We next set out to improve expression and purification conditions of Im9-KpsS1 to obtain enough pure enzyme for further study. Optimised production was achieved in E. coli BL21 (DE3) cells induced at OD600 0.8 and incubated at 30 °C for 4 h post induction, followed by lysis using 0.5% non-ionic detergent and nickel affinity purification using a two-step isocratic elution (Figure ). We subsequently performed PseT activity time-course assays using pure Im9-KpsS1 and a range of PNP-acceptors (Figure ). Over 120 min we again observed no turnover using PNP-Xylp 6, lacking the 6-OH. But we did observe increasing product formation over the time-course using PNP-Manp 7, albeit low level, and more rapid increased conversion to disaccharide product (approaching 20% at 120 min) for both PNP-Galp 8 and PNP-Glcp 5 acceptors. These results demonstrate that the purified fusion enzyme is active in vitro and also shows some substrate promiscuity with respect to glycosyl acceptors, with both PNP-Glcp and Galp acceptors used by the enzyme with comparable efficiency. We subsequently used a CMP-Glo™ assay to measure kinetic parameters of Im9-KpsS1(Supplementary Figures ) and determined the enzyme bound CMP-Pse5Ac7Ac 3 with a Km of approximately 105 µM, which is comparable to the donor affinity of other bacterial sialyltransferases. Although these enzyme activity assays provided evidence of turnover, the limited availability of pure CMP-Pse5Ac7Ac 3 donor restricted the scale on which these reactions could be performed and precluded NMR characterisation of the products. This was required to unequivocally assign the stereochemistry of the glycosidic linkage formed and therefore whether KpsS1 was indeed a retaining glycosyltransferase enzyme. We therefore chose to scale up the enzymatic glycosylation using Glc acceptor 5 to yield enough disaccharide product for full characterisation, and comparison to the unambiguous assignment of the Pse5Ac7Ac-a2,6-Glcp linkage in 4. To achieve this, we reconstituted the biosynthetic pathway for production of CMP-Pse5Ac7Ac 3 in vitro (Scheme 1). Starting from 30 mg of UDP-GlcNAc 10, using the five biosynthetic enzymes from C. jejuni, PseB, C, H, G and I and required co-factors, we synthesised Pse5Ac7Ac 1 in one pot in 6 h, with negative ion ESI LCMS confirming product formation and no remaining biosynthetic intermediates 11-14. To this reaction mixture we then added the CMP-Pse5Ac7Ac synthetase PseF from A. caviae, CTP and MgCl2 for 3 h until CMP-Pse5Ac7Ac 3 was observed. Finally, acceptor 6 and Im9-KpsS1 were then added to the reaction mixture before incubation for a further 72 h, when all donor was consumed. From this tandem seven enzyme two-pot synthesis the desired Pse5Ac7Ac-a2,6-PNP-b-DGlcp disaccharide 15 was synthesised in 16% yield. The stereochemistry of the newly formed glycosidic bond was unambiguously assigned as a based on the 0.54 ppm difference between the d values of the Pse5Ac7Ac H3ax and H3eq protons in 700 MHz 1 H-NMR analysis (reported as 0.55 ppm in the characterisation of repeating unit 4 at 600 MHz). Thus providing unequivocal evidence that KpsS1 acts as a retaining pseudaminyltransferase.