Characterization of a Novel Mutansucrase (MUT-I) from Leuconostoc pseudomesenteroides G29: Enzymatic Properties and Product Analysis

Glucansucrases are extracellular enzymes capable of synthesizing diverse α-glucan polymers and oligosaccharides, including the industrially relevant mutan. The mutI-encoded mutansucrase (MUT-I) from Leuconostoc pseudomesenteroides G29 was biochemically characterized as a robust biocatalyst for α-glucan engineering. Recombinant MUT-I (165.1 kDa) demonstrated optimal activity at pH 5.5/30 °C with a high substrate affinity (Km = 3.46 mM) and catalytic efficiency (kcat = 10.35 s⁻¹). It synthesized water-insoluble mutan (91% α-1,3 linkages; Mw = 644,083 g/mol) exhibiting exceptional thermal stability (degradation onset 287.65 °C) and compact fibrous morphology. The enzyme displayed dual functionality: (1) generating gluco-oligosaccharides through maltose-dependent acceptor reactions and (2) introducing α-1,3 branches into dextrans (74% and 14% branching efficiency for dextran T3 and T70, respectively). The deep learning tool AlphaFold 3 identified conserved catalytic residues (Asp476, Glu514, and Asp587) governing polymerization dynamics. This study establishes MUT-I's potential for sustainable production of structurally defined α-glucans as biodegradable alternatives to synthetic polymers in food and biomedical applications.