RESUMEN
Flavonoids are associated with health benefits, but most of them have poor oral bioavailability due to their extremely low aqueous solubility. Flavonoid O-phosphorylation suggests a potent modification to solve the problems. Here, we isolated, identified and characterized an unprecedented phosphotransferase, flavonoid phosphate synthetase (BsFPS), from B. subtilis. The enzyme catalyzes the ATP-dependent phosphorylation of flavonoid to generate flavonoid monophosphates, AMP and orthophosphate. BsFPS is a promiscuous phosphotransferase that efficiently catalyzes structurally-diverse flavonoids, including isoflavones, flavones, flavonols, flavanones and flavonolignans. Based on MS and NMR analysis, the phosphorylation mainly occurs on the hydroxyl group at C-7 of A-ring or C-4' of B-ring in flavonoid skeleton. Notably, BsFPS is regioselective for the ortho-3',4'-dihydroxy moiety of catechol-containing structures, such as luteolin and quercetin, to produce phosphate conjugates at C-4' or C-3' of B-ring. Our findings highlight the potential for developing biosynthetic platform to obtain new phosphorylated flavonoids for pharmaceutical and nutraceutical applications.
Asunto(s)
Flavanonas , Flavonas , Flavonolignanos , Isoflavonas , Adenosina Monofosfato , Adenosina Trifosfato , Bacillus subtilis , Catecoles , Flavonoides/química , Ligasas , Luteolina , Fosfatos , Fosfotransferasas , QuercetinaRESUMEN
Luteolin (LUT), a plant-derived flavone, exhibits various bioactivities; however, the poor aqueous solubility hampers its applications. Here, we revealed bioconversion of LUT by Bacillus subtilis BCRC 80517, yielding three water-soluble phosphate conjugates. These derivatives were identified as luteolin 4'-O-phosphate (L4'P), luteolin 3'-O-phosphate (L3'P), and luteolin 7-O-phosphate (L7P) by LC-ESI-MS/MS and NMR. Besides, we found that Bacillus subtilis BCRC 80517 was able to convert different levels of LUT but showed a limited conversion rate. By observing bacterial morphology with transmission electron microscopy and confocal fluorescence microscopy, we found that LUT disrupted the bacterial membrane integrity, which explained the incomplete conversion. Additionally, we revealed a spontaneous intramolecular transesterification of L4'P to L3'P, the thermodynamically more stable form, under acidic conditions and proposed the possible mechanism involving a cyclic phosphate as the intermediate. This study provides insight into development of a potent structural modification strategy to enhance the solubility of LUT through biophosphorylation.
Asunto(s)
Bacillus subtilis , Luteolina , Cromatografía Liquida , Luteolina/química , Fosfatos , Espectrometría de Masas en TándemRESUMEN
Glycidyl esters (GEs) and 3-chloroprapane-1,2-diol esters (3-MCPDEs) are processing contaminants in refined edible oils that have raised concerns globally owing to their potentially carcinogenic properties. Official analytical methods for GEs and 3-MCPDEs, such as AOCS Cd 29a-13 and AOCS Cd 29b-13, require up to 16 h for chemical hydrolysis. Also, parallel experiments should be conducted to correct for the conversion of analytes during hydrolysis in AOCS Cd 29b-13. For AOCS Cd 29c-13 with the shortest operating time, the reaction time (3.5-5.5 min) and temperature of alkaline hydrolysis should be carefully controlled, implying the accuracy may be influenced by human errors. Here, we propose a novel method based on Candida rugosa lipase hydrolysis and direct detection of free form GEs, glycidol, which was achieved by sample preparation with modified QuEChERS, to prevent side reactions in previous approaches, and also to shorten the overall sample preparation time. Glycidol was directly analyzed without halogenation and derivatization, whereas 3-MCPD required derivatization for analysis by GC-MS. Our method showed good accuracy and precision in terms of repeatability, intermediate precision, and reproducibility (inter-laboratory precision). The limit of detection (LOD) and limit of quantification (LOQ) for glycidol were 0.02 and 0.1 mg/kg, which is sufficient for practical applications. The proposed method was further compared with AOCS Cd 29c-13 by determination of GEs content in commercial oil samples and spiked samples. Our method with a streamlined procedure seems to possess potential advantage of reduced errors from operational factors. This proposed method based on direct detection of glycidol may serve as a simplified alternative for routine analysis of GEs and 3-MCPDEs in edible oils.
Asunto(s)
alfa-Clorhidrina , Cadmio/análisis , Ésteres/análisis , Contaminación de Alimentos/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos , Glicerol/análisis , Humanos , Hidrólisis , Lipasa , Aceites de Plantas/química , Reproducibilidad de los Resultados , alfa-Clorhidrina/análisisRESUMEN
The conversion of sesame lignans is of interest because the derived products may have potential applications. Here, in investigating the transformation of sesamin and sesamolin, main endogenous sesame lignans in sesame seeds, in both acidic aqueous and anhydrous systems, 7R,7'S-samin was identified as one of the major products of sesamolin in both systems catalyzed with common inorganic acids, but sesaminol was not generated. In investigating the effect of different oxidizing agents on the acid-catalyzed conversion of sesame lignans, 7R,7'S-samin was still the major product of sesamolin, whereas sesamolin as well as 7R,7'S-samin stereoselectively rendered 7R,7'R-samin in the presence of hydrogen peroxide. Hydrogen peroxide may play a role in stabilizing the transitional oxonium ions, derived from acid hydrolysis of sesamolin or 7R,7'S-samin by forming a seven-membered ring intermediate through hydrogen bonding, to consequently produce 7R,7'R-samin as the final product.