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. An 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 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, highlights 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. This article is protected by copyright. All rights reserved.

中文翻译：

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先前被认为是柠檬苦素 UDP-葡萄糖基转移酶的酶的功能表征和重新分类

背景技术橙子工业中的一个主要问题是“延迟”苦味，这是由柠檬苦素引起的，柠檬苦素是一种苦味化合物，在提取橙汁期间和之后由其无苦味的前体柠檬苦素酸 A 环内酯 (LARL) 产生。在橙子成熟过程中，柠檬苦素 UDP-葡萄糖基转移酶 (LGT) 将 LARL 糖苷化以形成无苦味的糖基柠檬苦素，已被证明是一种通过防止柠檬苦素形成去苦味的自然方式。结果 在此，评估了异质表达的葡萄糖基转移酶、麦芽糖结合蛋白 (MBP) 与来自 Citrus Unishiu Marc (MBP-cuGT) 的 cuGT 融合的去苦味潜力，该酶以前被认为是 LGT。建立了LC-MS方法来测定柠檬苦素及其衍生物的浓度。获得其潜在底物的协议，还开发了 LARL 和柠檬苦素（具有 A 和 D 环开环的柠檬苦素）。令人惊讶的是，当脐橙汁用作底物时，MBP-cuGT 对柠檬苦素降解没有表现出任何可检测的影响；MBP-cuGT 不能生物转化 LARL 或 limonoate 作为纯化的底物。然而，发现 MBP-cuGT 对类黄酮表现出广泛的活性谱，证实所产生的酶在体外评估的条件下具有活性。结论 我们基于 LC-MS 的结果表明 cuGT 功能被错误识别。其活性底物，包括各种黄酮类，但不包括柠檬苦素，强调需要进一步努力确定负责 LGT 活性的酶，以开发基于生物技术的方法，从传统上具有延迟苦味的品种生产橙汁。本文受版权保护。版权所有。