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

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, Zbasic2-CGT; sucrose synthase, Zbasic2-SuSy), each harboring a cationic binding module (Zbasic2) 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 (Zbasic2-CGT) and 0.19 U/mg (Zbasic2-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.

Organic acid cross-linked 3D printed cellulose nanocomposite bioscaffolds with controlled porosity, mechanical strength, and biocompatibility.

Herein, we fabricated chemically cross-linked polysaccharide-based three-dimensional (3D) porous scaffolds using an ink composed of nanofibrillated cellulose, carboxymethyl cellulose, and citric acid (CA), featuring strong shear thinning behavior and adequate printability. Scaffolds were produced by combining direct-ink-writing 3D printing, freeze-drying, and dehydrothermal heat-assisted cross-linking techniques. The last step induces a reaction of CA. Degree of cross-linking was controlled by varying the CA concentration (2.5-10.0 wt.%) to tune the structure, swelling, degradation, and surface properties (pores: 100-450 µm, porosity: 86%) of the scaffolds in the dry and hydrated states. Compressive strength, elastic modulus, and shape recovery of the cross-linked scaffolds increased significantly with increasing cross-linker concentration. Cross-linked scaffolds promoted clustered cell adhesion and showed no cytotoxic effects as determined by the viability assay and live/dead staining with human osteoblast cells. The proposed method can be extended to all polysaccharide-based materials to develop cell-friendly scaffolds suitable for tissue engineering applications.