UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

Characterization and multi-step transketolase-ω-transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube.

Abdul Halim, A; Szita, N; Baganz, F; (2013) Characterization and multi-step transketolase-ω-transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube. J Biotechnol , 168 (4) pp. 567-575. 10.1016/j.jbiotec.2013.09.001. Green open access

[img]
Preview
PDF
1-s2.0-S0168165613003921-main.pdf

Download (1MB)

Abstract

The concept of de novo metabolic engineering through novel synthetic pathways offers new directions for multi-step enzymatic synthesis of complex molecules. This has been complemented by recent progress in performing enzymatic reactions using immobilized enzyme microreactors (IEMR). This work is concerned with the construction of de novo designed enzyme pathways in a microreactor synthesizing chiral molecules. An interesting compound, commonly used as the building block in several pharmaceutical syntheses, is a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT). This chiral amino alcohol can be synthesized from simple achiral substrates using two enzymes, transketolase (TK) and transaminase (TAm). Here we describe the development of an IEMR using His6-tagged TK and TAm immobilized onto Ni-NTA agarose beads and packed into tubes to enable multi-step enzyme reactions. The kinetic parameters of both enzymes were first determined using single IEMRs evaluated by a kinetic model developed for packed bed reactors. The Km(app) for both enzymes appeared to be flow rate dependent, while the turnover number kcat was reduced 3 fold compared to solution-phase TK and TAm reactions. For the multi-step enzyme reaction, single IEMRs were cascaded in series, whereby the first enzyme, TK, catalyzed a model reaction of lithium-hydroxypyruvate (HPA) and glycolaldehyde (GA) to l-erythrulose (ERY), and the second unit of the IEMR with immobilized TAm converted ERY into ABT using (S)-α-methylbenzylamine (MBA) as amine donor. With initial 60mM (HPA and GA each) and 6mM (MBA) substrate concentration mixture, the coupled reaction reached approximately 83% conversion in 20min at the lowest flow rate. The ability to synthesize a chiral pharmaceutical intermediate, ABT in relatively short time proves this IEMR system as a powerful tool for construction and evaluation of de novo pathways as well as for determination of enzyme kinetics.

Type: Article
Title: Characterization and multi-step transketolase-ω-transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube.
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.jbiotec.2013.09.001
Publisher version: http://dx.doi.org/10.1016/j.jbiotec.2013.09.001
Additional information: © 2013 The Authors. Published by Elsevier B.V. All rights reserved. This is an open-access article distributed under the terms of the CreativeCommons Attribution License, which permits unrestricted use, distribution andreproduction in any medium, provided the original author and source are credited.
Keywords: Chiral amino alcohol, Immobilized enzyme, Microreactor, Transaminase, Transketolase
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/1408726
Downloads since deposit
209Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item