Details
| Original language | English |
|---|---|
| Pages (from-to) | 474-489 |
| Number of pages | 16 |
| Journal | Applied Biochemistry and Biotechnology |
| Volume | 178 |
| Issue number | 3 |
| Publication status | Published - 13 Oct 2015 |
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 KM and kcat were determined using a discontinuous kinetic assay.
Keywords
- Enzyme activity, Humulene, Purification, Recombinant expression, Sesquiterpene, Terpene synthase
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Chemical Engineering(all)
- Bioengineering
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
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In: Applied Biochemistry and Biotechnology, Vol. 178, No. 3, 13.10.2015, p. 474-489.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith
AU - Alemdar, Semra
AU - Hartwig, Steffen
AU - Frister, Thore
AU - König, Jan Christoph
AU - Scheper, Thomas
AU - Beutel, Sascha
PY - 2015/10/13
Y1 - 2015/10/13
N2 - 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.
AB - 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.
KW - Enzyme activity
KW - Humulene
KW - Purification
KW - Recombinant expression
KW - Sesquiterpene
KW - Terpene synthase
UR - http://www.scopus.com/inward/record.url?scp=84959518981&partnerID=8YFLogxK
U2 - 10.1007/s12010-015-1888-4
DO - 10.1007/s12010-015-1888-4
M3 - Article
C2 - 26463657
AN - SCOPUS:84959518981
VL - 178
SP - 474
EP - 489
JO - Applied Biochemistry and Biotechnology
JF - Applied Biochemistry and Biotechnology
SN - 0273-2289
IS - 3
ER -