Nano-fibrillated cellulose-based scaffolds for enzyme (co)-immobilization: Application to natural product glycosylation by Leloir glycosyltransferases.

Polysaccharide-based scaffolds are promising carriers for enzyme immobilization. Here, we demonstrate a porous scaffold prepared by direct-ink-writing 3D printing of an ink consisting of nanofibrillated cellulose, carboxymethyl cellulose and citric acid for immobilization application. Negative surface charge introduced by the components made the scaffold amenable for an affinity-like immobilization via the cationic protein module Zbasic2. Zbasic2 fusions of two sugar nucleotide-dependent glycosyltransferases (C-glycosyltransferase, Z-CGT; sucrose synthase, Z-SuSy) were immobilized individually, or co-immobilized, and applied to synthesize the natural C-glycoside nothofagin. The cascade reaction involved ß-C-glycosylation of phloretin (10 mM, ~90 % conversion) from UDP-glucose, provided from sucrose and catalytic amounts of UDP (1.0 mM). Enzymes were co-immobilized at ~65 mg protein/g carrier to receive activities of 9.5 U/g (Z-CGT) and 4.5 U/g (Z-SuSy) in 22-33 % yield (protein) and an effectiveness of 23 % (Z-CGT) and 13 % (Z-SuSy). The scaffold-bound enzymes were recyclable for 5 consecutive reactions.