Abstract
The transgalactosylations of serine/threonine derivatives were investigated using β-galactosidase from Escherichia coli as biocatalyst. Using ortho-nitrophenyl-β-d-galactoside as donor, the highest bioconversion yield of transgalactosylated N-carboxy benzyl l-serine benzyl ester (23.2%) was achieved in heptane:buffer medium (70:30), whereas with the lactose, the highest bioconversion yield (3.94%) was obtained in the buffer reaction system. The structures of most abundant galactosylated serine products were characterized by MS/MS. The molecular docking simulation revealed that the binding of serine/threonine derivatives to the enzyme’s active site was stronger (−4.6~−7.9 kcal/mol) than that of the natural acceptor, glucose, and mainly occurred through interactions with aromatic residues. For N-tert-butoxycarbonyl serine methyl ester (6.8%) and N-carboxybenzyl serine benzyl ester (3.4%), their binding affinities and the distances between their hydroxyl side chain and the 1′-OH group of galactose moiety were in good accordance with the quantified bioconversion yields. Despite its lower predicted bioconversion yield, the high experimental bioconversion yield obtained with N-carboxybenzyl serine methyl ester (23.2%) demonstrated the importance of the thermodynamically-driven nature of the transgalactosylation reaction.
Highlights
As the structural and molecular recognition roles of glycopeptides and glycoproteins in many biological systems are increasingly being recognized, the development of glycosylation strategies for the synthesis of these compounds has become of substantial importance in the fields of food, biology and pharmaceutical sciences
Heptane and heptanone were used as co-solvents in the biphasic reaction systems to investigate the effect of polarity of co-solvent on the transgalactosylation reaction
Using o-nitrophenyl β-D-galactopyranoside (ONPG) as donor, the highest yield of transgalactosylated N-Z-Ser-OMe (23.2%) and the fastest initial rate (4680.0 μmol product/L.h) were achieved in heptane:buffer reaction system, whereas with the lactose, the highest yield (3.9%) and the fastest initial rate (1600.0 μmol product/L.h) were obtained in the buffer reaction system. These results are attributed to the higher solubility of ONPG in the biphasic reaction system as compared to the aqueous reaction media
Summary
As the structural and molecular recognition roles of glycopeptides and glycoproteins in many biological systems are increasingly being recognized, the development of glycosylation strategies for the synthesis of these compounds has become of substantial importance in the fields of food, biology and pharmaceutical sciences. The improvements in the functional properties of carbohydrate conjugated food proteins, such as emulsifying activity, protein solubility, and thermal stability, have been previously demonstrated [3] These advantages emphasize the need for a highly specific method for the formation of glycosidic bonds between carbohydrates and amino acids/peptides/proteins. The first step of the mechanistic action of β-galactosidase-catalyzed reaction involves a cleavage of the glycosidic bond of the lactose or galactose-substituted molecules, and the formation of the covalent galactosyl-enzyme intermediate (Figure 1B) [7,8]. Limited studies have investigated the galactosylation of amino acid/peptide by β-galactosidase [10,11,12,13,14,15,16] None of these studies have used a structure-based computational approach to elucidate the binding of the amino acids at the galactosidase’s active site and to understand the effect of the amino acid’s blocking group on the synthesis of galactosylated amino acid derivatives.
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