Galactosyl-lactose sialylation using Trypanosoma cruzi trans-sialidase as the biocatalyst and bovine κ-casein-derived glycomacropeptide as the donor substrate.

trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(alpha 2-3) Gal(beta 1-x)-glycans (sialo-glycans) to Gal(beta 1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal)(n)Glc oligosaccharide mixtures (GOS) using bovine kappa-casein-derived glycomacropeptide (GMP) as the sialic acid donor, a kinetic study has been carried out with three components of GOS, i.e., 3'-galactosyl-lactose (beta 3'-GL), 4'-galactosyl-lactose (beta 4'-GL), and 6'-galactosyl-lactose (beta 6'-GL). This prebiotic GOS is prepared from lactose by incubation with suitable beta-galactosidases, whereas GMP is a side-stream product of the dairy industry. The trans-sialidase from Trypanosoma cruzi (TcTS) was expressed in Escherichia coli and purified. Its temperature and pH optima were determined to be 25 degrees C and pH 5.0, respectively. GMP [sialic acid content, 3.6% (wt/wt); N-acetylneuraminic acid (Neu5Ac), >99%; (alpha 2-3)-linked Neu5Ac, 59%] was found to be an efficient sialyl donor, and up to 95% of the (alpha 2-3)-linked Neu5Ac could be transferred to lactose when a 10-fold excess of this acceptor substrate was used. The products of the TcTS-catalyzed sialylation of beta 3'-GL, beta 4'-GL, and beta 6'-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated beta 3'-GL and beta 4'-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of beta 6'-GL, TcTS was shown to sialylate the 3 position of both the internal and terminal Gal moieties, yielding two different monosialylated products and a disialylated structure. Kinetic analyses showed that TcTS had higher affinity for the GL substrates than lactose, while the V-max and k(cat) values were higher in the case of lactose.