Abstract
Whole-cell biocatalysts are versatile tools in (industrial) production processes; though, the effects that impact the efficiency are not fully understood yet. One main factor that affects whole-cell biocatalysts is the surrounding medium, which often consists of organic solvents due to low solubility of substrates in aqueous solutions. It is expected that organic solvents change the biophysical and biochemical properties of the whole-cell biocatalysts, e.g. by permeabilising the cell membrane, and thus analysis of these effects is of high importance. In this work, we present an analysis method to study the impact of organic solvents on whole-cell biocatalysts by means of dielectrophoresis. For instance, we evaluate the changes of the characteristic dielectrophoretic trapping ratio induced by incubation of Escherichia coli, serving as a model system, in an aqueous medium containing isopropyl alcohol. Therefore, we could evaluate the impact on the electric polarisability of the cells. For this purpose, a special microchannel device was designed and Escherichia coli cells were genetically modified to reliably synthesise a green fluorescent protein. We could demonstrate that our method was capable of revealing different responses to small changes in isopropyl alcohol concentration and incubation duration. Complementary spectrophotometric UV-Vis (ultraviolet-visible light) absorbance analysis of released NAD(P)+/NAD(P)H cofactor and proteins confirmed our results. Based on our results, we discuss the biophysical effects taking place during incubation.Graphical abstract
Highlights
Escherichia coli (E. coli) cells are versatile organisms that are often used as host organisms in the construction of whole-cell biocatalysts
The plasmid pET21T5 ompA-co-SuperfolderGFP(-), that was used for expression of the green fluorescent protein (GFP), was constructed based on the plasmid pET21a(+) (Novagen/Merck) by exchange of the T7 promoter by the constitutive T5 promoter that can be recognised by E. coli enabling recombinant protein expression without induction resulting in the plasmid pET21aT5
The concept of the DEP analysis in this work is based upon the assumption that the dielectrophoretic migration behaviour of E. coli changes if the electrophysiology of the cell is changed by the organic solvents
Summary
Escherichia coli (E. coli) cells are versatile organisms that are often used as host organisms in the construction of whole-cell biocatalysts. Their stable and well-controlled microenvironment yields very good conditions for enzymes; the intracellular matrix further stabilises enzymes. E. coli consist of numerous intrinsic cofactors that are important for a large number of industrially used enzymes, e.g. alcohol dehydrogenase or monooxygenase. Biocatalysis in whole-cell biocatalysts can be more stable and reliable as in batch reactors with free enzymes. About 60% of the industrial biocatalytic reactions are performed with whole-cell biocatalysts as reported in 2002 by Straathof et al [1]
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