3D Printed Porous Nanocellulose-Based Scaffolds As Carriers for Immobilization of Glycosyltransferases

Abstract

Biocatalysis is increasingly becoming an alternative method for the synthesis of industrially relevant complex molecules. This can be realized by using enzyme immobilized polysaccharide-based 3D scaffolds as compatible carriers, with defined properties. Especially, immobilization of either single or multiple enzymes on a 3D printed polysaccharide scaffold, exhibiting well-organized interconnected porous structure and morphology, is a versatile approach to access the performance of industrially important enzymes. Here, we demonstrated the use of nanocellulose-based 3D porous scaffolds for the immobilization of glycosyltransferases, responsible for glycosylation in natural biosynthesis. The scaffolds were produced using an ink containing nanofibrillated cellulose (NFC), carboxymethyl cellulose (CMC), and citric acid. Direct-ink-writing 3D printing followed by freeze-drying and dehydrothermal treatment at elevated temperature resulted in chemically cross-linked scaffolds, featuring tunable negative charges (2.2−5.0 mmol/g), pore sizes (10−800 μm), fluid uptake capacity, and exceptional dimensional and mechanical stability in the wet state. The negatively charged scaffolds were applied to immobilize two sugar nucleotide-dependent glycosyltransferases (C-glycosyltransferase, Z basic2 -CGT; sucrose synthase, Z basic2 -SuSy), each harboring a cationic binding module (Z basic2 ) to promote charge-based enzyme adsorption. Both enzymes were immobilized at ∼30 mg of protein/g of dry carrier (∼20% yield), independent of the scaffold used. Their specific activities were 0.50 U/mg (Z basic2 -CGT) and 0.19 U/mg (Z basic2 -SuSy), corresponding to an efficacy of 37 and 18%, respectively, compared to the soluble enzymes. The glycosyltransferases were coimmobilized and shown to be active in a cascade reaction to give the natural C-glycoside nothofagin from phloretin (1.0 mM; ∼95% conversion). All enzyme bound scaffolds showed reusability of a maximum of 5 consecutive reactions. These results suggest that the 3D printed and cross-linked NFC/CMC-based scaffolds could present a class of solid carriers for enzyme (co)-immobilization, with promising applications in glycosyltransferase-catalyzed synthesis and other fields of biocatalysis.

3D-Printed Collagen–Nanocellulose Hybrid Bioscaffolds with Tailored Properties for Tissue Engineering Applications

“…9,54 CMC, on the other hand, showed a broad diffraction peak at 2θ = 20°and an amorphous structure (see Figure S1). 55 Interestingly, although the typical diffraction peaks of NFC were found, the main peak of Coll and CMC at 2θ = 20−21°F was covered by the diffraction peak of NFC in all bioscaffolds and no new peaks were present, indicating that the structural properties of the bulk phase of all components were preserved after DHT treatment. Figure 5d, e shows the results of the TGA and its derivative (dTG, mass loss rate) of the neat Coll and the bioscaffolds with different Coll concentrations between 40 and 900 °C.…”

“…For NFC, four main diffraction peaks were found at 2θ = 14.6°(110), 20.1°(020), 34.2°(004) and a diffraction pattern corresponding to crystalline cellulose I. , CMC, on the other hand, showed a broad diffraction peak at 2θ = 20° and an amorphous structure (see Figure S1). Interestingly, although the typical diffraction peaks of NFC were found, the main peak of Coll and CMC at 2θ = 20–21° was covered by the diffraction peak of NFC in all bioscaffolds and no new peaks were present, indicating that the structural properties of the bulk phase of all components were preserved after DHT treatment.…”