Abstract: | |
An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in Escherichia coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to ∼325 mg/L−1 were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni2+-IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg2+ containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC–MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis–Menten model, kinetic parameters of KM = 1.111 μM (±0.113), vmax = 0.3245 μM min−1 (±0.0035), kcat = 2.95 min−1, as well as a catalytic efficiency kcat/KM = 4.43 × 104 M−1 s−1 were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution.
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License of this version: | CC BY-NC-ND 4.0 Unported - https://creativecommons.org/licenses/by-nc-nd/4.0/ |
Publication type: | Article |
Publishing status: | acceptedVersion |
Publication date: | 2015 |
Keywords english: | Enzyme kinetics, Khusimene, Sesquiterpenes, SUMO, Terpene synthase, Zizaene, Chrysopogon zizanioides extract, farnesyl diphosphate, plant extract, SUMO protein, unclassified drug, zizaene synthase, hybrid protein, isoprenoid phosphate, khusimol, sesquiterpene, SUMO 1 protein, terpene synthase, transferase, cold shock response, enzyme mechanism, enzyme synthesis, Escherichia coli, gel electrophoresis, gene expression system, isoelectric point, mass fragmentography, Michaelis Menten kinetics, molecular weight, nonhuman, priority journal, protein analysis, protein binding, protein cleavage, protein domain, protein purification, substrate concentration, enzymology, genetics, isolation and purification, metabolism, molecular cloning, Vetiveria, Alkyl and Aryl Transferases, Cloning, Molecular, Escherichia coli, Polyisoprenyl Phosphates, Recombinant Fusion Proteins, Sesquiterpenes, Vetiveria |
DDC: | 500 | Naturwissenschaften, 570 | Biowissenschaften, Biologie, 540 | Chemie |
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