title: Galactosyl-lactose sialylation using Trypanosoma cruzi trans-sialidase as the biocatalyst and bovine κ-casein-derived glycomacropeptide as the donor substrate.
abstract: 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)nGlc oligosaccharide mixtures (GOS) using bovine κ-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 Vmax and kcat values were higher in the case of lactose.
authors: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors: Maarten H
authors: MH
authors: Wilbrink
authors: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors: Geert A
authors: GA
authors: ten Kate
authors: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors: Sander S
authors: SS
authors: van Leeuwen
authors: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors: Peter
authors: P
authors: Sanders
authors: Royal FrieslandCampina Innovation Center, Wageningen, The Netherlands.
authors: Erik
authors: E
authors: Sallomons
authors: Darlingii, Son, The Netherlands.
authors: Johannes A
authors: JA
authors: Hage
authors: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands l.dijkhuizen@rug.nl.
authors: Lubbert
authors: L
authors: Dijkhuizen
authors: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors: Johannis P
authors: JP
authors: Kamerling
mesh_terms: D000818:Animals; D044822:Biodiversity; D002364:Caseins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007785:Lactose / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008892:Milk / Q000737:chemistry / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis / Q000378:metabolism*; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D002364:Caseins; D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; D010446:Peptide Fragments; D012794:Sialic Acids; C065989:caseinomacropeptide; C020251:kappa-casein glycomacropeptide; C069715:trans-sialidase; D009439:Neuraminidase; D001616:beta-Galactosidase; D019158:N-Acetylneuraminic Acid; D007785:Lactose