期刊
NEW BIOTECHNOLOGY
卷 47, 期 -, 页码 31-38出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.nbt.2017.12.004
关键词
Microfluidics; Poly(methyl methacrylate) (PMMA); Enzyme immobilisation; Histidine-tagged enzyme; Transketolase
资金
- European Union [608104]
- Biotechnology and Biological Sciences Research Council, UK (BBSRC) [BB/L000997/1]
- BBSRC [BB/L000997/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/L000997/1] Funding Source: researchfish
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.
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