Characterization of the First α-(1 → 3) Branching Sucrases of the GH70 Family

Leuconostoc citreum NRRL B-742 has been known for years to produce a highly alpha-(1 -> 3)-branched dextran for which the synthesis had never been elucidated. In this work a gene coding for a putative alpha-transglucosylase of the GH70 family was identified in the reported genome of this bacteria and functionally characterized. From sucrose alone, the corresponding recombinant protein, named BRS-B, mainly catalyzed sucrose hydrolysis and leucrose synthesis. However, in the presence of sucrose and a dextran acceptor, the enzyme efficiently transferred the glucosyl residue from sucrose to linear alpha-(1 -> 6) dextrans through the specific formation of alpha-(1 -> 3) linkages. To date, BRS-B is the first reported alpha-(1 -> 3) branching sucrase. Using a suitable sucrose/dextran ratio, a comb-like dextran with 50% of alpha-(1 -> 3) branching was synthesized, suggesting that BRS-B is likely involved in the comb-like dextran produced by L. citreum NRRL B-742. In addition, data mining based on the search for specific sequence motifs allowed the identification of two genes putatively coding for branching sucrases in the genome of Leuconostoc fallax KCTC3537 and Lactobacillus kunkeei EFB6. Biochemical characterization of the corresponding recombinant enzymes confirmed their branching specificity, revealing that branching sucrases are not only found in L. citreum species. According to phylogenetic analyses, these enzymes are proposed to constitute a new subgroup of the GH70 family.