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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

  • Martin Schilling
  • Frauke Patett
  • Wilfried Schwab
  • Jens Schrader
Applied Genetics and Molecular Biotechnology

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 β-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.

Keywords

Carotenoid cleavage dioxygenase Micellar reaction system Fusion protein Natural flavours Hydrophobic substrate 

Notes

Acknowledgment

This work was supported by the Federal Ministry for Economy and Technology of Germany via the AiF ZUTECH program (project no.110 ZN). Holger Schmidt is thanked for providing the AtCCD1 gene.

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Martin Schilling
    • 1
  • Frauke Patett
    • 1
  • Wilfried Schwab
    • 2
  • Jens Schrader
    • 1
  1. 1.Biochemical Engineering GroupDECHEMA e.V.FrankfurtGermany
  2. 2.Biomolecular Food TechnologyTechnical University MunichMunichGermany

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