Tanaka H, Ito F, Iwasaki T
Technical Research Institute, Snow Brand Milk Products Co., Ltd., Saitama, Japan.
Biochem Biophys Res Commun. 1992 Nov 30;189(1):524-9. doi: 10.1016/0006-291x(92)91589-i.
A sialidase from Bacteroides fragilis SBT3182 was purified 2,240-fold to apparent homogeneity by ammonium sulfate precipitation and sequential chromatographies on DEAE-Toyopearl 650M, Hydroxyapatite, MonoS and Superose6 columns. The N-terminal amino acid sequence of this sialidase, Ala-Asp-X-Ile-Phe-Val-Arg-Glu-Thr-Arg-Ile-Pro-, was determined. Substrate specificity of this enzyme using a variety of sialoglycoconjugates showed a 1.5- and 2.2-fold preference for sialyl alpha 2-8 linkages when compared with alpha 2-3 and alpha 2-6 bound sialic acids, respectively. The native sialidase had a molecular weight of 165kDa, as determined by Superose6 gel filtration chromatography and consisted of three subunits each of 55kDa by SDS-polyacrylamide gel electrophoresis. This enzyme had optimal activity at pH6.1 with colominic acid as substrate.
通过硫酸铵沉淀以及在DEAE- Toyopearl 650M、羟基磷灰石、MonoS和Superose6柱上的连续层析，从脆弱拟杆菌SBT3182中纯化出一种唾液酸酶，纯化倍数达2240倍，达到表观均一。测定了该唾液酸酶的N端氨基酸序列：Ala-Asp-X-Ile-Phe-Val-Arg-Glu-Thr-Arg-Ile-Pro-。使用多种唾液酸糖缀合物对该酶的底物特异性研究表明，与α2-3和α2-6连接的唾液酸相比，该酶对α2-8连接的唾液酸的偏好性分别为1.5倍和2.2倍。通过Superose6凝胶过滤色谱法测定，天然唾液酸酶的分子量为165kDa，SDS-聚丙烯酰胺凝胶电泳显示其由三个55kDa的亚基组成。以共聚唾液酸为底物时，该酶在pH6.1时具有最佳活性。
