Kinetic Studies on Endo-.BETA.-galactosidase by a Novel Colorimetric Assay and Synthesis Poly-N-acetyllactosamines Using Its Transglycosylation Activity
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概要
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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 enzymatically synthesized 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 analogs 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-acetylglucosaminidase 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 almost equal to that for keratan sulfate, indicating that 2 is very suitable as a sensitive substrate for analytical use in an endo-β-galactosidase assay. In addition, the hydrolytic action of the enzyme toward 2 has shown to be remarkably promoted by the presence of 2-acetamide group adjacent to p-nitrophenyl group in comparison with 4. In addition, enzymatic synthesis of GlcNAc-terminated poly-N-acetyllactosamine β-glycosides GlcNAcβ1,3 (Galβ1,4GlcNAcβ1,3)n Galβ1,3GlcNAcβ-pNP (n=1-5) has been demonstrated using a transglycosylation reaction of E. freundii endo-β-galactosidase. The enzyme catalyzed a transglycosylation reaction on 1, which served both as a donor and an acceptor, and converted 1 into p-nitrophenyl β-glycosides GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)nGalβ1,4GlcNAcβ-pNP (9, n=1; 10, n=2; 11, n=3; 12, n=4; 13, n=5). The efficiency of production of poly-N-acetyllactosamines by E. freundii endo-β-galactosidase was significantly enhanced by the addition of BSA and by a low temperature condition.
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