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. 2020 Oct;100(13):4870-4878.
doi: 10.1002/jsfa.10547. Epub 2020 Jun 29.

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

Youtian Cui  1 , Steven D Allmon  2 , Justin B Siegel  1   3
Affiliations

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

Youtian Cui et al. J Sci Food Agric. 2020 Oct.

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.

Keywords: delayed bitterness; flavonoids; glycosyltransferase; limonin; limonoid UDP-glucosyltransferase.

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Figures

Figure 1.
Figure 1.
Biosynthetic pathway of limonin and its related enzymatic reaction. LARL, the precursor of limonin, could be catalyzed by LGT to form non-bitter glucosyl-limonin, which serves as the naturally debittering approach.
Figure 2.
Figure 2.
Expression and purification of cuGT with MBP tag (MBP-cuGT), and evaluation of its activity towards Navel orange juices. (A) LGT with co-expression of MBP tag at N-terminus was solubly expressed in E. coli and purified by IMAC as shown by 12 % SDS-PAGE gel (B) The quantification of limonin by reverse phase HPLC ESI+ LC-MS spectrum. Selected ion mode (SIM) in ESI+ at m/z 471.2 corresponding to [M + H]+ of limonin was executed and various concentrations of limonin were sampled to test the sensitivity and accuracy of this method. The standard curve of limonin was drawn by the ion intensity calibrated with internal standard Podophyllotoxin at m/z 397.2 (Figure S2 of Supporting Information). (C) LGT activity assay with Navel orange juice as substrate in different conditions. Purified MBP-cuGT was blended with 1mM UDP-glucose and diluted Navel orange juice in which the pH was adjusted to 4.0, 5.5 and 7.0 and temperature held at either 20 °C and 37 °C for 24 hours. Half of the sample was directly analyzed for limonin concentration, and the second half acidified and after a 24 h incubation was evaluated for limonin. The increase in limonin observed in acidified samples was assumed to be a result of the cyclization of LARL into limonin. No significant decrease in levels of limonin and LARL was observed between MBP-cuGT-treated and control orange juice, as indicated by p-value > 0.1 calculated by the t-test. Error bars indicate the standard deviation of the triplicate samples
Figure 3.
Figure 3.
Preparation of LARL and limonoate as substrates and corresponding MBP-cuGT activity assays. (A) LARL could be purified by SPE (See materials and methods) and converted into limonin sequentially by strong acid, enabling its quantification by the same HPLC-MS method for limonin. Purified LARL only contained a small amount of residual limonin as contamination (< 5%). (B) HPLC-MS trace of purified LARL treated with 1mM UDP-glucose and MBP-cuGT (1 mg/mL) and the LARL control. No decrease in the observed limonin after incubation 37 °C, 24 hours with UDP-glucose and MBP-cuGT is observed, indicating that no LGT activity was observed. (C) Preparation of limonoate by strong base treatment. Because limononate does not occur naturally in citrus juice, limonoate (506.5 Da) can be synthesized by incubating limonin (470.5 Da) under basic conditions for an extended period of time. (D) HPLC-MS trace of limonin converted from limonoate by strong acid, which is used to quantify concentrations of limonoate. Limonoate (>10 ppm) was mixed with UDP-glucose (1 mM) and MBP-cuGT (1 mg/mL) under the assay condition 37 °C for 24 hours in tris buffer. As observed, no significant change in the level of limonoate is observed after incubation with MBP-cuGT, indicating the lack of LGT enzymatic activity.
Figure 4.
Figure 4.
Activity profile of MBP-cuGT towards flavonoids and related chemicals. The substrates were mixed with MBP-cuGT (1 mg/mL) and UDP-glucose (1 mM) at 37 °C for 24 hours. The qualitative activity was obtained by comparing the spectrums between the reaction samples and the corresponding controls without enzyme added. (A) The flavonoids accepted by LGT were enclosed by the red box while not active substrates were in the blue box. (B) HPLC spectrum of biotransformation of 7-hydroxyflavone by MBP-cuGT as representative of active flavonoid substrates. The product of flavone-7-o-glucoside was then verified by MS. (C) HPLC spectrum of biotransformation of apigenin by MBP-cuGT. Apigenin possessing three hydroxyl functional groups and two of them were glycosylated, resulting in two different products.
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References

    1. Jayaprakasha GK, Patil BS, In vitro evaluation of the antioxidant activities in fruit extracts from citron and blood orange. Food Chem 101:410–418 (2007).
    1. Paula BMD, Raithore S, Manthey JA et al., Active taste compounds in juice from oranges symptomatic for Huanglongbing (HLB) citrus greening disease. LWT – Food Sci Technol 91:518–525 (2018).
    1. Raithore SAK, Johannes and Manthey, John A. and Plotto, Anne and Bai, Jinhe and Zhao, Wei and Baldwin, Elizabeth, Mitigation of Off-flavor in Huanglongbing-affected Orange Juice Using Natural Citrus Non-volatile Compounds. J Agric Food Chem 68:1038–1050 (2020). - PubMed
    1. Narnoliya LK, Jadaun JS, Biotechnological Avenues for Fruit Juices Debittering. Green Bio-processes 119–149 (2019).
    1. Hasegawa S, Limonin bitterness in citrus juices. Flavor Chemistry (1999).

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