Dielectrophoretic analysis of the impact of isopropyl alcohol on the electric polarisability of Escherichia coli whole-cells.

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.

High-Throughput Feasible Screening Tool for Determining Enzyme Stabilities against Organic Solvents Directly from Crude Extracts.

Biotransformations in organic chemistry frequently suffer from limitations caused by low water-solubility of substrates and product inhibition. Both, usually are addressed by the addition of organic cosolvents, which often accompanies at the expense of enzyme stability. A common method for measuring enzyme stability is to determine the melting temperature (Tm ) of the enzyme. However, current methods are limited to the application of purified enzymes. Herein, for the first time, an easy and fast (<1 h) high-throughput feasible method to determine enzyme stabilities directly from crude extracts is reported. In pure buffer, the Tm value measured in the crude extract was identical to that obtained for the purified enzyme. Through the addition of different organic compounds, the Tm values in the crude extract differed by up to 2.4 °C from that of the purified enzymes due to the presence of the host-cell proteins. Thus, the measurement of enzyme stabilities in crude extracts appears to represent conditions in whole-cell catalysts even better. The applied nano differential scanning fluorimetry technology is further proven to be suitable for whole-cell catalysts with two overexpressed enzymes; thus representing a tool for the rapid screening of natural and mutant enzyme libraries in terms of process stability for challenging biotransformations.