Department of Food Chemistry and Phytochemistry, Institute of Applied Biosciences , Karlsruhe Institute of Technology (KIT) , Karlsruhe 76131 , Germany.
Lehrstuhl für Technische Mikrobiologie , Technische Universität München (TUM) , Freising 80333 , Germany.
J Agric Food Chem. 2019 Jun 19;67(24):6856-6866. doi: 10.1021/acs.jafc.9b01822. Epub 2019 Jun 7.
The water kefir organism Leuconostoc citreum TMW 2.1194 forms highly branched dextrans with O3- and O4-bound side chains. To obtain detailed information on the enzymatic synthesis of these polymers, the four glucansucrases encoded by Leuconostoc citreum TMW 2.1194 were cloned, heterologously expressed, and used for polysaccharide production. Molecular and macromolecular structure of the synthesized glucans were analyzed by methylation analysis, two-dimensional NMR spectroscopy, oligosaccharide analysis after partial hydrolysis, and asymmetric flow field-flow fractionation. It was demonstrated that two glucansucrases form insoluble glucans with variously branched dextran sections and varying portions of consecutive, 1,3-linked glucose units. In contrast, the other two glucansucrases synthesized O3- (Lc6255) and O4-branched (Lc1785) soluble dextrans. Analysis, isolation, and characterization of enzymatically liberated oligosaccharides showed that monomeric and elongated side chains are abundant in both polysaccharides. From the structures and size distributions it was concluded that Lc1785 is mainly responsible for synthesis of fermentatively produced soluble dextrans.
水苏糖生物体乳杆菌属柠檬酸盐 TMW 2.1194 形成具有 O3- 和 O4-结合侧链的高度分支葡聚糖。为了获得关于这些聚合物酶促合成的详细信息，克隆了乳杆菌属柠檬酸盐 TMW 2.1194 编码的四种葡聚糖蔗糖酶，进行了异源表达，并用于多糖生产。通过甲基化分析、二维 NMR 光谱、部分水解后的寡糖分析和不对称流场流分离分析，研究了合成葡聚糖的分子和大分子结构。结果表明，两种葡聚糖蔗糖酶形成具有不同分支葡聚糖部分和不同比例连续 1,3-连接葡萄糖单元的不溶性葡聚糖。相比之下，另外两种葡聚糖蔗糖酶合成了 O3-（Lc6255）和 O4-支链（Lc1785）可溶性葡聚糖。酶解释放的寡糖的分析、分离和表征表明，两种多糖中均含有丰富的单体和伸长侧链。从结构和尺寸分布来看，Lc1785 主要负责发酵生产的可溶性葡聚糖的合成。
