Immobilization protects enzymes from plasma-mediated inactivationNon-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.
]]>biocatalysishydrogen peroxideImmobilizationperoxygenaseprotein protectionPlasma in Liquidsb4491a5f-46b6-48e4-92b6-b40c1baed60a2023-12-06T09:54:30+01:002024-04-28T11:28:52+02:00en-USApplied MicrobiologyFigure 2 HRP residual activitiesResidual activity of immobilized HRP after 900 s of plasma treatment. Immobilization was carried out using Lifetech ECR (Purolite) carriers according to the manufacturers' instructions. For HRP, only non-directional resins were used for immobilization, because the enzyme did not have a His-tag. The specific activity of the respective untreated immobilized enzyme was set to 100%. Means and standard deviations of three independent biological replicates are shown.
]]>2023-12-06T10:07:34+01:002024-04-25T11:22:11+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx245015https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/e2935b94-88c2-4a79Figure 2 GapA Residual activities Residual activity of immobilized GapA after 180 s of plasma treatment. Immobilization was carried out using Lifetech ECR (Purolite) and EziG (EnginZyme) carriers according to the manufacturers' instructions. The specific activity of the respective untreated immobilized enzyme was set to 100%. Means and standard deviations of three independent biological replicates are shown
]]>2023-12-06T10:13:38+01:002024-04-25T11:22:21+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx410476https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/087429ca-964f-4e1eFigure 2 LacZ Residual activitiesResidual activity of immobilized LacZ after 900 s of plasma treatment. Immobilization was carried out using Lifetech ECR (Purolite) and EziG (EnginZyme) carriers according to the manufacturers' instructions. The specific activity of the respective untreated immobilized enzyme was set to 100%. Means and standard deviations of three independent biological replicates are shown.
]]>2023-12-06T10:22:03+01:002024-04-25T11:22:06+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx413738https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/375046fe-b4c2-4598Figure 2 LdhA Residual activityResidual activity of immobilized LdhA after 900 s of plasma treatment. Immobilization was carried out using Lifetech ECR (Purolite) and EziG (EnginZyme) carriers according to the manufacturers' instructions. The specific activity of the respective untreated immobilized enzyme was set to 100%. Means and standard deviations of three independent biological replicates are shown.
]]>2023-12-06T10:32:25+01:002024-04-25T11:21:55+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx422719https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/348b743e-af81-466dFigure 2 VCPO Residual activityResidual activity of immobilized VCPO after 900 s of plasma treatment . Immobilization was carried out using Lifetech ECR (Purolite) and EziG (EnginZyme) carriers according to the manufacturers' instructions. The specific activity of the respective untreated immobilized enzyme was set to 100%. Means and standard deviations of three independent biological replicates are shown.
]]>2023-12-06T12:27:06+01:002024-04-25T11:21:52+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx422013https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/094c2f25-0f53-4ec4Figure 3 GapA Residual activities of free enzyme and enzyme immobilized on the most efficiently protecting carrier material (either amino or epoxy-butyl based Lifetech ERC resins (Purolite)) after different plasma treatment times. GapA on amino based ERC resin. Plasma treatment times of up to 3600 s were used. The specific activity of untreated enzyme was set to 100% activity. Means and standard deviations of three independent biological replicates are shown.
]]>2023-12-06T12:29:36+01:002024-04-25T11:22:29+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx275169https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/1f82fa25-6c15-4ef1Figure 3 HRPResidual activities of free enzyme and enzyme immobilized on the most efficiently protecting carrier material (either amino or epoxy-butyl based Lifetech ERC resins (Purolite)) after different plasma treatment times. HRP immobilized on epoxy-butyl based ERC resin. Plasma treatment times of up to 3600 s were used. The specific activity of untreated enzyme was set to 100% activity. Data of free HRP are taken from Yayci et al.. Means and standard deviations of three independent biological replicates are shown.
]]>2023-12-06T12:30:21+01:002024-04-25T11:21:59+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx129337https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/01ccfd52-92cb-4986Figure 3 LacZResidual activities of free enzyme and enzyme immobilized on the most efficiently protecting carrier material (either amino or epoxy-butyl based Lifetech ERC resins (Purolite)) after different plasma treatment times. LacZ on epoxy-butyl based ERC resin. Plasma treatment times of up to 3600 s were used. The specific activity of untreated enzyme was set to 100% activity. Means and standard deviations of three independent biological replicates are shown.
]]>2023-12-06T12:31:15+01:002024-04-25T11:22:23+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx129457https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/72345181-91e4-4d6aFigure 3 LdhAResidual activities of free enzyme and enzyme immobilized on the most efficiently protecting carrier material (either amino or epoxy-butyl based Lifetech ERC resins (Purolite)) after different plasma treatment times. LdhA on amino based ERC resin. Plasma treatment times of up to 3600 s were used. The specific activity of untreated enzyme was set to 100% activity. Means and standard deviations of three independent biological replicates are shown.
