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.
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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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