Holck, Jesper4; Larsen, Dorte Møller5; Michalak, Malwina1; Li, Haiying6; Kjærulff, Louise5; Kirpekar, Finn6; Gotfredsen, Charlotte Held4; Forssten, Sofia7; Ouwehand, Arthur C.7; Mikkelsen, Jørn Dalgaard1; Meyer, Anne S.1
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 Organic Chemistry, Department of Chemistry, Technical University of Denmark4 Department of Chemistry, Technical University of Denmark5 Department of Systems Biology, Technical University of Denmark6 University of Southern Denmark7 DuPont Nutrition and Health
A Trypanosoma cruzi trans-sialidase (E.C. 18.104.22.168) was cloned into Pichia pastoris and expressed. The pH and temperature optimum of the enzyme was determined as pH 5.7 and 30°C. Using casein glycomacropeptide (CGMP) and lactose as sialyl-donor and acceptor respectively, the optimal donor/acceptor ratio for the trans-sialidase catalysed 3′-sialyllactose production was found to be 1:4. Quantitative amounts of 3′-sialyllactose were produced from CGMP and lactose at a yield of 40mg/g CGMP. The 3′-sialyllactose obtained exerted a stimulatory effect on selected probiotic strains, including different Bifidobacterium strains in single culture fermentations. The trans-sialidase also catalysed the transfer of sialic acid from CGMP to galacto-oligosaccharides (GOS) and to the human milk oligosaccharide (HMO) backbone lacto-N-tetraose (LNT) to produce 3′-sialyl-GOS, including doubly sialylated GOS products, and 3′-sialyl-LNT, respectively. This work thus provides proof of the concept of producing 3′-sialyllactose and potentially other sialylated HMOs as well as sialylated GOS enzymatically by trans-sialidase activity, while at the same time providing valorisation of CGMP, a co-processing product from cheese manufacture.
New Biotechnology, 2014, Vol 31, Issue 2, p. 156-165