Discovery, functional verification and enzymatic properties of β-carotene dioxygenases with aroma-producing capability

To screen carotenoid cleavage dioxygenases (CCDs) specifically capable of degrading β-carotene to produce aroma substances, a bioinformatic search based on structural similarity was employed with Petunia hybrida PhCCD1 as the reference sequence, 10 novel CCDs candidates were identified from the public metaproteomic database MGnify. Heterologous expression in Escherichia coli confirmed successful production of 9 out of 11 recombinant CCD proteins, with PhCCD1 and CCD-11 exhibiting soluble expression. In vitro enzymatic results showed that both PhCCD1 and CCD-11 displayed strong degradation activity toward β-carotene and zeaxanthin. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that these enzymes catalyzed the cleavage production of β-ionone from β-carotene, with yields reaching 637.35 and 523.92 μg/mL, respectively. In addition, both enzymes demonstrated lycopene degradation capability. Further characterization showed that CCD-11 demonstrated optimal activity at 50 ℃ with a pH value of 5.0, and its enzymatic activity was significantly enhanced by Fe²⁺ and Tween-80. The Michaelis constant (K_m) of CCD-11 was 4.60 mol/L, and the maximum enzymatic reaction rate (V_max) was 3.47 μmol/(min·mg). This study demonstrates that the novel CCD-11 possesses the ability to produce the aroma substance β-ionone, providing a research foundation for its application in tobacco aroma enhancement.