Abstract: The low efficiency and high cost of traditional methods for producing high-molecular-mass levan limit its industrial-scale utilization. To overcome this limitation, two levansucrase genes, Lc-SacB1 and Lc-SacB2, were identified in Leuconostoc citreum BD1707 through genome mining in this study. Heterologous expression and functional analysis revealed that Lc-SacB2 (with a molecular mass of 130 kDa, the known levansucrase with the highest molecular mass) possessed both transfructosylation and hydrolytic activities, with a high total catalytic efficiency (Kcat/Km = 0.048 L/(s·mmol)), whereas Lc-SacB1 only had hydrolytic activity with a lower catalytic efficiency (Kcat/Km = 0.029 L/(s·mmol)). Enzymatic characterization showed that the optimal reaction conditions of Lc-SacB2 (pH 5.5 and 30 ℃) were close to those of Lc-SacB1 (pH 6.0 and 30 ℃). Furthermore, Ca2+ significantly enhanced the activities of both enzymes, increasing the relative activity of Lc-SacB2 by 134%, and the activating effect on Lc-SacB1 was more pronounced (155%). The product obtained using Lc-SacB2 was identified as a high-molecular-mass β-(2,6)-fructan (4.0 × 106 Da) through nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). Structural analysis suggested that the steric hindrance in the loop region at the substrate channel entrance of Lc-SacB1 likely inhibited the elongation of the fructan chain, causing Lc-SacB1 to be unable to catalyze the formation of levan and leading to functional differentiation between the two enzymes. This study provides a theoretical foundation for the development and molecular engineering of novel enzyme resources, accelerating the industrialization of high-efficiency levan biomanufacturing.

Key words: Leuconostoc citreum; levansucrase; functional differentiation; high-molecular-mass levan