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Fig. 8 | Microbial Cell Factories

Fig. 8

From: Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans

Fig. 8

F10-ORF19 hexosaminidase activity on N-glycans. A F10-ORF19 hexosaminidase substrate specificity. F10-ORF19 was assayed on a pool of human APTS-labelled immunoglobulin A (hIgA) N-glycans pretreated with sialidase and β-galactosidase (black electropherogram). Substrate and product were analyzed by xCGE-LIF. The activity of F10-ORF19 (blue electropherogram) is indicated with a blue arrow that shows the migration time shift of the FA2G0-SO4 peak from ~ 150 MTU″ to the FA1G0 peak at ~ 245 MTU″ due to the loss of a sulfated GlcNAc. The activity of β-N-acetylhexosaminidase S (β-GlcNAcase S; pink electropherogram) is shown with 3 pink arrows illustrating the collapse of all structures to paucimannose at (~ 178 MTU″) and fucosylated paucimannose (~ 207 MTU″), respectively. Structure assignment was enabled by matching the normalized migration times with those of a N-glycan database and confirmed by exoglycosidase digests (Cajic S, Hennig R, Grote V, Reichl U and Rapp E; manuscript in preparation). B F10-ORF19 optimal pH. Optimal pH for F10-ORF19 was determined using the hIgA-APTS labelled glycan pool as substrate and following product formation by xCGE-LIF. The x-axis (time) of the electropherograms was normalized to two internal standards by glyXtoolCE, resulting in double normalized migration time units (MTU″). Signal Intensities were normalized to the total peak height, resulting in relative peak height proportions (%). Glycans are represented using SNFG nomenclature [69]

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