葡聚糖蔗糖酶在食品级乳酸菌中重组表达

Recombinant Expression of Dextransucrase in Food-grade Lactic Acid Bacteria

为研究葡聚糖在乳酸菌中的合成情况以及结构分析,本研究将葡聚糖蔗糖酶DsrB与带有SP45分泌信号肽的乳酸乳球菌表达载体pNZ8148质粒载体进行连接,电转入乳酸乳球菌NZ9000中进行异源表达。经由一系列醇沉、三氯乙酸去除蛋白质、二次醇沉、透析以及纯化等步骤得到多糖溶液。采用高效液相色谱法检测单糖组成;利用HPSEC测定葡聚糖的分子量;通过傅里叶红外技术、核磁共振技术以及扫描电镜分析多糖的结构并观察多糖表面特征。结果表明,将葡聚糖蔗糖酶在乳酸菌中进行表达,在蔗糖浓度为10%的条件下,葡聚糖产量为10.54 g/L;单糖组成显示仅含有葡萄糖一种单糖;胞外多糖分子量测定结果为2.4×10^(6) Da;由傅里叶红外和核磁联合表明体外合成的胞外多糖仅含有α-(1,6)糖苷键;扫描电镜表示胞外多糖表面呈现为多孔状结构。在乳酸菌中异源表达,实现了葡聚糖的食品级生产,为葡聚糖于食品中应用提供了理论基础。

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 2.4×10^(6) Da.The combination of FT-IR and NMR indicated that the extracellular polysaccharide synthesized in vitro contained only theα-(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.