Premium
Functional characterization and reclassification of an enzyme previously proposed to be a limonoid UDP ‐glucosyltransferase
Author(s) -
Cui Youtian,
Allmon Steven D,
Siegel Justin B
Publication year - 2020
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.10547
Subject(s) - limonin , limonoid , glucosyltransferase , chemistry , enzyme , orange (colour) , orange juice , biochemistry , chromatography , stereochemistry , food science
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 cu GT from Citrus unishiu Marc (MBP‐ cu GT), 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‐ cu GT did not exhibit any detectable effect on limonin degradation when Navel orange juice was used as the substrate; MBP‐ cu GT was unable to biotransform either LARL or limonoate as purified substrates. However, it was found that MBP‐ cu GT 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 cu GT 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