A potential Carotenoid Cleavage Dioxygenase (CCD) gene was identified among a Vitis vinifera L. EST collection and a full-length cDNA (VvCCD1) was isolated. Recombinant expression of VvCCD1 confirmed that the gene encoded a functional CCD. Experimental evidence was obtained that VvCCD1 cleaves zeaxanthin symmetrically yielding 3-hydroxy-beta-ionone, a C(13)-norisoprenoidic compound, and a C(14)-dialdehyde. Expression of the gene was studied by real-time PCR at different developmental stages of grape berries from Muscat of Alexandria and Shiraz cultivars. A significant induction of the gene expression approaching véraison was observed in both cultivars. In parallel, the C(13)-norisoprenoid level increased from véraison to maturity in both cultivars.

A recombinant carotenoid cleavage dioxygenase from Vitis vinifera L. was produced by Escherichia coli as a fusion with the glutathione-S-transferase (GST) protein under different bacterial growth conditions. The enzyme production was monitored by a GST assay. Addition of Triton X-100 prior to bacterial cell disruption doubled the release of soluble protein. A simple spectrophotometric enzyme assay was developed to measure carotenoid cleavage activity using lutein as substrate. Enzyme activity showed a 26-fold increase with the addition of 10% (v/v) acetone in the reaction mixture.

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