Journal article
A Pasteurella multocida sialyltransferase displaying dual trans-sialidase activities for production of 3′-sialyl and 6′-sialyl glycans
tThis study examined a recombinant Pasteurella multocida sialyltransferase exhibiting dual trans-sialidase activities. The enzyme catalyzed trans-sialylation using either 2-O-(p-nitrophenyl)--d-N-acetylneuraminic acid or casein glycomacropeptide (whey protein) as the sialyl donor and lactose asthe acceptor, resulting in production of both 3-sialyllactose and 6-sialyllactose.
This is the first studyreporting -2,6-trans-sialidase activity of this sialyltransferase (EC 2.4.99.1 and 2.4.99.4). A responsesurface design was used to evaluate the effects of three reaction parameters (pH, temperature, andlactose concentration) on enzymatic production of 3- and 6-sialyllactoses using 5% (w/v) casein glyco-macropeptide (equivalent to 9 mM bound sialic acid) as the donor.
The maximum yield of 3-sialyllactose(2.75 ± 0.35 mM) was achieved at a reaction condition with pH 6.4, 40◦C, 100 mM lactose after 6 h; andthe largest concentration of 6-sialyllactose (3.33 ± 0.38 mM) was achieved under a condition with pH5.4, 40◦C, 100 mM lactose after 8 h. 6-sialyllactose was presumably formed from -2,3 bound sialic acidin the casein glycomacropeptide as well as from 3-sialyllactose produced in the reaction.
The kcat/Kmvalue for the enzyme using 3-sialyllactose as the donor for 6-sialyllactose synthesis at pH 5.4 and 40◦Cwas determined to be 23.22 ± 0.7 M−1s−1. Moreover, the enzyme was capable of catalyzing the synthesisof both 3- and 6-sialylated galactooligosaccharides, when galactooligosaccharides served as acceptors.
| Language: | English |
|---|---|
| Year: | 2014 |
| Pages: | 60-67 |
| ISSN: | 18734863 and 01681656 |
| Types: | Journal article |
| DOI: | 10.1016/j.jbiotec.2013.11.013 |
| ORCIDs: | Jers, Carsten , Meyer, Anne S. and Mikkelsen, Jørn Dalgaard |
2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid 3'-sialyllactose 4-methylumbelliferyl β-d-lactoside 6'-sialyllactose ATP Bacterial Proteins CMP-NeuAc CTP CV Casein glycomacropeptide as source for sialic acid Enzyme expression and purification GOS GalNAc Glycoproteins HMOs HPAEC-PAD Human milk oligosaccharides Humans Hydrogen-Ion Concentration Kinetics of trans-sialidase LC–MS LacMu Lactose MALDI-MS Milk Proteins N-Acetylneuraminic Acid N-His6-tag N-acetylgalactosamine N-acetylneuraminic acid N-terminal His(6)-tag NDV Neu5Ac Neuraminidase Newcastle disease virus Oligosaccharides PEP PGC Pasteurella multocida Pasteurella multocida sialyltransferase PmST RSM Recombinant Proteins Response surface SDS-PAGE Sia-GOS Sialyltransferases Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization TLC Temperature Whey Proteins adenosine-5′-triphosphate cGMP capillary liquid chromatography/mass spectrometry casein glycomacropeptide column volume cytidine-5′-monophospho-N-acetylneuraminic acid cytidine-5′-triphosphate galactooligosaccharides high-performance anion exchange chromatography human milk oligosaccharides matrix-assisted laser desorption/ionization mass spectrometry pNP-Neu5Ac phosphoenolpyruvate porous graphitic carbon response surface methodology sialylated galactooligosaccharides sodium dodecyl sulfate polyacrylamide gel electrophoresis thin-layer chromatography trans-sialidase
