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The juvenile hormones (JH) in plants are suggested to act as a form of plant defensive strategy especially against insect herbivory. The oxidation of farnesol to farnesoic acid is a key step in the juvenile hormone biosynthesis pathway. We herein present the purification and characterisation of the recombinant Theobroma cacao farnesol dehydrogenase enzyme that catalyses oxidation of farnesol to farnesal. The recombinant enzyme was purified to apparent homogeneity by affinity chromatography. The purified enzyme was characterised in terms of its deduced amino acid sequences, phylogeny, substrate specificity, kinetic parameters, structural modeling, and docking simulation. The phylogenetic analysis indicated that the T. cacao farnesol dehydrogenase (TcFolDH) showed a close relationship with A. thaliana farnesol dehydrogenase gene. The TcFolDH monomer had a large N-terminal domain which adopted a typical Rossmann-fold, harboring the GxxGxG motif (NADP(H)-binding domain) and a small C-terminal domain. The enzyme was a homotrimer comprised of subunits with molecular masses of 36 kDa. The TcFolDH was highly specific to NADP+ as coenzyme. The substrate specificity studies showed trans, trans -farnesol was the most preferred substrate for the TcFolDH, suggesting that the purified enzyme was a NADP+-dependent farnesol dehydrogenase. The docking of trans, trans -farnesol and NADP+ into the active site of the enzyme showed the important residues, and their interactions involved in the substrate and coenzyme binding of TcFolDH. Considering the extensive involvement of JH in both insects and plants, an in-depth knowledge on the recombinant production of intermediate enzymes of the JH biosynthesis pathway could help provide a potential method for insect control. • NADP+ dependent farnesol dehydrogenase enzyme of T. cacao (TcFolDH) was expressed, purified and biochemically characterized. • TcFolDH enzyme was the first homotrimer reported among farnesol dehydrogenases. • The TcFolDH was highly specific to NADP+ as coenzyme and trans, trans-farnesol was the most preferred substrate. • TcFolDH gene can serve as a novel target for pest control based on the JH-dependent regulation. [ABSTRACT FROM AUTHOR]