Immobilization of CMP-Sialic Acid Synthetase and α2,3-Sialyltransferase for Cascade Synthesis of 3′-Sialyl β-D-Galactoside with Enzyme Reuse

Sabine Schelch, Romana Koszagova, Jürgen Kuballa, Bernd Nidetzky*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Sialo-oligosaccharides are often synthesized via cascade reaction of CMP-sialic acid synthetase (CSS) and sialyltransferase (SiaT). Here, we studied individual enzyme immobilization to develop solid-supported preparations of the CSS from Neisseria meningitis (NmCSS) and the α2,3-SiaT from Pasteurella dagmatis (PdSiaT). Oriented immobilization via N-terminal His-tag as well as “random” (orientationally uncontrolled) immobilization via multipoint covalent coupling gave catalyst preparations of each enzyme with low activity and effectiveness factor (η). We therefore constructed N-terminal fusions of NmCSS and PdSiaT with the cationic binding module Zbasic2 and show individual immobilization of even the unpurified enzymes on sulfonate carrier (ReliSorb SP400) in excellent yields (≥95 %) and binding selectivities. For both enzymes in individual reactions, the initial η (Z-NmCSS: 90 %; Z-PdSiaT: 25 %) declined sharply with increasing enzyme loading and the maximum immobilized activity was 110 U/g carrier (η=27 %) for Z-NmCSS, 7.5 U/g (η≤5 %) for Z-PdSiaT. Supplied with neuraminic acid and cytidine 5′-triphosphate (20 mM each), the immobilized enzymes promoted α2,3-sialylation of the model substrate 4-nitrophenyl β-D-galactoside (20 mM) in 85 % yield and could be recycled 5 times with only small loss in their overall synthetic activity (≤10 %).

Original languageEnglish
Article numbere202101860
JournalChemCatChem
Volume14
Issue number9
DOIs
Publication statusPublished - 6 May 2022

Keywords

  • CMP-Neu5Ac synthetase
  • glycosyltransferase cascade
  • immobilization
  • sialo-oligosaccharide
  • sialyltransferase

ASJC Scopus subject areas

  • Catalysis
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

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