Departamento de Ingeniería Bioquímica, Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Avenida Wilfrido Massieu s/n, Unidad Profesional Adolfo López Mateos, Ciudad de Mexico 07738, Mexico.
Departamento de Microbiología, Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Prolongación de Carpio y Plan de Ayala s/n, Colonia Santo Tomás, Ciudad de Mexico 11340, Mexico.
Biomolecules. 2023 Sep 20;13(9):1423. doi: 10.3390/biom13091423.
The yeast excretes a brilliant yellow fluorescent compound into its growth culture. In this study, we isolated and identified this compound using reverse-phase high-performance liquid chromatography-diode array detector (RP-HPLC-DAD) as well as H NMR and UV-Vis spectroscopy. Two of the three RP-HPLC-DAD methods used successfully separated the fluorescent compound and involved (1) a double separation step with isocratic flow elution, first on a C18 column and later on a cyano column, and (2) a separation with a linear gradient elution on a phenyl column. The wavelengths of maximum absorption of the fluorescent compound-containing HPLC fractions (~224, 268, 372, and 446 nm) are in good agreement with those exhibited by flavins. The H NMR spectra revealed methyl ( 2.30 and 2.40) and aromatic proton ( 7.79 and 7.77) signals of riboflavin. The H NMR spectra of the samples spiked with riboflavin confirmed that the brilliant yellow fluorescent compound is riboflavin. The maximum excitation and emission wavelengths of the fluorescent compound were 448 and 528 nm, respectively, which are identical to those of riboflavin.
酵母将一种亮黄色荧光化合物分泌到其生长培养基中。在这项研究中，我们使用反相高效液相色谱-二极管阵列检测器（RP-HPLC-DAD）以及 1H NMR 和 UV-Vis 光谱法对该化合物进行了分离和鉴定。所使用的三种 RP-HPLC-DAD 方法中的两种成功地分离了荧光化合物，涉及（1）两次使用等度洗脱的分离步骤，首先在 C18 柱上，然后在氰基柱上，以及（2）在苯基柱上进行线性梯度洗脱的分离。含荧光化合物的 HPLC 馏分的最大吸收波长（~224、268、372 和 446nm）与黄素表现出的波长非常吻合。1H NMR 谱显示核黄素的甲基（2.30 和 2.40）和芳质子（7.79 和 7.77）信号。用核黄素加标的样品的 1H NMR 谱证实亮黄色荧光化合物是核黄素。荧光化合物的最大激发和发射波长分别为 448nm 和 528nm，与核黄素相同。
