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

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 %).