Enzymatic synthesis of poly-N-acetyllactosamines as potential substrates for endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions.
Enzymatic synthesis of GlcNAc-terminated poly-N-acetyllactosamine beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(n)Galbeta1,4GlcNAcbeta-pNP (n=1-4) was demonstrated using a transglycosylation reaction of Escherichia freundii endo-beta-galactosidase. The enzyme catalyzed a transglycosylation reaction on GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (1), which served both as a donor and an acceptor, and converted 1 into p-nitrophenyl beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(1)Galbeta1,4GlcNAcbeta-pNP (2), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(2)Galbeta1,4GlcNAcbeta-pNP (3), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(3)Galbeta1,4GlcNAcbeta-pNP (4) and GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(4)Galbeta1,4GlcNAcbeta-pNP (5). When 2 was used as an initial substrate, it led to the preferential synthesis of nonasaccharide beta-glycoside 4 to heptasaccharide beta-glycoside 3. This suggests that 4 is directly synthesized by transferring the tetrasaccharide unit GlcNAcbeta1,3Galbeta1,4GlcNAcbeta1,3Gal to nonreducing end GlcNAc residue of 2 itself. The efficiency of production of poly-N-acetyllactosamines by E. freundii endo-beta-galactosidase was significantly enhanced by the addition of BSA and by a low-temperature condition. Resulting 2 and 3 were shown to be useful for studying endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions.
Murata
Takeomi
T
Department of Applied Biological Chemistry, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan. actmura@shizuoka.ac.jp
Honda
Hiroki
H
Hattori
Takeshi
T
Usui
Taichi
T
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002240:Carbohydrate Sequence; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D011134:Polysaccharides; C037199:poly-N-acetyllactosamine; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase