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

Gulsim Kulsharova, Nikolay Dimov, Marco P.C. Marques, Nicolas Szita, Frank Baganz

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)31-38
Number of pages8
JournalNew Biotechnology
Volume47
DOIs
Publication statusPublished - Dec 25 2018
Externally publishedYes

Keywords

  • Enzyme immobilisation
  • Histidine-tagged enzyme
  • Microfluidics
  • Poly(methyl methacrylate) (PMMA)
  • Transketolase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Molecular Biology

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