Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine ?-Casein-Derived Glycomacropeptide as the Donor Substrate

• Published in: Applied and environmental microbiology 2014-10, Vol.80 (19), p.5984
• Main Authors: Wilbrink, Maarten H, ten Kate, Geert A, S van Leeuwen, Sander, Sanders, Peter, Sallomons, Erik, Hage, Johannes A, Dijkhuizen, Lubbert, Kamerling, Johannis P
• Format: Article
• Language: English
• Subjects: Biocatalysts; Biochemistry; Enzymes; Gene expression; Peptides; Protozoa; Spectrum analysis
• ISSN: 0099-2240; 1098-5336

trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(α2-3)Gal(β1-x)-glycans (sialo-glycans) to Gal(β1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal)...Glc oligosaccharide mixtures (GOS) using bovine ...-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 (β3'-GL), 4'-galactosyl-lactose (β4'-GL), and 6'-galactosyl-lactose (β6'-GL). This prebiotic GOS is prepared from lactose by incubation with suitable β-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...C and pH 5.0, respectively. GMP [sialic acid content, 3.6% (wt/wt); N-acetylneuraminic acid (Neu5Ac), >99%; (α2-3)-linked Neu5Ac, 59%] was found to be an efficient sialyl donor, and up to 95% of the (α2-3)-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 β3'-GL, β4'-GL, and β6'-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated β3'-GL and β4'-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of β6'-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... and k... values were higher in the case of lactose. (ProQuest: ... denotes formulae/symbols omitted.)