Recombinant Expression of Dextransucrase in Food-grade Lactic Acid Bacteria

Abstract

In order to study the synthesis of dextran in Lactobacillus and to analyze its structure, in this study, the dextransucrase DsrB was ligated with the Lactococcus lactis expression vector pNZ8148 plasmid vector with SP45 secretion signal peptide, and electrotransferred into L. lactis NZ9000 for heterologous expression. The polysaccharide solution was obtained through a series of steps including alcohol precipitation, protein removal by trichloroacetic acid, secondary alcohol precipitation, dialysis and purification. The composition of monosaccharides was detected by high performance liquid chromatography (HPLC). The molecular weight of dextran was determined by high-performance size exclusion chromatography (HPSEC). The structure of polysaccharides was analyzed by fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR) and scanning electron microscopy (SEM), and the surface characteristics of polysaccharides were observed. The results showed that dextransucrase was expressed in lactic acid bacteria, and the glucan yield was 10.54 g/L at a sucrose concentration of 10%. The monosaccharide composition showed that it contained only one monosaccharide, glucose. The molecular weight of the extracellular polysaccharide was determined to be 2.4×106 Da. The combination of FT-IR and NMR indicated that the extracellular polysaccharide synthesized in vitro contained only the α-(1,6) glycosidic bond, and SEM indicated that the surface of the extracellular polysaccharide showed a porous structure. The heterologous expression in lactic acid bacteria realized the food-grade production of dextran and would provide a theoretical basis for the application of dextran in food.

Keywords

• dextransucrase
• dextran
• extracellular polysaccharides
• food-grade production
• recombinant expression