Decoding microbial fructosyltransferases: Structural insights, molecular engineering, and biotechnological applications.

Fructosyltransferase (FTF), classified within glycoside hydrolase families GH32 and GH68 of the GH-J clan, plays an indispensable catalytic role in the biosynthesis of fructooligosaccharides (FOS) and fructan polymers (such as inulin and levan). These enzymes specifically catalyze fructosyl transfer reactions, constructing structurally diverse carbohydrates through β-(2 → 1) or β-(2 → 6) glycosidic linkages: FOS (linear oligosaccharides with DP 2-10 and a terminal glucose) and fructan polymers (high-molecular-weight polysaccharides). The resulting products not only exhibit excellent physicochemical properties but also demonstrate multiple physiological benefits including prebiotic activity, dietary fiber functionality, and regulation of glucose/lipid metabolism, showing broad application prospects in functional foods and pharmaceutical fields. This review systematically summarizes the microbial source diversity, three-dimensional structural characteristics, substrate recognition mechanisms and the molecular basis for the diversity of catalytic products of microbial FTF, including levansucrase, inulosucrase and β-fructofuranosidase. Focusing on enzyme molecular engineering strategies such as directed evolution, rational design, and semi-rational design, this review clarifies the intrinsic relationship between the catalytic properties of FTF and the structural and functional characteristics of their products, and discusses their application value in the synthesis of functional carbohydrates. This paper provides important theoretical support for the fundamental research of FTF and the development of precision synthesis technologies for FOS and fructans.