Microbial production of oligosialic acid

微生物生产低聚唾液酸

基本信息

批准号：
07455327
负责人：
IIJIMA Shinji
金额：
$ 4.74万
依托单位：
Nagoya University, School of Engineering
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07455327/
关键词：
sialic acid selectin microbial production sugar technology metastasis phage phage suger technology

项目摘要

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.

糖在生物过程中的分析和应用尚未得到广泛研究。在这方面，我们研究了功能性糖的微生物生产，例如寡唾液酸和唾液酸乳糖（唾液酸路易斯糖的类似物）。以大肠杆菌K1菌株为宿主细菌，从污水中分离出七种不同的噬菌体。这些噬菌体对宿主荚膜多糖、α-2,8-连接的聚唾液酸表现出特定的降解活性。对于最小底物尺寸，每种酶都有其自己的特异性。在七种分离的噬菌体中，四种酶表现出新的内切-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其中一种酶。我们还开发了一种针对可溶性神经氨酸酶的抗体。除此之外，还在大肠杆菌中克隆了神经氨酸酶基因并纯化了重组蛋白。通过免疫印迹，这3种神经氨酸酶在免疫学上是相同的，它们的分子量均为90kD。通过使用正常内皮细胞，我们能够检测来自无乳链球菌的类型特异性多糖对癌细胞与内皮细胞粘附的抑制作用，这是必不可少的癌症转移的步骤。这种抑制可能是由于细菌多糖的特定结构，因为这些糖的结构与癌症特异性唾液酸Lewis碳水化合物（唾液酸Le^a和Le^x）的结构非常相似，后者与内皮细胞的ELAM-1结合。在无乳链球菌菌株特异性荚膜糖中，配体（唾液酸乳糖）从主链（Gal-GluNAc 的重复单元）上脱色。在主链的每个重复单元上都观察到支化。我们尝试用β-半乳糖苷酶消化细菌糖并产生唾液酸乳糖低聚物。