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.

中文翻译：

---

多糖序列影响葡萄糖醛酸C5-表异构酶的特异性和催化活性。

肝素是从动物组织生产的一种广泛使用的生物治疗剂。但是，依靠分离大肠杆菌K5胶囊肝素及其化学转化成N-硫杂庚烷（NSH）的方法，可以使用多酶工艺生产生物工程版本。最早作用于NSH的酶葡糖醛酸C5-表异构酶催化葡糖醛酸（GlcA）向艾杜糖酸（IdoA）的可逆转化。使用全长NSH，其中包含不同量的N-乙酰基葡糖胺（GlcNAc）残基，我们证明了C5-表异构酶的特异性与多糖序列有关，特别是GlcNAc残基在链中的位置。我们利用氘交换和新的β-葡萄糖醛酸苷酶乙酰肝素酶BP，其在GlcA残基处裂解。部分或完全的乙酰肝素酶BP消化产物的液相色谱-质谱分析和凝胶渗透色谱分析表明，这些NSH底物经C5-表异构酶处理后，可提供有关C5-表异构酶活性和作用方式的信息。该研究为优化生物工程肝素的大规模生产提供了见识。