Enzyme catalysed production of sialylated human milk oligosaccharides and galactooligosaccharides by Trypanosoma cruzi trans-sialidase

Holck, Jesper; Larsen, Dorte Møller; Michalak, Malwina; Li, Haiying; Kjaerulff, Louise; Kirpekar, Finn; Gotfredsen, Charlotte Held; Forssten, Sofia D.; Ouwehand, Arthur C.; Mikkelsen, Jørn Dalgaard; Meyer, Anne S.

doi:10.1016/j.nbt.2013.11.006

Cited by 39 publications

(30 citation statements)

References 45 publications

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“…Anion exchange chromatography for the purification of sialylated compounds prior to MALDI-TOF and NMR analysis was performed with an ÄKTA purifier 100 (GE Healthcare, Uppsala, Sweden) equipped with a SepharoseQ column (GE Healthcare, Uppsala, Sweden) as described previously [ 18 ].…”

Section: Methodsmentioning

confidence: 99%

“…rangeli (TrSA), which has a sequence identity of 70% to TcTS, has recently been engineered to primarily carry out trans-sialylation under certain reaction conditions [ 10 , 16 , 17 ]. Trans-sialidases are also of commercial interest as it has been demonstrated that both TcTS and mutants of TrSA can efficiently sialylate a variety of glycans, enabling the production of sialylated human milk oligosaccharides [ 10 , 16 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

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It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity

et al. 2016

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Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified through an iterative alignment from 2909 native sialidases available in GenBank, which were cloned and expressed in Escherichia coli. Of these, one enzyme, SialH, derived from Haemophilus parasuis had an aromatic sandwich structure on the protein surface facing the end of the catalytic site (Phe168; Trp366), and was indeed found to exhibit trans-sialidase activity. SialH catalyzed production of the human milk oligosaccharide 3’-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor. The findings corroborated that Tyr119 and Trp312 in the T. cruzi trans-sialidase are part of an aromatic sandwich structure that confers trans-sialylation activity for lactose sialylation. The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases. The approach moreover provided data that help understand structure-function relations of trans-sialidases.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity

et al. 2016

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“…Traditionally, when the production of SL has been performed at small scale, separation of lactose and SL has been achieved by anion exchange chromatography [5,4,3,2]. However, considering the economy of a large scale production process and the fact that anion exchange chromatography relies on the addition of buffers and eluents -which would then need to be removed before the SL can be considered for consumption -an alternative process for the efficient purification of SL from lactose is necessary for the addition of SL to infant formula to become a reality.…”

Section: Introductionmentioning

confidence: 99%

Separation of 3′-sialyllactose and lactose by nanofiltration: A trade-off between charge repulsion and pore swelling induced by high pH

Nordvang

Luo

Zeuner

et al. 2014

Separation and Purification Technology

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“…To upgrade the TcTS-mediated production of 3=-SL from lactose and a suitable, low-cost sialyl donor substrate, several protocols have been reported and patented. Attention has been paid especially to the use of the glycoproteins fetuin (30) and -casein-derived glycomacropeptide (31,32), both rich in (␣2-3)-linked sialic acid and available in relatively large amounts at relatively low cost, as sialyl donor substrates.…”

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confidence: 99%

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

Wilbrink

Kate

Leeuwen

et al. 2014

Appl Environ Microbiol

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c trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(␣2-3)Gal(␤1-x)-glycans (sialo-glycans) to Gal(␤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 -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 (␤3=-GL), 4=-galactosyl-lactose (␤4=-GL), and 6=-galactosyl-lactose (␤6=-GL). This prebiotic GOS is prepared from lactose by incubation with suitable ␤-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%; (␣2-3)-linked Neu5Ac, 59%] was found to be an efficient sialyl donor, and up to 95% of the (␣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 ␤3=-GL, ␤4=-GL, and ␤6=-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated ␤3=-GL and ␤4=-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of ␤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.

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Enzyme catalysed production of sialylated human milk oligosaccharides and galactooligosaccharides by Trypanosoma cruzi trans-sialidase

Cited by 39 publications

References 45 publications

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity

Separation of 3′-sialyllactose and lactose by nanofiltration: A trade-off between charge repulsion and pore swelling induced by high pH

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

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