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

• PmST, a Pasteurella multocida sialyltransferase expressed in Escherichia coli exhibited both α-2,3- and α-2,6-trans-sialidase activities.
• Optimum conditions were identified for maximum production of 3′- and 6′-sialyllactoses by PmST-catalyzed trans-sialylation using casein glycomacropeptide as the donor for sialic acid.
• The regioselective synthesis of sialyllactose could be achieved by regulating reaction time.
• The kinetic parameter for α-2,6-trans-sialidase activity of PmST was estimated.
• PmST catalyzed α-2,3 as well as α-2,6 sialylation of prebiotic galactooligosaccharides.

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 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; and the largest concentration of 6′-sialyllactose (3.33 ± 0.38 mM) was achieved under a condition with pH 5.4, 40 °C, 100 mM lactose after 8 h. 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 ± 0.7 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.