Determination of glucansucrase encoding gene in Leuconostoc mesenteroides

Abstract

A glucansucrase encoding gene was cloned into pET-28a(+) vector and expression in Escherichia coli BL21(DE3). An about 160 kDa recombinant glucansucrase was purified with a yield of 50.73% and a 4.02-fold increase in activity. The 1464 amino acid residue enzyme belongs to the GH70 subfamily and shares 90% similarity with Leuconostoc sp. glucansucrase. The optimal temperature and pH were 30 °C and pH 5.5, and 80% of activity was retained after incubation at 10-30 °C and pH 5-7. Enzyme activity was strongly activated by Ca²⁺ and Mn²⁺ and inhibited by various metal ions and chemical agents, and a high affinity for sucrose (K_m = 11.6 mM, V_max = 8.1 mmol/(mL·min)). Circular dichroism (CD) and Raman spectra collectively indicated a high proportion of random coil structure.

Keywords: Characterization; Cloning; Glucansucrase; Leuconostoc mesenteroides; Structural analysis.