1 Department of Chemical and Biochemical Engineering, Technical University of Denmark2 Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark3 Department of Systems Biology, Technical University of Denmark4 University of Southern Denmark
tThis study examined a recombinant Pasteurella multocida sialyltransferase exhibiting dual trans-sialidase activities. The enzyme catalyzed trans-sialylation using either 2-O-(p-nitrophenyl)--d-N-acetylneuraminic acid or casein glycomacropeptide (whey protein) as the sialyl donor and lactose asthe acceptor, resulting in production of both 3-sialyllactose and 6-sialyllactose. This is the first studyreporting -2,6-trans-sialidase activity of this sialyltransferase (EC 184.108.40.206 and 220.127.116.11). A responsesurface design was used to evaluate the effects of three reaction parameters (pH, temperature, andlactose concentration) on enzymatic production of 3- and 6-sialyllactoses using 5% (w/v) casein glyco-macropeptide (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; andthe largest concentration of 6-sialyllactose (3.33 ± 0.38 mM) was achieved under a condition with pH5.4, 40◦C, 100 mM lactose after 8 h. 6-sialyllactose was presumably formed from -2,3 bound sialic acidin the casein glycomacropeptide as well as from 3-sialyllactose produced in the reaction. The kcat/Kmvalue for the enzyme using 3-sialyllactose as the donor for 6-sialyllactose synthesis at pH 5.4 and 40◦Cwas determined to be 23.22 ± 0.7 M−1s−1. Moreover, the enzyme was capable of catalyzing the synthesisof both 3- and 6-sialylated galactooligosaccharides, when galactooligosaccharides served as acceptors.