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

Florian Lackner, Hui Liu, Andreja Dobaj Štiglic, Matej Bračič, Rupert Kargl, Bernd Nidetzky, Tamilselvan Mohan*, Karin Stana Kleinschek*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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. copy; 2022 American Chemical Society.

Original languageEnglish
Pages (from-to)5728-5740
Number of pages13
JournalACS Applied Bio Materials
Volume5
Issue number12
Early online date5 Dec 2022
DOIs
Publication statusPublished - 19 Dec 2022

Keywords

  • carboxymethyl cellulose
  • citric acid
  • cross-linking
  • direct-ink-writing 3D printing
  • enzyme immobilization
  • glycosyltransferase
  • nanofibrillated cellulose
  • nothofagin

ASJC Scopus subject areas

  • Biochemistry, medical
  • Chemistry(all)
  • Biomedical Engineering
  • Biomaterials

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