]]>2023-12-06T12:31:50+01:002024-04-25T11:22:01+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx524214https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/bacbbbdc-63f0-49f5Figure 3 VCPOResidual activities of free enzyme and enzyme immobilized on the most efficiently protecting carrier material (either amino or epoxy-butyl based Lifetech ERC resins (Purolite)) after different plasma treatment times. VCPO on epoxy-butyl based ERC resin. Plasma treatment times of up to 3600 s were used . The specific activity of untreated enzyme was set to 100% activity. Means and standard deviations of three independent biological replicates are shown.
]]>2023-12-06T12:32:38+01:002024-04-25T11:22:14+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx237584https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/f6fe6f7a-51f5-4049Figure 4 Protection FactorTreatment times at which residual enzyme activity of free and immobilized HRP, VCPO, LdhA, LacZ and GapA reached 70%. HRP, VCPO, and LacZ were immobilized on epoxy-butyl-functionalized resin, LdhA and GapA on amino-functionalized resin following manufacturers' instructions. The treatment time at which residual activity reached 70% was calculated based on the respective inactivation curve regression.
]]>2023-12-06T12:33:44+01:002024-04-25T11:22:12+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx15549https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/b82c0485-6650-43a2Figure 5 b-e DegradationProtein degradation under plasma treatment conditions were investigated using Ni-NTA agarose, to which enzyme is bound non-covalently. Proteins VCPO, LdhA, LacZ, or GapA were immobilized on Ni-NTA agarose and treated with plasma or left untreated. Using ninhydrin, after plasma treatment times of up to 600 s, the concentration of terminal amino groups as an indicator of protein degradation was determined for free enzyme and enzyme immobilized on Ni-NTA agarose. Means and standard deviations of three independent biological replicates are shown.
]]>2023-12-06T12:35:38+01:002024-04-25T11:22:02+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx53708https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/c3e91ae8-1927-44f5Figure 5a Plasma-mediated protein detachmentPlasma-mediated protein detachment from the carrier material were investigated using Ni-NTA agarose, to which enzyme is bound non-covalently. Proteins VCPO, LdhA, LacZ, or GapA were immobilized on Ni-NTA agarose and treated with plasma or left untreated. After 300 s of plasma treatment, the amount of protein in the supernatant was determined using a Bradford assay and compared to protein concentrations in the untreated control.
]]>2023-12-06T12:36:50+01:002024-04-25T11:22:25+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx315601https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/6e1735be-f5d6-4a87Figure 6 Residual activity reversibility assayResidual activity of VCPO, LdhA, LacZ, or GapA reversibly immobilized on Ni-NTA agarose after 300 s of plasma treatment. After plasma treatment, enzyme was eluted from the carrier with imidazole and washed with buffer. Protein concentration was determined using Bradford assay. Enzyme activity was measured using 5 µl of the eluted enzyme in its respective activity assay. Activities were normalized to the respective protein concentrations and activity of the untreated enzyme was set to 100%. Means and standard deviations represent three independent biological replicates.
]]>2023-12-06T12:37:29+01:002024-04-25T11:21:50+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx30305https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/f0391f20-1e77-400fSuppl. Figure 2 GapA Binding efficiency Binding efficiencies of GapA on different resins after immobilization. Immobilization was carried out using Lifetech ECR (Purolite) and EziG (EnginZyme) carriers according to manufacturers’ instructions.
]]>2023-12-06T12:39:29+01:002024-04-25T11:22:26+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx87378https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/53aebb63-ff08-472aSuppl. Figure 2 HRP Binding efficiency Binding efficiencies of HRP on different resins after immobilization. Immobilization was carried out using Lifetech ECR (Purolite) carriers according to manufacturers’ instructions.
]]>2023-12-06T12:40:04+01:002024-04-25T11:22:09+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx68191https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/15d6deec-97f4-48f6Suppl. Figure 2 LacZ Binding efficiency Binding efficiencies of LacZ on different resins after immobilization. Immobilization was carried out using Lifetech ECR (Purolite) and EziG (EnginZyme) carriers according to manufacturers’ instructions.
]]>2023-12-06T12:40:44+01:002024-04-25T11:22:07+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx109501https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/37279037-4950-403eSuppl. Figure 2 LdhA Binding efficiency Binding efficiencies of LdhA on different resins after immobilization. Immobilization was carried out using Lifetech ECR (Purolite) and EziG (EnginZyme) carriers according to manufacturers’ instructions.
]]>2023-12-06T12:41:23+01:002024-04-25T11:22:28+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx78095https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/c43991a7-3bc3-43bcSuppl. Figure 2 VCPO Binding efficiencyBinding efficiencies of VCPO, on different resins after immobilization. Immobilization was carried out using Lifetech ECR (Purolite) and EziG (EnginZyme) carriers according to manufacturers’ instructions.
]]>2023-12-06T12:41:59+01:002024-04-25T11:22:04+02:00application/vnd.openxmlformats-officedocument.spreadsheetml.sheetxlsx173466https://rdpcidat.rub.de/dataset/immobilization-protects-enzymes-plasma-mediated-inactivation/resource/53a1e69e-df54-4ee0DKANhttps://rdpcidat.rub.de