Purification and characterization of endo-beta-galactosidase from Escherichia freundii induced by hog gastric mucin.

作者：Michiko N. Fukuda · 发表于：Journal of Biological Chemistry · 年份：1981 · DOI：10.1016/s0021-9258(19)69543-8 · 被引用次数：98 · 研究领域：Seaweed-derived Bioactive Compounds、Carbohydrate Chemistry and Synthesis、Microbial Metabolites in Food Biotechnology

A new procedure for inducing and purifying endo-beta-galactosidase from Escherichia freundii was described. The enzyme was found to be induced with high efficiency in culture medium containing Smith-degraded hog gastric mucin, which was prepared from a commercially available starting material. Endo-beta-galactosidase was then purified by ammonium sulfate fractionation, DEAE-Sephadex chromatography, and affinity chromatography on Sepharose conjugated with the Smith-degraded mucin. The enzyme thus purified by only three steps showed no other glycosidase or protease activities and had higher specific activity compared to the previous method. This new method has a great advantage since the gastric mucin is abundantly available and the efficiency of enzyme production was high without significant induction of exoglycosidase. The hydrolysis of oligosaccharides, glycosphingolipid, and keratansulfate was studied by using this newly purified enzyme. Kinetic data indicate that hydrolyzability of these substrates is largely affected by substrate concentration, enzyme concentration and the structure of substrates. Based on these results, the specificity of E. freundii endo-beta-galactosidase was discussed.