title: Influence of solubility-enhancing fusion proteins and organic solvents on the in vitro biocatalytic performance of the carotenoid cleavage dioxygenase AtCCD1 in a micellar reaction system.
abstract: The influence of the solubility-enhancing fusion proteins glutathione-S-transferase (GST) and NusA on the heterologous expression and in vitro biocatalytic performance of the carotenoid cleavage dioxygenase AtCCD1 from Arabidopsis thaliana was investigated. A micellar dispersion of the water-insoluble model substrate beta-apo-8'-carotenal in combination with Triton X-100 was used for the spectrophotometric in vitro assays. Specific activity in the cellular extract was twofold increased by the use of GST as a carrier protein, whereas it was decreased by 70% when fused with NusA. Reduced molar activity of the purified fusion proteins was observed, which could not be regained by proteolytic removal of the carrier protein. The addition of organic solvents in the form of short-chain aliphatic alcohols positively influenced the enzyme activity. Optimization of the reaction medium led to an 18-fold activation, and a clear correlation could be found between the organic solvent concentration required for maximum activation and the log P of the solvent. The results provide a foundation for the development towards the application of carotenoid cleavage dioxygenases as in vitro biocatalysts for the production of norisoprenoids and apocarotenals from carotenoids.
authors.affiliation: Biochemical Engineering Group, DECHEMA e.V., Theodor-Heuss-Allee 25, 60486, Frankfurt, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Schilling
authors.forename: Frauke
authors.initials: F
authors.lastname: Patett
authors.forename: Wilfried
authors.initials: W
authors.lastname: Schwab
authors.forename: Jens
authors.initials: J
authors.lastname: Schrader
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D017830:Octoxynol / Q000032:analysis; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D012997:Solvents / Q000737:chemistry*; D013053:Spectrophotometry; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D035602:Transcriptional Elongation Factors
chemical_list: D029681:Arabidopsis Proteins; D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D012997:Solvents; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D017830:Octoxynol; D049308:Dioxygenases; C431939:carotenoid cleavage dioxygenase 1; D005982:Glutathione Transferase