Heparin is a widely used biotherapeutic produced from animal tissues. However, it might be possible to produce a bioengineered version using a multienzyme process, relying on the isolation of the E. coli K5 capsule heparosan and its chemical conversion to N-sulfoheparosan, NSH. Glucuronyl C5-epimerase, the first enzyme that acts on NSH, catalyzes the reversible conversion of glucuronic acid (GlcA) to iduronic acid (IdoA). Using full-length NSH, containing different amounts of N-acetylglucosamine (GlcNAc) residues, we demonstrate that C5-epimerase specificity relates to polysaccharide sequence, particularly the location of GlcNAc residues within the chain. We leveraged the deuterium exchange and the novel β-glucuronidase heparanase BP, which cleaves at the GlcA residue. Liquid chromatography-mass spectrometry and gel permeation chromatography of partial/complete heparanase BP digestion products from various NSH substrates treated with C5-epimerase provide information on C5-epimerase activity and action pattern. This study provides insight into optimizing the large-scale production of bioengineered heparin.