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Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices

Kulsharova, G; Dimov, N; Marques, MPC; Szita, N; Baganz, F; (2017) Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices. New Biotechnology 10.1016/j.nbt.2017.12.004. Green open access

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Abstract

Poly(methyl methacrylate) (PMMA) microfluidic devices have become promising platforms for a wide range of applications. Here we report a simple method for immobilising histidine-tagged enzymes suitable for PMMA microfluidic devices. The 1-step-immobilisation described is based on the affinity of the His-tag/Ni-NTA interaction and does not require prior amination of the PMMA surface, unlike many existing protocols. We compared it with a 3-step immobilisation protocol involving amination of PMMA and linking NTA via a glutaraldehyde cross-linker. These methods were applied to immobilise transketolase (TK) in PMMA microfluidic devices. Binding efficiency studies showed that about 15% of the supplied TK was bound using the 1-step method and about 26% of the enzyme was bound by the 3-step method. However, the TK-catalysed reaction producing l-erythrulose performed in microfluidic devices showed that specific activity of TK in the device utilising the 1-step immobilisation method was approximately 30% higher than that of its counterpart. Reusability of the microfluidic device produced via the 1-step method was tested for three cycles of enzymatic reaction and at least 85% of the initial productivity was maintained. The device could be operated for up to 40 h in a continuous flow and on average 70% of the initial productivity was maintained. The simplified immobilisation method required fewer chemicals and less time for preparation of the immobilised microfluidic device compared to the 3-step method while achieving higher specific enzyme activity. The method represents a promising approach for the development of immobilised enzymatic microfluidic devices and could potentially be applied to combine protein purification with immobilisation.

Type: Article
Title: Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.nbt.2017.12.004
Publisher version: http://doi.org/10.1016/j.nbt.2017.12.004
Language: English
Additional information: Copyright © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/)
Keywords: Microfluidics, Poly(methyl methacrylate) (PMMA)Enzyme immobilisation, Histidine-tagged enzyme, 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/10042534
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