Enzyme-based glycosylation is of great interest in the context of natural products decoration. Yet, its industrial application is hindered by optimisation difficulties and hard-to-standardise productivities. In this study, five sugar nucleotide-dependent glucosyltransferases from different origins (bacterial, plant and fungal) were coupled with soy sucrose synthase (SuSy) to create a set of diverse cascade biocatalysts for flavonoid glucosylation, which evaluation brought new insights into the field. Investigations into co-expression conditions and reaction settings enabled to define optimal induction temperature (25 °C) and uridine diphosphate (UDP) concentration (0.5 mM) for all tested pairs of enzymes. Moreover, the influence of pH and substrate concentration on the monoglucosylated product distribution was detected and analysed. The utilisation of crude protein extracts as a cost-effective source of catalysts unveiled their glycosidase activity against flavonoid glucosides, resulting in decreased productivity, which, to our knowledge, has not previously been discussed in such a context. Additionally, examination of the commercially available EziG immobilisation resins showed that selection of suitable carrier for solid catalyst production can be problematic and not only enzyme’s but also reagent’s properties have to be considered. Flavonoids, due to their complexation and hydrophobic properties, can adsorb on different types of surfaces, including divalent metal ions required for IMAC based immobilisation, necessitating detailed examination of the resins while the catalysis design.

中文翻译：

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基于蔗糖合酶的黄酮类修饰糖基化级联的产生和反应条件的研究

基于酶的糖基化在天然产品装饰领域引起了极大的兴趣。然而，其工业应用受到优化困难和难以标准化的生产率的阻碍。在这项研究中，来自不同来源（细菌、植物和真菌）的五种糖核苷酸依赖性葡萄糖基转移酶与大豆蔗糖合酶（SuSy）偶联，创建了一套用于黄酮类葡萄糖基化的多样化级联生物催化剂，该评估为该领域带来了新的见解。对共表达条件和反应设置的研究能够确定所有测试酶对的最佳诱导温度 (25 °C) 和尿苷二磷酸 (UDP) 浓度 (0.5 mM)。此外，检测并分析了pH和底物浓度对单葡萄糖基化产物分布的影响。利用粗蛋白质提取物作为一种经济高效的催化剂来源，揭示了它们针对类黄酮苷的糖苷酶活性，导致生产率下降，据我们所知，以前从未在这种情况下讨论过这一点。此外，对市售 EziG 固定树脂的检查表明，选择合适的固体催化剂生产载体可能存在问题，不仅需要考虑酶的特性，还必须考虑试剂的特性。黄酮类化合物由于其络合和疏水特性，可以吸附在不同类型的表面上，包括基于 IMAC 的固定化所需的二价金属离子，因此需要在催化设计时对树脂进行详细检查。