Production of a recombinant carotenoid cleavage dioxygenase from grape and enzyme assay in water-miscible organic solvents.
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.
Mathieu
Sandrine
S
UMR IR2B, ENSAM-INRA, Université Montpellier II, 34060, Montpellier cedex 1, France.
Bigey
Frédéric
F
Procureur
Jérôme
J
Terrier
Nancy
N
Günata
Ziya
Z
D000096:Acetone / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D049308:Dioxygenases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development; D005982:Glutathione Transferase / Q000378:metabolism; D014975:Lutein / Q000737:chemistry / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D009930:Organic Chemicals / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012995:Solubility / Q000187:drug effects; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D027843:Vitis / Q000201:enzymology* / Q000235:genetics; D014867:Water / Q000737:chemistry*
D009930:Organic Chemicals; D011993:Recombinant Fusion Proteins; D012997:Solvents; D014867:Water; D000096:Acetone; D017830:Octoxynol; D049308:Dioxygenases; C431939:carotenoid cleavage dioxygenase 1; D005982:Glutathione Transferase; D014975:Lutein