Abstract
Phenolic composition of virgin olive oil is determined by the enzymatic and/or chemical reactions that take place during olive fruit processing. Of these enzymes, β-glucosidase activity plays a relevant role in the transformation of the phenolic glycosides present in the olive fruit, generating different secoiridoid derivatives. The main goal of the present study was to characterize olive fruit β-glucosidase genes and enzymes responsible for the phenolic composition of virgin olive oil. To achieve that, we have isolated an olive β-glucosidase gene from cultivar Picual (OepGLU), expressed in Nicotiana benthamiana leaves and purified its corresponding recombinant enzyme. Western blot analysis showed that recombinant OepGLU protein is detected by an antibody raised against the purified native olive mesocarp β-glucosidase enzyme, and exhibits a deduced molecular mass of 65.0 kDa. The recombinant OepGLU enzyme showed activity on the major olive phenolic glycosides, with the highest levels with respect to oleuropein, followed by ligstroside and demethyloleuropein. In addition, expression analysis showed that olive GLU transcript level in olive fruit is spatially and temporally regulated in a cultivar-dependent manner. Furthermore, temperature, light and water regime regulate olive GLU gene expression in olive fruit mesocarp. All these data are consistent with the involvement of OepGLU enzyme in the formation of the major phenolic compounds present in virgin olive oil.
Highlights
Olive (Olea europaea L.) is one of the first plants grown as oil crop
Alignment of the deduced amino acid sequence of OepGLU from cultivar Picual with other plant β-glucosidase protein sequences (Supplementary Figure S1) suggests that it codes for a β-glucosidase enzyme because it showed the conserved sequence motifs characteristic of the glycosyl hydrolases family 1 (GH1) T(F/L/M)NEP and Y(I/V)TENG, which include the two glutamic acid residues involved in the catalytic mechanism (Esen, 2003)
The band corresponding to the recombinant olive β-glucosidase was observed by SDS-PAGE only when the enzymatic preparation purified by affinity chromatography was applied, but not in the case of the purified preparation isolated from tobacco leaves infiltrated with untransformed Agrobacterium cells used as control, or when the crude extract was loaded onto the gel
Summary
Olive (Olea europaea L.) is one of the first plants grown as oil crop. Olive oil is one of the oldest known plant oils and it can be consumed as virgin olive oil (VOO). In the Mediterranean diet, this oil constitutes the main lipid source and it has been related with several beneficial nutritional properties which are mainly associated to its phenolic components (Konstantinidou et al, 2010; Visioli and Bernardini, 2011). Phenolic compounds are relevant because their nutritional properties, and due to their organoleptic characteristics. Phenolic compounds are being currently used as a trait in new Oleuropein β-Glucosidase Olive Oil Phenolics cross breeding programs (León et al, 2011), and as VOO quality markers, because of their health promoting and organoleptic properties
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