Endo galactosidases (EC 3.2.1.103) were discovered as keratan sulfate degrading enzymes, so called keratanases, in culture filtrates of E. freundii, 1) Coccobacillus sp., 2) Pseudomonas sp., 3) Flavobacterium keratolyticus 4,5) and Bacteroides fragilis.
6)E. freundii keratanase was found to have hydrolyzing activity for a wide range of nonsulfated oligosaccharides isolated from human milk and carbohydrate moieties of glycoproteins and glycolipids.7 10) The use of endo galactosidase has been expanded to detection of poly N acetyllactosamine chains in a variety of complex glycoconjugates in addition to keratan sulfate. Bacteroides fragilis endo galactosidase has properties similar to those of E. freundii endo galactosidase.11 13) Therefore, the endo galactosidases from E. freundii and B. fragilis have been widely used as tools for structural and functional analyses of glycans involved in glycoconjugates. An assay using keratan sulfate as a substrate has been widely used for estimation of endo galactosidase activity. However, this method is not always reproducible because of lack of uniformity of the polymer. Methods using low molecular weight substrates defined their structures have been preferred and recommended for accurate determination of endoglycosidases such as amylase, 14) lysozyme, 15) and endo N acetyl glucosaminidase, 16) because the purity of the substrate and the reaction pattern can be determined exactly. Therefore, a series of chromogenic substances having a partial substituted unit of poly N acetyllactosamine were designed as substrate analogs for the enzyme.Poly N acetyllactosamine has been shown to be present on membrane glycoconjugates, 17,18) and has been identified as a precursor of Lewis X, sialyl Lewis X, and blood group antigens.19) The amounts of poly N acetyllactosamine chains have been shown to be changed during cellular differentiation and malignant transformation of cells. 20,21) Furthermore, B16 melanoma cells that expressed sialyl Lewis X on long poly N acetyllactosamine were highly metastatic, while cells expressing even more sialyl Lewis X on short poly N acetyllactosamine were not metastatic.
22)In addition, poly N acetyllactosamines have been shown to be directly recognized with high affinity by galectins, 23) and to be involved in apoptosis.24) These findings suggest 1,4GlcNAcβ -pNP (9, n 1; 10, n 2; 11, n 3; 12, n 4; 13, n 5). The efficiency of production of poly-Nacetyllactosamines by E. freundii endo-β -galactosidase was significantly enhanced by the addition of BSA and by a low temperature condition.