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Biochemical characterisation of the neuraminidase pool of the human gut symbiont Akkermansia muciniphila

“…The synthesis of sialylated 5-bromo-4-chloro-indolyl-β-dgalactopyranosides (X-gal sialosides) in milligram scale followed procedures previously described by Knorst and Fessner, 34 Cao et al 35 and Reyes Martinez et al 26 For the synthesis of X-gal-α2,6-Neu5Ac and X-gal-α2,6-Neu5Gc, the reaction mixtures (31 mL each) contained 22 mg Neu5Ac or 23 mg Neu5Gc; 38 mg CTP, 25 mg X-gal, 2 mM MgCl2, 40 mM MES buffer pH 6.5, α2,6-sialyltransferase (PdST6, ≥7 mU), CMP-sialic acid synthase (NmCTT, ≥7 mU) in distilled water. The mixtures were incubated at 37°C until 80% product formation was reached (as judged by HPLC analysis).…”

Flexibility of Substrate Binding of Cytosine‐5′‐Monophosphate‐N‐Acetylneuraminate Synthetase (CMP‐Sialate Synthetase) from Neisseria meningitidis: An Enabling Catalyst for the Synthesis of Neo‐sialoconjugates

“…In the quest for a more flexible direct synthetic approach to both natural sialoconjugates and neo-sialoconjugates containing various non-natural sialic acids we previously cloned and overproduced the CSS from Neisseria meningitidis, and qualitatively demonstrated by TLC reaction screening and by preparative synthesis of various CMP-activated sialic acid derivatives that this enzymes unusually broad substrate tolerance also allows for the activation of non-natural Neu5Ac analogues and derivatives. [8] Allowable structural modifications included N-acyl variation as well as altered substitution pattern, backbone size, and inver-sion of stereoconfiguration. This capacity of CSS is a key feature that ought to strongly facilitate direct synthetic access also to those natural sialic acid conjugates that otherwise would need additional steps for post-synthetic enzymatic modification, making larger libraries of conjugates readily available along a common synthetic pathway by using a standard set of identical enzyme activities.…”

Direct Enzymatic Branch‐End Extension of Glycocluster‐Presented Glycans: An Effective Strategy for Programming Glycan Bioactivity

“…[17,18] The pyrophosphate side product was hydrolyzed to inorganic phosphate in situ by adding ap yrophosphatase to prevent inhibition. [17,18] The pyrophosphate side product was hydrolyzed to inorganic phosphate in situ by adding ap yrophosphatase to prevent inhibition.…”

“…SPR experiments were performed with aBiacore 3000. [17,18] VAAw as purified by affinity chromatography on lactosylated Sepharose 4B, obtained after activation by divinyl sulfone, checked for purity by one-and two-dimensional gel electrophoresis and for activity by haemagglutination, solid-phase assays and cell toxicity. Eur.J.…”