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 Infection Microbiology, Department of Biotechnology and Biomedicine, Technical University of Denmark
Enzymatic treatment of biomass is an environmentally friendly method to obtain a range of value- added products, such as biofuels, animal feed or food ingredients. The objective of this PhD study was to biocatalytically produce biofunctional food ingredients – human milk oligosaccharides decorated with sialic acid from casein glycomacropeptide obtained from dairy side streams. In addition, the biocatalysts employed in this study, i.e. , a sialyltransferase and a sialidase, were subjected to protein engineering to alter the enzyme’s regioselectivity and to improve hydrolase activity, respectively. A recombinant Pasteurella multocida sialyltransferase (EC 2.4.99.-), namely PmST, exhibiting promiscuous trans-sialidase activities was examined. The enzyme catalysed α-2,3- and α-2,6- sialylation of lactose using either 2- O -(p-nitrophenyl)-α- D - N -acetylneuraminic acid or casein glycomacropeptide as a sialyl donor. This is the first study reporting α-2,6-trans-sialidase activity of this enzyme. Using response surface design allowed identification of two differently optimised conditions for PmST-catalysed production of 3'-sialyllactose and 6'-sialyllactose, giving maximum yields of 2.8 mM and 3.3 mM from casein glycomacropeptide (9 mM bound sialic acid), respectively. The k cat / K m value for PmST catalysing 6'-sialyllactose synthesis using 3'-sialyllactose as donor was 23.2±0.7 M -1 s -1 . Further, the enzyme was capable of catalysing synthesis of both 3'- and 6'-sialylated galactooligosaccharides with use of galactooligosaccharides as acceptors. Secondly, we examined the regioselectivity of five designed mutants of PmST catalysing synthesis of 3'- and 6'-sialyllactoses using casein glycomacropeptide and lactose as substrates. The mutants PmST E271F , PmST R313Y and PmST E271F/R313Y preferentially catalysed synthesis of 3'-sialyllactose over 6'-sialyllactose. The best mutant PmST E271F/R313Y for α-2,3-trans-sialylation gave a maximum 3'-sialyllactose yield of 4.5 mM from casein glycomacropeptide (9 mM bound sialic acid). Another mutant PmST P34H displayed a distinct preference for 6'-sialyllactose synthesis throughout the reaction, though the total sialyllactose yield was consistently and significantly lower than that using the wild type enzyme. PmST P34H had a 980-fold increase in α-2,6-sialyltransferase activity compared to the wild type enzyme, while its α-2,3-sialyltransferase activity was almost abolished. The k cat / K m value for PmST P34H catalysing 6'-sialyllactose synthesis using 3'-sialyllactose as donor was 31.2 M -1 s -1 . Moreover, both the wild type enzyme and PmST P34H were capable of catalysing the hydrolysis and transfer of α-2,6 bound sialic acid.
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Mikkelsen, Jørn Dalgaard, Jers, Carsten
Technical University of Denmark, Department of Chemical Engineering, 2014