Functional characterization and reclassification of an enzyme previously proposed to be a limonoid UDP‐glucosyltransferase

Abstract

BACKGROUND A major problem in the orange industry is ‘delayed’ bitterness, which is caused by limonin, a bitter compound developing from its non‐bitter precursor limonoate A‐ring lactone (LARL) during and after extraction of orange juice. The glucosidation of LARL by limonoid UDP‐glucosyltransferase (LGT) to form non‐bitter glycosyl‐limonin during orange maturation has been demonstrated as a natural way to debitter by preventing the formation of limonin. RESULT Here, the debittering potential of heterogeneously expressed glucosyltransferase, maltose‐binding protein (MBP) fused to cuGT from Citrus unishiu Marc (MBP‐cuGT), which was previously regarded as LGT, was evaluated. A liquid chromatography – mass spectrometry (LC–MS) method was established to determine the concentration of limonin and its derivatives. The protocols to obtain its potential substrates, LARL and limonoate (limonin with both A and D ring open), were also developed. Surprisingly, MBP‐cuGT did not exhibit any detectable effect on limonin degradation when Navel orange juice was used as the substrate; MBP‐cuGT was unable to biotransform either LARL or limonoate as purified substrates. However, it was found that MBP‐cuGT displayed a broad activity spectrum towards flavonoids, confirming that the enzyme produced was active under the conditions evaluated in vitro. CONCLUSION Our results based on LC–MS demonstrated that cuGT functionality was incorrectly identified. Its active substrates, including various flavonoids but not limonoids, highlight the need for further efforts to identify the enzyme responsible for LGT activity to develop biotechnology‐based approaches for producing orange juice from varietals that traditionally have a delayed bitterness. © 2020 Society of Chemical Industry

Neutral loss scan in complement with high‐resolution MS/MS: Combination of detection methods for flavonoid and limonoid glycosides analysis

“…Although these are the most abundant limonoids in citrus, there are also other types of limonoids and their corresponding glycosides which have been less studied, as many of these standards are not readily available commercially 25 . Despite this, both limonoids and their glycosides are of research interest as they can contribute to the understanding of metabolic pathways and secondary metabolite formation in the citrus fruits, which can lead to applications such as debittering of citrus juices 26 …”

“…25 Despite this, both limonoids and their glycosides are of research interest as they can contribute to the understanding of metabolic pathways and secondary metabolite formation in the citrus fruits, which can lead to applications such as debittering of citrus juices. 26 Although there have been some studies characterising the tandem MS properties of both limonoids and their glycosides, they have not explored the use of neutral loss scans for the detection of these glycosides. 11 Unlike the aglycones, limonoid glycosides have been adduct.…”