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Immobilization protects enzymes from plasma-mediated inactivation

Non-thermal plasmas are used in various applications to inactivate biological agents or biomolecules. A complex cocktail of reactive species, (vacuum) UV radiation and in some cases exposure to an electric field together cause the detrimental effects. In contrast to this disruptive property of technical plasmas, we have shown previously that it is possible to use non-thermal plasma-generated species such as H2O2 as cosubstrates in biocatalytic reactions. One of the main limitations in plasma-driven biocatalysis is the relatively short enzyme lifetime under plasma-operating conditions. This challenge could be overcome by immobilizing the enzymes on inert carrier materials. Here, we tested whether immobilization is suited to protect proteins from inactivation by plasma. To this end, using a dielectric barrier discharge device (PlasmaDerm), plasma stability was tested for five enzymes immobilized on ten different carrier materials. A comparative analysis of the treatment times needed to reduce enzyme activity of immobilized and free enzyme by 30% showed a maximum increase by a factor of 44. Covalent immobilization on a partly hydrophobic carrier surface proved most effective. We conclude from the study, that immobilization universally protects enzymes under plasma-operating conditions, paving the way for new emerging applications.

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Plasma Source Name
Plasma Source Application
Plasma Source Specification
Plasma Source Properties
V(RMS) = 13.5 kV, trigger frequency = 300 Hz, diameter = 20 mm
English (United States)
Plasma Source Procedure
Droplets of protein samples (containing either 100 µl of 1 mg ml−1 free enzyme or 100 µl with 10 mg protein-loaded ECR carrier or 0.5 mg protein-loaded EziG carrier) were applied onto a glass slide partially coated with polytetrafluoroethylene (PTFE; supplier: VWR), such that the droplets sat on glass and were confined by the PTFE coating. The glass slide was placed onto a grounded metal plate. Plasma treatment of the samples was carried out using the PlasmaDerm dielectric barrier discharge (DBD) (Cinogy) with an electrode diameter of 20 mm, VRMS = 13.5 kV, and a trigger frequency of 300 Hz. The distance of the sample to the driven electrode was approx. 1 mm (± 1 mm). For experiments using Ni-NTA agarose as immobilization carrier, plasma treatment was carried out with in-house built metal plates, which are formed in such a way that they can be placed on reaction tubes in place of the lid. This allowed for complete recovery of the samples by centrifugation.
Plasma Medium Name
Plasma Medium Properties
Plasma Target Name
Contact Name
Bandow, Julia E.
Plasma Target Properties
Horseradish peroxidase (P8375); Lactate dehydrogenase A (LdhA), β-Galactosidase (LacZ) and Glyceraldehyde-3-phosphate dehydrogenase A (GapA) purified from Escherichia coli expression strain; all enzymes were stored in 10 mM Kai buffer, pH 7
Plasma Target Procedure
HRP was purchased from Sigma-Aldrich (P8375, RZ > 2.5) and stored in 100 mM potassium phosphate buffer (KPi), pH 7. The enzymes LdhA, LacZ and GapA were purified as N-terminal His6-tag fusion proteins from E. coli BL21 strains harbouring the plasmids pCA24N::ldhA, pCA24N::lacZ, and pCA24N::gapA, respectively. Cells were grown in LB medium containing 50 µg ml−1 chloramphenicol. For overexpression, overnight cultures were diluted to an OD600 of 0.05 and incubated at 37°C. At OD600 of 0.5, overexpression was induced with 100 µM isopropyl β-D-thiogalactoside (IPTG). Cultures were shifted to 30°C and harvested after 4 h of growth following the shift. Cell pellets were resuspended in lysis buffer (20 mM sodium phosphate, 500 mM NaCl, 0.2 mg ml−1 DNase, 0.2 mg ml−1 RNase, 0.35 mg ml−1 lysozyme, cOmplete protease inhibitor (Roche); all other components acquired from Sigma-Aldrich). After sonication and centrifugation for 30 min (21 000g), the supernatant was loaded onto a HisTrap FF crude 5 ml column (GE Healthcare). Purification of the His6-tagged proteins was carried out with an ÄKTA pure25 system (GE Healthcare). Proteins were eluted with a linear gradient from 20 to 500 mM imidazole in 20 mM sodium phosphate buffer and 500 mM NaCl. Elution fractions of interest were pooled, concentrated using centrifugal filter units (cut-off 10 kDa), aliquoted, and stored in potassium phosphate buffer (100 mM, pH 7) at −80°C.
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Project Contact Name: 
Bandow, Julia E.