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

This study examined a recombinant Pasteurella multocida sialyltransferase exhibiting dual transsialidase activities. The enzyme catalyzed trans-sialylation using either 2-O-(p-nitrophenyl)-alpha-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 alpha-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 9 mM bound sialic acid) as the donor. The maximum yield of T-sialyllactose (2.75 +/- 0.35 mM) was achieved at a reaction condition with pH 6.4, 40 degrees C, 100 mM lactose after 6h; and the largest concentration of &-sialyllactose (3.33 +/- 0.38 mM) was achieved under a condition with pH 5.4, 40 degrees C, 100 mM lactose after 8 h. 6'-sialyllactose was presumably formed from alpha-2,3 bound sialic acid in the casein glycomacropeptide as well as from T-sialyllactose produced in the reaction. The k(cat)/K-m value for the enzyme using T-sialyllactose as the donor for &-sialyllactose synthesis at pH 5.4 and 40 degrees C was determined to be 23.22 +/- 0.7 M-1 s(-1). Moreover, the enzyme was capable of catalyzing the synthesis of both 3'- and 6'-sialylated galactooligosaccharides, when galactooligosaccharides served as acceptors. (C) 2013 Elsevier B.V. All rights reserved.