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Immobilised enzyme microreactor for screening of multi-step bioconversions: Characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols

Paper ID Volume ID Publish Year Pages File Format Full-Text
24067 43493 2011 10 PDF Available
Title
Immobilised enzyme microreactor for screening of multi-step bioconversions: Characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols
Abstract

Complex molecules are synthesised via a number of multi-step reactions in living cells. In this work, we describe the development of a continuous flow immobilized enzyme microreactor platform for use in evaluation of multi-step bioconversion pathways demonstrating a de novo transketolase/ω-transaminase-linked asymmetric amino alcohol synthesis. The prototype dual microreactor is based on the reversible attachment of His6-tagged enzymes via Ni-NTA linkage to two surface derivatised capillaries connected in series. Kinetic parameters established for the model transketolase (TK)-catalysed conversion of lithium-hydroxypyruvate (Li-HPA) and glycolaldehyde (GA) to l-erythrulose using a continuous flow system with online monitoring of reaction output was in good agreement with kinetic parameters determined for TK in stop-flow mode. By coupling the transketolase catalysed chiral ketone forming reaction with the biocatalytic addition of an amine to the TK product using a transaminase (ω-TAm) it is possible to generate chiral amino alcohols from achiral starting compounds. We demonstrated this in a two-step configuration, where the TK reaction was followed by the ω-TAm-catalysed amination of l-erythrulose to synthesise 2-amino-1,3,4-butanetriol (ABT). Synthesis of the ABT product via the dual reaction and the on-line monitoring of each component provided a full profile of the de novo two-step bioconversion and demonstrated the utility of this microreactor system to provide in vitro multi-step pathway evaluation.

Keywords
Microfluidics; Enzyme immobilisation; Microreactor; Asymmetric synthesis; Transketolase; Transaminase
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Immobilised enzyme microreactor for screening of multi-step bioconversions: Characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 155, Issue 3, 20 September 2011, Pages 320–329
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us