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

Abdul Halim, Amanatuzzakiah and Szita, Nicolas and Baganz, Frank (2013) Characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube. Journal of Biotechnology, 168 (4). pp. 567-575. ISSN 0168-1656

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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)--methylbenzylamine (MBA) as amine donor. With initial 60 mM (HPA and GA each) and 6 mM (MBA) substrate concentration mixture, the coupled reaction reached approximately 83% conversion in 20 min 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.

Item Type:	Article (Journal)
Additional Information:	5355/47769
Uncontrolled Keywords:	Microreactor,transketolase,transaminase,immobilized enzyme,chiral amino alcohol
Subjects:	T Technology > TP Chemical technology > TP155 Chemical engineering
Kulliyyahs/Centres/Divisions/Institutes (Can select more than one option. Press CONTROL button):	Kulliyyah of Engineering > Department of Biotechnology Engineering
Depositing User:	ms Amanatuzzakiah Abdul Halim
Date Deposited:	23 Feb 2016 15:27
Last Modified:	23 Feb 2016 15:27
URI:	http://irep.iium.edu.my/id/eprint/47769

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