García-Limones Carmen, Schnäbele Kerstin, Blanco-Portales Rosario, Luz Bellido M, Caballero Jose Luis, Schwab Wilfried, Muñoz-Blanco Juan
Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, 14071 Córdoba, Spain.
J Agric Food Chem. 2008 Oct 8;56(19):9277-85. doi: 10.1021/jf801096t. Epub 2008 Sep 9.
A gene encoding a carotenoid cleavage dioxygenase class 1 enzyme (FaCCD1) was identified among a strawberry fruit expressed sequence tag collection. The full-length cDNA was isolated, and the expression profiles along fruit receptacle development and ripening, determined by quantitative real time polymerase chain reaction, showed that FaCCD1 is a ripening-related gene that reaches its maximal level of expression in the red fully ripe stage. FaCCD1 was expressed in Escherichia coli, and the products formed by the recombinant protein through oxidative cleavage of carotenoids were identified by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analyses. The FaCCD1 protein cleaves zeaxanthin, lutein, and beta-apo-8'-carotenal in vitro. Although beta-carotene is not a good substrate for FaCCD1 in vitro, the expression of FaCCD1 in an engineered carotenoid-producing E. coli strain caused the degradation of beta-carotene in vivo. Additionally, the carotenoid profile in strawberry was analyzed by high-performance liquid chromatography-photodiode detection, and a correlation between the increase of the expression level of FaCCD1 during ripening and the decrease of the lutein content suggests that lutein could constitute the main natural substrate of FaCCD1 activity in vivo.
在草莓果实表达序列标签文库中鉴定出一个编码类胡萝卜素裂解双加氧酶1类酶（FaCCD1）的基因。分离出全长cDNA，并通过定量实时聚合酶链反应确定其在果实花托发育和成熟过程中的表达谱，结果表明FaCCD1是一个与成熟相关的基因，在完全成熟的红色阶段达到其最大表达水平。FaCCD1在大肠杆菌中表达，通过液相色谱 - 质谱联用和气相色谱 - 质谱联用分析鉴定了重组蛋白通过类胡萝卜素氧化裂解形成的产物。FaCCD1蛋白在体外可裂解玉米黄质、叶黄素和β-阿朴-8'-胡萝卜醛。虽然β-胡萝卜素在体外不是FaCCD1的良好底物，但FaCCD1在工程化产类胡萝卜素的大肠杆菌菌株中的表达导致体内β-胡萝卜素的降解。此外，通过高效液相色谱 - 光电二极管检测分析了草莓中的类胡萝卜素谱，并且成熟过程中FaCCD1表达水平的增加与叶黄素含量的减少之间的相关性表明，叶黄素可能是体内FaCCD1活性的主要天然底物。
