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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.

SUBMITTER: Lackner F

PROVIDER: S-EPMC9768809 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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

Lackner Florian F Liu Hui H Štiglic Andreja Dobaj AD Bračič Matej M Kargl Rupert R Nidetzky Bernd B Mohan Tamilselvan T Kleinschek Karin Stana KS

ACS applied bio materials 20221205 12

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 i ...[more]

PMID: 36469033

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Project description:Over the past two decades, electrospinning has emerged as a common technique to produce biomedical scaffolds composed of ultrafine fibers formed from many natural and synthetic polymers. A major advantage of this technique is the ability to produce scaffolds that resemble the native extracellular matrix in physical, chemical, and topological properties. However, scaffolds fabricated via electrospinning are not formed with a controlled architecture and typically do a poor job of directing cell growth into prescribed structures for tissue/organ development. To address these weaknesses, 3D bioprinting has recently been used to develop scaffolds that have a highly organized and precise global topology. Unfortunately, these 3D bioprinted scaffolds do not typically resemble the native extracellular matrix in physical properties, such as porosity, fiber diameter, and pore size (e.g., the microarchitecture). Thus, the goal of the current study was to develop a technique that harnesses the intrinsic advantages of both conventional electrospinning and 3D bioprinting techniques to produce scaffolds that have the potential to be used within biomedical applications. The physical properties of formed 3D printed electrospun scaffolds were compared with conventional electrospun and 3D printed scaffolds. Further, we conducted initial proof-of-concept biocompatibility studies to illustrate the applicability of the scaffolds within vascular applications. Our results illustrate that 3D printed electrospun scaffolds can be developed, via our technique, that have highly tailored and organized arbitrary geometries with scaffold properties in the range of the innate extracellular matrix. In addition, these scaffolds were shown to support endothelial cell growth. Therefore, we illustrate the development and testing of a novel bioscaffold fabrication technique that may be used for many tissue engineering and regenerative medicine applications, which allows for the direct printing of electrospun scaffolds into well-defined macro-scale geometries that also retain the micro-structures commonly observed in electrospun scaffolds.

Dataset Information

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

Publications

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

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