Enabling quantification of 2′-fucosyllactose via ligand-dependent thermal stabilization of BoGT6a

Abstract

2′-Fucosyllactose (2′-FL) is a major component of Human Milk Oligosaccharides (HMOs) that plays a crucial role in developing the neonatal immune system and modulating gut microbiota. Due to its health benefits, 2′-FL has gained industrial importance as a key ingredient in probiotic products and functional foods. Although quantifying 2′-FL is crucial for its economical production and nutritional management, conventional methods require expensive equipment and skilled personnel, making high-throughput quantification challenging. In this study, we present a simple and cost-effective method for 2′-FL quantification by utilizing the thermal stability of BoGT6a, a glycosyltransferase derived from Bacteroides ovatus that specifically binds to 2′-FL. Initially, the binding of BoGT6a and 2′-FL was confirmed, and we demonstrated that 2′-FL-bound BoGT6a is protected from thermal stress. To achieve rapid detection of 2′-FL, we fused BoGT6a with the fluorescent protein mCherry, resulting in mCherry-BoGT6a, and investigated its thermal stability and fluorescence in response to varying 2′-FL concentrations. Finally, we developed a 2′-FL quantification device that measures protein precipitation with the change of electrical voltage. These results demonstrate the reliability and industrial applicability of BoGT6a-based 2′-FL quantification technology.