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Appl Biochem Biotechnol (2016) 178:474489 DOI 10.1007/s12010-015-1888-4
Semra Alemdar1 & Steffen Hartwig1 & Thore Frister1 & Jan Christoph Knig1 & Thomas Scheper1 &Sascha Beutel1
Received: 9 July 2015 /Accepted: 4 October 2015 / Published online: 13 October 2015# Springer Science+Business Media New York 2015
Abstract The -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--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 -humulene (~94.5 %) and -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 Terpene synthase . Sesquiterpene . Humulene . Recombinant expression . Purification . Enzyme activity
Introduction
Natural products continue to be the most important source of lead compounds for the pharmaceutical industry since the past century [1]. Many natural products have been successfully developed for the clinical use to treat human diseases in a wide range of therapeutic areas [2]. Although synthetic chemistry has also produced many new bioactive substances and combinatorial techniques have considerably expanded the
* Sascha Beutel [email protected]
1 Institute of Technical Chemistry, Gottfried Wilhelm Leibniz University of Hannover, Callinstr. 5,
30167 Hannover, Germany
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Web End = Heterologous Expression, Purification, and Biochemical Characterization of -Humulene Synthase from Zingiber zerumbet Smith
Appl Biochem Biotechnol (2016) 178:474489 475
number of compounds available for tests, natural products and their derivatives represent over 50 % of drugs in clinical use [3].
With over 30,000 characterized substances, terpenes are the largest and most structurally diverse class of natural products. Terpenes are widely distributed in nature occurring in plants, bacteria, fungi, and marine organisms. However, most of the isolated and identified terpene structures are of herbal origin. As primary metabolites, they...