One-step enzymatic synthesis of medium molecular weight dextran using engineered dextransucrase DarM from Leuconostoc...

The dextransucrase DarM from Leuconostoc citreum CBA3623, a large GH70 family enzyme of approximately 213 kDa, was refractory to full-length expression in E. coli. Based on the predicted domain architecture, two catalytically active truncation variants-DarM-ΔV1 (146 kDa) and DarM-ΔV2 (124 kDa), were rationally designed and expressed in soluble form. Both variants exhibited optimal activity under acidic conditions (pH 4.5) but differed in their temperature optima and kinetic parameters. Although these variants produced glucans from high-molecular-weight (HMW) dextran to oligosaccharides, the molecular weight distribution was highly dependent on reaction conditions. Notably, DarM-ΔV1 uniquely enabled one-step synthesis of a medium-molecular-weight (MMW) dextran fraction (~45 kDa). In silico structural modeling and site-directed mutagenesis suggested that a putative sugar-binding pocket (V-C) located at the C-terminal region may contribute to the chain length control during dextran synthesis. Disruption of this pocket via the Y1283A mutation impaired MMW dextran synthesis and shifted the product distribution toward oligosaccharides. Guided by this structural insight, we further tuned the reaction temperature (from 35 °C to 5 °C) to expand its accessible MMW window to 27.8-75.2 kDa. Collectively, this work identifies DarM-ΔV1 as a practical and convenient biocatalyst scaffold for MMW dextran production and provides clues to the catalytic mechanism underlying controlled dextran synthesis in GH70 dextransucrase.