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.

Keywords: 2 ´ -Fucosyllactose; BoGT6a; Human milk oligosaccharides (HMOs); Ligand-dependent protein stabilization.