Determination of glucansucrase encoding gene in Leuconostoc mesenteroides.

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. The presented study described the gene cloning, expression, purification, and characterization of glucansucrase from Leuconostoc mesenteroides TDS2-19. Sequence analysis indicated that it attaches to glucansucrase family: GH 70 family. The recombinant enzyme was purified to 4.02 fold with a specific activity of 35.85 U/mg and the molecular weight was about 160 kDa. The enzyme demonstrated its optimum activity at pH 5.5 and 30 °C. Enzyme activity was strongly activated by Ca2+ and Mn2+ and inhibited by various metal ions and chemical agents. Circular dichroism (CD) and Raman spectra collectively indicated a high proportion of random coil structure. Unlabelled Image • Gene encoding dextransucrase from Leuconostoc mesenteroides was cloned. • A 160 kDa recombinant dextransucrase was purified with a 4.02-fold increase in activity. • This optimal temperature and pH of recombinant dextransucrase were 30 °C and pH 5.5. • The dextransucrase was a random coil protein. [ABSTRACT FROM AUTHOR]