Production of a recombinant carotenoid cleavage dioxygenase from grape and enzyme assay in water-miscible organic solvents
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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.
KeywordsCarotenoid cleavage dioxygenase C13-norisoprenoids Spectrophotometric assay Water-miscible solvents
We gratefully thank F.X. Sauvage and R. Ratomahenina for their helpful suggestions. This work was supported by a grant from the French “Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche”.
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