Microbial production of oligosialic acid

糖在生物过程中的分析和应用研究尚不广泛。在这方面，我们研究了微生物生产的功能糖，如低聚硅酸和硅基乳糖，一种类似于硅基刘易斯糖。以大肠杆菌K1菌株为宿主菌，从污水中分离出7种不同的噬菌体。这些噬菌体对宿主荚膜多糖α -2,8-链聚唾液酸具有特异性降解活性。对于最小底物大小，每种酶都有自己的特异性。在七个分离的噬菌体中，有四个酶显示出新的内切- n -乙酰神经氨酸酶活性。我们从噬菌体颗粒和受感染微生物培养物的可溶性部分中纯化了一种酶。我们还开发了一种针对可溶性神经氨酸酶的抗体。此外，在大肠杆菌中克隆了神经氨酸酶基因，并纯化了重组蛋白。免疫印迹结果表明，这3种神经氨酸酶在免疫学上完全相同，分子量均为90kD。通过正常内皮细胞，我们可以检测到无乳链球菌类型特异性多糖对癌细胞粘附内皮细胞的抑制作用，这是癌细胞转移的必要步骤。抑制作用可能是由于细菌多糖的特殊结构，因为糖的结构非常类似于与内皮细胞的ELAM-1结合的癌症特异性sialyl Lewis碳水化合物（sialyl Le^a和Le^x）。在S.agalactiae菌株特异性荚膜糖中，从主链（Gal-GluNAc的重复单元）上脱去一个配体（唾液基乳糖）。在主链的每个重复单元上都观察到支链。我们尝试用-半乳糖苷酶来消化细菌糖，并生产了唾液酰乳糖低聚物。

Analyzes and application of sugar in biological process have not been studied extensively. In this regard, we studied microbial production of functional sugar such as oligosialic acids and sialyl lactose, an analog of sialyl Lewis sugar.Seven different bacteriophages were isolated from sewage with Escherichia coli K1 strains as host bacteria. These phages showed specific degrading activity for the host capsular polysaccharide, alpha-2,8-linked polysialic acid. With respect to the minimum substrate size, each enzyme had its own specificity. Of the seven isolated phages, four enzyme showed novel endo-N-acetylneuraminidase activities. We purified one of the enzyme from both phage particle and soluble fraction of infected microbial culture. We also develop an antibody against soluble neuraminidase. In addition to this, the neuraminidase gene was cloned in E.coli and the recombinant protein was purified. By immunoblotting, these 3 neuraminidase species were immunologically identical and their molecular weigh was 90kD.By using normal endothelial cells, we were able to detect inhibitory effects of type specific polysaccharides from Streptococcus agalactiae on adhesion of cancer cells to endothelial cells, which is an essential step of cancer metastasis. The inhibition was probably due to specific structures of the bacterial polysaccharides, since the structures of the saccharides are very similar to those of cancer specific sialyl Lewis carbohydrates (sialyl Le^a and Le^x) which bind to ELAM-1 of endothelial cells.In S.agalactiae strain specific capsular sugar, a ligand (sialyl lactose) was blanched from main chain (repeating units of Gal-GluNAc). The branchings are observed every repeating unit of main chain. We tried to digest the bacterial sugar with a beta-galactosidase and produced sialyl lactose oligomer.