Expanding the enzymatic toolbox for ß-glucosylation of natural products using cellobiose phosphorylase and ß-glucosidase

Glycosylation plays a fundamental role in modifying the physicochemical properties of natural products, influencing their solubility, stability, and bioactivity. While enzymatic ¿-glucosylation has been widely explored, ß-glucosylation remains underutilized despite its potential for generating novel bioactive compounds with improved pharmacological and industrial applications. Building upon previous investigations, our research has focused on cellobiose phosphorylase from Clostridium thermocellum and ß-glucosidases from Talaromyces amestolkiaefor the selective ß-glucosylation of diverse natural products. The former shows activity towards polyphenols and terpenoids, despite the traditionally low affinity of phosphorylases for non-carbohydrate acceptors, while the latter shows broad acceptor promiscuity, successfully glycosylating phenolic compounds such as vanillin, hydroquinone, and hydroxytyrosol. Among the target molecules, geraniol and nerol¿key aroma constituents of wine¿were selected due to the lack of these ß-glucoside standards. The concentration of these compounds serves as an indicator of the wine's aromatic potential and its evolution over time. Measuring geraniol and nerol glucosides allows winemakers and analysts to obtain a more precise evaluation of the character, authenticity, and commercial value of the final product. This work seeks to expand the enzymatic toolbox for ß-glucosylation beyond conventional strategies. The reactions were monitored by UHPLC-MS, observing the formation of one product, and the reaction conditions and kinetics were studied. Additionally, nuclear magnetic resonance (NMR) was used to determine the product structure. The reactions were scaled up, and the products were purified by flash columna chromatography. Our results support the potential of enzymatic glucosylation for the modification of terpenoids and phenolic compounds, paving the way for applications in food chemistry, pharmaceuticals, and cosmetics.