1. A Pasteurella multocida sialyltransferase displaying dual trans-sialidase activities for production of 3'-sialyl and 6'-sialyl glycans.
- Author
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Guo Y, Jers C, Meyer AS, Arnous A, Li H, Kirpekar F, and Mikkelsen JD
- Subjects
- Bacterial Proteins metabolism, Humans, Hydrogen-Ion Concentration, Lactose metabolism, Recombinant Proteins metabolism, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Temperature, Whey Proteins, Glycoproteins metabolism, Lactose analogs & derivatives, Milk Proteins metabolism, N-Acetylneuraminic Acid metabolism, Neuraminidase metabolism, Oligosaccharides metabolism, Pasteurella multocida enzymology, Sialyltransferases metabolism
- 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., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2014
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