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

被引：21

作者：

Alemdar, Semra ^{[1
]}

Hartwig, Steffen ^{[1
]}

Frister, Thore ^{[1
]}

Koenig, Jan Christoph ^{[1
]}

Scheper, Thomas ^{[1
]}

Beutel, Sascha ^{[1
]}

机构：

[1] Leibniz Univ Hannover, Inst Tech Chem, Callinstr 5, D-30167 Hannover, Germany

来源：

APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY | 2016年 / 178卷 / 03期

关键词：

Terpene synthase; Sesquiterpene; Humulene; Recombinant expression; Purification; Enzyme activity; MOLECULAR-CLONING; TERPENE SYNTHASES; KEY ENZYME; BIOSYNTHESIS; EVOLUTIONARY; VOLATILES; PLANTS; GENES;

D O I：

10.1007/s12010-015-1888-4

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

070307 [化学生物学]; 071010 [生物化学与分子生物学];

摘要：

The alpha-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-beta-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 alpha-humulene (similar to 94.5 %) and beta-caryophyllene (similar to 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.

引用

页码：474 / 489

页数：16

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