Olive Polyphenol Oxidase Gene Family

Abstract

The phenolic compounds containing hydroxytyrosol are the minor components of virgin olive oil (VOO) with the greatest impact on its functional properties and health benefits. Olive breeding for improving the phenolic composition of VOO is strongly dependent on the identification of the key genes determining the biosynthesis of these compounds in the olive fruit and also their transformation during the oil extraction process. In this work, olive polyphenol oxidase (PPO) genes have been identified and fully characterized in order to evaluate their specific role in the metabolism of hydroxytyrosol-derived compounds by combining gene expression analysis and metabolomics data. Four PPO genes have been identified, synthesized, cloned and expressed in Escherichia coli, and the functional identity of the recombinant proteins has been verified using olive phenolic substrates. Among the characterized genes, two stand out: (i) OePPO2 with its diphenolase activity, which is very active in the oxidative degradation of phenols during oil extraction and also seems to be highly involved in the natural defense mechanism in response to biotic stress, and (ii) OePPO3, which codes for a tyrosinase protein, having diphenolase but also monophenolase activity, which catalyzes the hydroxylation of tyrosol to form hydroxytyrosol.

Extraction, Purification, and Characterization of Olive (Olea europaea L., cv. Chemlal) Polyphenol Oxidase

“…This mass matches the reported mass (∼55 kDa) for the mature protein as documented by Sańchez et al 11 The electrophoretic profile of OePPO on the SDS-PAGE gel, appearing as a thick band (double band), has also been observed in pure recombinant OePPO1. 11 This characteristic electrophoretic pattern has been reported for other plant PPOs as well. 24 The presence of OePPO as a double band suggests potential modifications in the enzyme's folding 11,24 or the possible existence of multiple isoforms of the enzyme that share similar physicochemical properties but differ slightly in their molecular weight.…”

“…Components of the buffer, through their ionic strength and nature, can directly influence the catalytic process of the enzyme and its activity . The optimal pH value (7.5) in the presence of SDS is slightly higher than the reported optimal pH of 7.0 for recombinant Oe PPO1 …”

“…35 The optimal pH value (7.5) in the presence of SDS is slightly higher than the reported optimal pH of 7.0 for recombinant OePPO1. 11 On the other hand, in the absence of SDS (Figure 3A), OePPO displayed a preference for acidic pH, remaining active even at pH 1.5 with 0.8% relative activity. The enzyme's activity peaked at pH 4.0, showing a relative activity of 19.2%, which was five times higher than its activity in the presence of SDS (4.4%).…”

“…PPO can exist in multiple variants in olive fruits, with the sequence of these variants showing significant differences between various cultivars . Furthermore, these variants display notable distinctions in biochemical properties to the extent that the enzyme may either lack or possess monophenolase activity .…”

“…PPO can exist in multiple variants in olive fruits, with the sequence of these variants showing significant differences between various cultivars. 11 Furthermore, these variants display notable distinctions in biochemical properties to the extent that the enzyme may either lack or possess monophenolase activity. 11 Therefore, for the accurate identification of our purified enzyme and to facilitate the interpretation of mass spectrometry results, it was necessary to clone OePPO genes and obtain the gene that encodes the isolated protein, as the sequences present in the database are from different olive cultivars.…”