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<p>Carotenoid cleavage dioxygenases (CCDs) have a crucial role in the biosynthesis of apocarotenoids. This study focuses on the molecular cloning and biochemical characterization of <i>Bixa orellana</i> (achiote) carotenoid cleavage dioxygenase (BoCCD4-4), an underexplored woody plant species. CCDs are crucial for the production of apocarotenoids, such as bixin, which have significant industrial applications due to their bioactive properties. Previous research indicated that BoCCD4-4 can cleave lycopene to produce bixin aldehyde and norbixin, suggesting its role in bixin synthesis. The BoCCD4-4 gene was successfully cloned and expressed in <i>E. coli</i>. Biochemical characterization revealed optimal enzyme expression at 25&#xa0;°C with 0.5&#xa0;mM IPTG (β-<span>d</span>-1-thiogalactopyranoside). The recombinant BoCCD4-4, with a theoretical molecular weight of 69.72&#xa0;kDa and an isoelectric point of 8.58, primarily accumulated in insoluble fractions, forming inclusion bodies in <i>E. coli</i>. The purified protein yield was 1&#xa0;mg/ml. The resulting sequencing showed 82% coverage of the expected sequence and 100% identity with the reported <i>B. orellana</i> CCD4-4 sequences. When BoCCD4-4 was incubated with lycopene, its optimal enzymatic activity was detected with 75&#xa0;µg of purified protein. Enzymatic assays confirmed that purified BoCCD4-4 is active, transforming lycopene into other products, with a direct proportionality between enzyme activity and increasing protein concentrations (25–75&#xa0;µg). Molecular docking analysis indicated a higher affinity of BoCCD4-4 for lycopene than BoCCD4-3, supporting its role in bixin accumulation. This research offers valuable insights into bixin biosynthesis, providing a foundation for further elucidation.</p>

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Biochemical characterization of carotenoid cleavage dioxygenase (BoCCD4-4) from Bixa orellana L.

  • 期刊论文

  • 发表日期：

    2026年5月20日

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  • Guadalupe Hernández-Reséndiz,
  • Ignacio Islas-Flores,
  • Victor Carballo-Uicab,
  • Margarita Aguilar-Espinosa,
  • Diana Sierra-Ulín,
  • Alba A. Vallejo-Cardona,
  • Renata Rivera-Madrid

摘要

Carotenoid cleavage dioxygenases (CCDs) have a crucial role in the biosynthesis of apocarotenoids. This study focuses on the molecular cloning and biochemical characterization of Bixa orellana (achiote) carotenoid cleavage dioxygenase (BoCCD4-4), an underexplored woody plant species. CCDs are crucial for the production of apocarotenoids, such as bixin, which have significant industrial applications due to their bioactive properties. Previous research indicated that BoCCD4-4 can cleave lycopene to produce bixin aldehyde and norbixin, suggesting its role in bixin synthesis. The BoCCD4-4 gene was successfully cloned and expressed in E. coli. Biochemical characterization revealed optimal enzyme expression at 25 °C with 0.5 mM IPTG (β-d-1-thiogalactopyranoside). The recombinant BoCCD4-4, with a theoretical molecular weight of 69.72 kDa and an isoelectric point of 8.58, primarily accumulated in insoluble fractions, forming inclusion bodies in E. coli. The purified protein yield was 1 mg/ml. The resulting sequencing showed 82% coverage of the expected sequence and 100% identity with the reported B. orellana CCD4-4 sequences. When BoCCD4-4 was incubated with lycopene, its optimal enzymatic activity was detected with 75 µg of purified protein. Enzymatic assays confirmed that purified BoCCD4-4 is active, transforming lycopene into other products, with a direct proportionality between enzyme activity and increasing protein concentrations (25–75 µg). Molecular docking analysis indicated a higher affinity of BoCCD4-4 for lycopene than BoCCD4-3, supporting its role in bixin accumulation. This research offers valuable insights into bixin biosynthesis, providing a foundation for further elucidation.