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

Youtian Cui, Steven D. Allmon, Justin B. Siegel

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
JournalJournal of the Science of Food and Agriculture
DOIs
StateAccepted/In press - 2020

Keywords

  • delayed bitterness
  • flavonoids
  • glycosyltransferase
  • limonin
  • limonoid UDP-glucosyltransferase

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Agronomy and Crop Science
  • Nutrition and Dietetics

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