title: 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.
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, 2800 Kgs. Lyngby, Denmark.
authors.forename: Yao
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, 2800 Kgs. Lyngby, Denmark.
authors.forename: Carsten
authors.initials: C
authors.lastname: Jers
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, 2800 Kgs. Lyngby, Denmark.
authors.forename: Anne S
authors.initials: AS
authors.lastname: Meyer
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, 2800 Kgs. Lyngby, Denmark.
authors.forename: Anis
authors.initials: A
authors.lastname: Arnous
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark.
authors.forename: Haiying
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark.
authors.forename: Finn
authors.initials: F
authors.lastname: Kirpekar
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, 2800 Kgs. Lyngby, Denmark. Electronic address: jdm@kt.dtu.dk.
authors.forename: Jørn D
authors.initials: JD
authors.lastname: Mikkelsen
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000031:analogs & derivatives* / Q000378:metabolism; D008894:Milk Proteins / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009439:Neuraminidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D016979:Pasteurella multocida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D000067816:Whey Proteins
chemical_list: C421467:3'-sialyllactose; C403777:6'-sialyllactose; D001426:Bacterial Proteins; D006023:Glycoproteins; D008894:Milk Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000067816:Whey Proteins; D012799:Sialyltransferases; C069715:trans-sialidase; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid; D007785:Lactose
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