Kinetic studies on endo‐β‐galactosidase by a novel colorimetric assay and synthesis of N‐acetyllactosamine‐repeating oligosaccharide β‐glycosides using its transglycosylation activity

Novel chromogenic substrates for endo‐β‐galactosidase were designed on the basis of the structural features of keratan sulfate. Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ‐pNP (2), which consists of two repeating units of N‐acetyllactosamine, was synthesized enzymatically by consecutive additions of GlcNAc and Gal residues to p‐nitrophenyl β‐N‐acetyllactosaminide. In a similar manner, GlcNAcβ1‐3Galβ1‐4GlcNAcβ‐pNP (1), GlcNAcβ1‐3Galβ1‐4Glcβ‐pNP (3), Galβ1‐4GlcNAcβ1‐3Galβ1‐4Glcβ‐pNP (4), Galβ1‐3GlcNAcβ1‐3Galβ1‐4Glcβ‐pNP (5), and Galβ1‐6GlcNAcβ1‐3Galβ1‐4Glcβ‐pNP (6) were synthesized as analogues of 2. Endo‐β‐galactosidases released GlcNAcβ‐pNP or Glcβ‐pNP in an endo‐manner from each substrate. A colorimetric assay for endo‐β‐galactosidase was developed using the synthetic substrates on the basis of the determination of p‐nitrophenol liberated from GlcNAcβ‐pNP or Glcβ‐pNP formed by the enzyme through a coupled reaction involving β‐N‐acetylhexosaminidase (β‐NAHase) or β‐d‐glucosidase. Kinetic analysis by this method showed that the value of Vmax/Km of 2 for Escherichia freundii endo‐β‐galactosidase was 1.7‐times higher than that for keratan sulfate, indicating that 2 is very suitable as a sensitive substrate for analytical use in an endo‐β‐galactosidase assay. Compound 1 still acts as a fairly good substrate despite the absence of a Gal group in the terminal position. In addition, the hydrolytic action of the enzyme toward 2 was shown to be remarkably promoted compared to that of 4 by the presence of a 2‐acetamide group adjacent to the p‐nitrophenyl group. This was the same in the case of a comparison of 1 and 3. Furthermore, the enzyme also catalysed a transglycosylation on 1 and converted it into GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ‐pNP (9) and GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ‐pNP (10) as the major products, which have N‐acetyllactosamine repeating units.