Expression, purification and characterization of recombinant ( E)-β-farnesene synthase from Artemisia annua

A cDNA clone encoding the sesquiterpene synthase ( E)-β-farnesene synthase has been isolated from Artemisia annua L. The recombinant enzyme produced in Escherichia coli has been characterized. A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, ( E)-β-farnesene synthase, has been isolated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated p I = 5.03. The deduced amino acid sequence is 30–50% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, β-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the K m- and k cat-values for farnesyl diphosphate, is 2.1 μM and 9.5 × 10 −3 s −1, respectively resulting in the efficiency 4.5 × 10 −3 M −1 s −1. The enzyme exhibits substantial activity in the presence of Mg 2+, Mn 2+ or Co 2+ but essentially no activity when Zn 2+, Ni 2+ or Cu 2+ is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and