesquiterpenes
Biotechnology
Chromatography
Gas
Farnesyl diphosphate
Bioengineering
Ionization of gases
03 medical and health sciences
Recombinant expression
Column chromatography
Affinity chromatography
Zingiberaceae
ddc:570
Escherichia coli
Gas chromatography-flame ionization detections
Molecular Biology
Substrate (chemistry)
biology.organism_classification
Enzyme assay
Monocyclic Sesquiterpenes
Kinetics
030104 developmental biology
Zingiber zerumbet
biology.protein
Heterologous expression
Zdroj: Applied Biochemistry and Biotechnology 178 (2016), Nr. 3
ISSN: 1559-0291
0273-2289
DOI: 10.1007/s12010-015-1888-4
Popis: 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 KM and kcat were determined using a discontinuous kinetic assay. The final publication is available at Springer via http://dx.doi.org/10.1007/s12010-015-1888-4. EFRE/ZW-8-80130940
Databáze: OpenAIRE
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