Engineering of continuous bienzymatic cascade process using monolithic microreactors – In flow synthesis of trehalose

Daria Kowalczykiewicz, Marta Przypis, Luuk Mestrom, Antje Kumpf, Dirk Tischler, Peter Leon Hagedoorn, Ulf Hanefeld, Andrzej Jarzębski, Katarzyna Szymańska*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

1 Citation (Scopus)

Abstract

Here, we present a two-step continuous flow enzymatic synthesis process in monolithic microreactors using basic sugars as substrates. In the first step UDP-glucose pyrophosphorylase (TaGalU) catalyses the synthesis of uridine-diphosphate-glucose (UDP-Glc) using uridine triphosphate (UTP) and glucose-1-phosphate (Glc-1-P). This is followed by the trehalose transferase (mCherry-TuTreT) catalysed reaction of UDP-Glc and Glc, to obtain trehalose. First, procedures for immobilisation of both enzymes on functionalised silica supports were studied and it was found that covalent bonding by amino groups using a glutaraldehyde linker gives highly active biocatalysts. Due to a drastic difference in temperature range of activity and stability of the immobilised enzymes a bi-reactor cascade was rationally the best solution. Depending on the applied flow rate and hence reaction (residence) time (1.5–10 min) the space-time-yield values varied, respectively, from 1.9 to 14.4 and 8.3 to 49.6 gproduct·L-1·h−1·mgprotein-1, for UDP-glucose pyrophosphorylase and trehalose transferase catalysed reactions. Prolonged (100 h) continuous flow operation showed that the system is operationally stable, but owing to neutral pH, it is prone to microbiological infections. They can be eliminated applying an antibacterial/antifungal therapy or preventive actions by storing and washing the reactors with a NaN3 solution. The presented process paves the way for the continuous in flow synthesis of natural and non-natural trehalose analogues and disaccharides.

Original languageEnglish
Article number131439
Number of pages8
JournalChemical Engineering Journal
Volume427
DOIs
Publication statusPublished - 2022

Bibliographical note

Green Open Access added to TU Delft Institutional Repository 'You share, we take care!' - Taverne project https://www.openaccess.nl/en/you-share-we-take-care
Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.

Keywords

  • Modular production system
  • Monolithic microreactors
  • Multi-enzyme cascade
  • NDP-glucose
  • Trehalose
  • UDP-glucose

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