Enzymatic Production of Difructose Anhydrides

The enzymatic production of difructose anhydrides (DFAs) harnesses specific fructotransferases and glycoside hydrolases to convert natural fructan polymers or sucrose into cyclic d-fructose disaccharides via intramolecular dehydration. Major isomers—DFA I, DFA III and DFA IV—derive from inulin or levan substrates through the coordinated action of inulin fructotransferase, levansucrase and levan fructotransferase. Structural studies have elucidated active-site architectures and catalytic mechanisms, enabling protein engineering to enhance isomer selectivity and reaction yield. Concurrently, metabolic engineering of microbial hosts has established consolidated bioprocesses for direct one-pot conversion of feedstocks to DFAs. These advances underpin the growing global interest in DFAs as low-calorie sweeteners, prebiotic ingredients and versatile building blocks for functional foods and pharmaceuticals.

Research from Nature Portfolio

Recent studies have demonstrated direct one-step bioproduction of DFA IV by co-cultivation of recombinant yeasts engineered to secrete levansucrase and levan fructotransferase. Optimised translational fusion partners facilitated secretion titres of 3.8 U/mL (levansucrase) and 16.0 U/mL (fructotransferase), while fine-tuning of sucrose concentration and cell ratios yielded up to 64.3 g/L crude DFA IV from 244.7 g/L sucrose in a single fermentation. This consolidated approach represents a significant improvement in process efficiency and scalability for industrial DFA IV manufacturing.

Enzymatic Production of Difructose Anhydrides publication trend

The graph below shows the total number of articles in enzymatic production of difructose anhydrides across all publications each year (not limited to Nature Index journals).

Technical terms

Difructose anhydrides (DFAs): Cyclic spiroketal disaccharides formed by dehydration of two fructose units, existing as multiple linkage isomers such as DFA I, III and IV.

Inulin fructotransferase (IFTase): A glycoside hydrolase (GH91) enzyme that catalyses intramolecular transfer of fructose units in inulin to release difructose anhydrides.

Levansucrase: A fructosyltransferase that polymerises fructose from sucrose to form the levan polymer, serving as a precursor substrate for DFA IV synthesis.

Levan fructotransferase: An enzyme that cleaves and transfers fructose units within levan to generate DFA IV via intramolecular cyclisation.

Glycoside hydrolase families: Classification of enzymes sharing sequence and structural motifs that catalyse glycosidic bond hydrolysis or intramolecular transfer reactions.

References

1. Degradation mechanism of difructose dianhydride III in Blautia species. Applied Microbiology and Biotechnology (2024).
2. Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family. Journal of Biological Chemistry (2021).
3. Structural and Functional Insights into Intramolecular Fructosyl Transfer by Inulin Fructotransferase*. Journal of Biological Chemistry (2006).
4. Direct Production of Difructose Anhydride IV from Sucrose by Co-fermentation of Recombinant Yeasts. Scientific Reports (2019).