A Pasteurella multocida sialyltransferase displaying dual trans-sialidase activities for production of 3'-sialyl and 6'-sialyl glycans

Abstract

This study examined a recombinant Pasteurella multocida sialyltransferase exhibiting dual trans-sialidase activities. The enzyme catalyzed trans-sialylation using either 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid or casein glycomacropeptide (whey protein) as the sialyl donor and lactose as the acceptor, resulting in production of both 3'-sialyllactose and 6'-sialyllactose. This is the first study reporting α-2,6-trans-sialidase activity of this sialyltransferase (EC 2.4.99.1 and 2.4.99.4). A response surface design was used to evaluate the effects of three reaction parameters (pH, temperature, and lactose concentration) on enzymatic production of 3'- and 6'-sialyllactoses using 5% (w/v) casein glycomacropeptide (equivalent to 9mM bound sialic acid) as the donor. The maximum yield of 3'-sialyllactose (2.75±0.35mM) was achieved at a reaction condition with pH 6.4, 40°C, 100mM lactose after 6h; and the largest concentration of 6'-sialyllactose (3.33±0.38mM) was achieved under a condition with pH 5.4, 40°C, 100mM lactose after 8h. 6'-sialyllactose was presumably formed from α-2,3 bound sialic acid in the casein glycomacropeptide as well as from 3'-sialyllactose produced in the reaction. The kcat/Km value for the enzyme using 3'-sialyllactose as the donor for 6'-sialyllactose synthesis at pH 5.4 and 40°C was determined to be 23.22±0.7M(-1)s(-1). Moreover, the enzyme was capable of catalyzing the synthesis of both 3'- and 6'-sialylated galactooligosaccharides, when galactooligosaccharides served as acceptors.

Keywords: 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid; 4-methylumbelliferyl β-d-lactoside; ATP; CMP-NeuAc; CTP; CV; Casein glycomacropeptide as source for sialic acid; Enzyme expression and purification; GOS; GalNAc; HMOs; HPAEC-PAD; Human milk oligosaccharides; Kinetics of trans-sialidase; LC–MS; LacMu; MALDI-MS; N-His6-tag; N-acetylgalactosamine; N-acetylneuraminic acid; N-terminal His(6)-tag; NDV; Neu5Ac; Newcastle disease virus; PEP; PGC; Pasteurella multocida sialyltransferase; PmST; RSM; Response surface; SDS-PAGE; Sia-GOS; TLC; 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.