This README_Supplementary_Data.txt file was generated on 2025-03-04 by Kathrin Marina Eckert (kathrin.eckert@tuhh) ------------------- GENERAL INFORMATION ------------------- Name: Kathrin Marina Eckert (ORCID: 0000-0002-8454-4886) Role/Function: Data collector (main contact person) Institution: Institute of Thermal Separation Processes, Hamburg University of Technology Address: Eißendorfer Straße 38, 21073 Hamburg, Germany Email: kathrin.eckert@tuhh.de Name: Kayla Reata Dittmer (ORCID: 0009-0005-4841-9438) Role/Function: Data collector Institution: Institute of Technical Biocatalysis, Hamburg University of Technology Address: Denickestraße 15, 21073 Hamburg, Germany Email: kayla.dittmer@tuhh.de Name: Sherliana Setiawan Role/Function: Data collector Institution: Institute of Technical Biocatalysis, Hamburg University of Technology Address: Denickestraße 15, 21073 Hamburg, Germany Email: sherliana.setiawan@tuhh.de Name: Daniel Ohde (ORCID: 0000-0002-5482-3534) Role/Function: Supervision Institution: Institute of Technical Biocatalysis, Hamburg University of Technology Address: Denickestraße 15, 21073 Hamburg, Germany Email: daniel.ohde@tuhh.de Name: Andreas Liese (ORCID: 0000-0003-4503-4039) Role/Function: Principal Investigator (alternative contact person) Institution: Institute of Technical Biocatalysis, Hamburg University of Technology Address: Denickestraße 15, 21073 Hamburg, Germany Email: liese@tuhh.de Name: Irina Smirnova (ORCID: 0000-0003-4503-4039) Role/Function: Principal Investigator (alternative contact person) Institution: Institute of Thermal Separation Processes, Hamburg University of Technology Address: Eißendorfer Straße 38, 21073 Hamburg, Germany Email: irina.smirnova@tuhh.de Date of data collection: December 2024 - May 2025 Location of data collection: Institute of Technical Biocatalysis and Institute of Thermal Separation Processes, Hamburg University of Technology, Hamburg, Germany Funding: This project is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SFB 1615 – 503850735. --------------------------- SHARING/ACCESS INFORMATION --------------------------- Title of data set: "Enzyme Immobilization on Smart Gels, Part B" DOI of data set: https://doi.org/10.15480/882.16797 This data set is part of a second data set: https://doi.org/10.15480/882.16752 Keywords: biocatalysis, bioreactor, stimuli-responsive, hydrogels, PFR, plug-flow, continuous reactor --------------------- DATA & FILE OVERVIEW --------------------- ### 1. Table_S1.csv (creation date: 27.02.2026, version: 01) - **Description:** Reaction yield for varying flow rates, comparing different hydrogel carrier materials in the plug-flow reactor (PFR). - **Columns:** - Hydrogel: Polymer formulation (p(HEMA-co-IA), p(NIPAM-co-IA), pNIPAM) - Flow Rate (mL/min): Applied volumetric flow rate in the reactor - Relative Yield (-): Reaction yield for the applied conditions. ### 2. Table_S2.csv (creation date: 27.02.2026, version: 01) - **Description:** Relative yield of temperature-responsive hydrogels and silica (non-responsive reference) under constant and fluctuating temperature conditions (25 °C and 40 °C).. - **Columns:** - Hydrogel: Polymer formulation (pNIPAM, p(NIPAM-co-IA), silica) - Time (h): Reaction time - Relative Yield at Fluctuating Conditions (-): Yield under alternating temperature conditions - Relative Yield at Constant Conditions (-): Relative Yield under constant temperature conditions ### 3. Table_S3.csv (creation date: 27.02.2026, version: 01) - **Description:** Relative yield of pH-responsive hydrogels and silica (non-responsive reference) under constant and fluctuating temperature conditions (pH 8 and pH 4). - **Columns:** - Hydrogel: Polymer formulation (p(HEMA-co-IA), p(NIPAM-co-IA), silica) - Time (h): Reaction time - Relative Yield at Fluctuating Conditions (-): Yield under alternating temperature conditions - Relative Yield at Constant Conditions (-): Relative Yield under constant temperature conditions ### 4. Table_S4.csv (creation date: 27.02.2026, version: 01) - **Description:** Relative yield of dual-responsive hydrogel p(NIPAM-co-IA) at 25 °C and pH 4. - **Columns:** - Hydrogel: Polymer formulation (p(NIPAM-co-IA)) - Time (h): Reaction time - Relative Yield (-): Relative Yield at 25 °C and pH 4. --------------------------- METHODOLOGICAL INFORMATION --------------------------- 1. Gel Synthesis: The synthesis is performed as described in Part A: https://doi.org/10.15480/882.16752. 2. Hydrogel Preparation: Copolymeric systems contained 10 mol% itaconic acid. After polymerization in syringes (0.5 cm inner diameter), gels were cut into cylindrical monoliths (0.5 cm height) and washed extensively with deionized water to remove residual monomers. Commercial silica beads (2–5 mm) were sieved (> 4 mm fraction) to obtain particles comparable in size to the hydrogel monoliths. Silica carriers were pre-wetted prior to enzyme immobilization to avoid fracture due to capillary effects. 3. Enzyme Immobilization: Formate dehydrogenase (FDH) was immobilized on hydrogel monoliths via EDC/NHS-mediated covalent coupling following the protocol established in Part A (https://doi.org/10.15480/882.16752). Silica carriers were functionalized by physical adsorption due to the absence of reactive groups for covalent attachment. 4. Plug-Flow Reactor: Catalytic performance was evaluated in a vertically oriented glass plug-flow reactor packed with hydrogel monoliths and glass beads. The carrier bed height was kept constant (4 cm) across all formulations to ensure comparable hydrodynamic conditions. A substrate solution containing 200 mM sodium formate and 1 mM NAD⁺ in 50 mM potassium phosphate buffer (pH 8 or pH 4) was continuously supplied using a syringe pump. Flow rates were individually adjusted based on the void volume of each packed reactor to maintain a fixed residence time of 20 min. Samples were collected every 30 min and analyzed spectrophotometrically (340 nm) to quantify NADH formation. 5. Variation of Flow Rate: To determine appropriate operating conditions, flow rates between 0.25 and 2.3 mL·min⁻¹ were investigated. Based on the comparison of residence time and conversion, a residence time of 20 min was selected for subsequent fluctuating condition experiments. 6. Operation Under Fluctuating Conditions: Reactor experiments were conducted under both constant and alternating environmental conditions: Temperature fluctuation: 25 °C → 40 °C → 25 °C (2 h per stage) pH fluctuation: pH 8 → pH 4 → pH 8 (2 h per stage) These sequences simulate dynamic process environments. Non-responsive silica carriers were tested under identical conditions to distinguish effects caused by material responsiveness from intrinsic enzyme deactivation. 7. Extreme Conditions: The dual-responsive hydrogel p(NIPAM-co-IA) was operated continuously at pH 4 and 40 °C to evaluate protective effects under prolonged harsh conditions. ----------------- RESEARCH CONTEXT ----------------- This study investigates stimuli-responsive hydrogels as smart carrier materials for enzyme immobilization. Three polymer systems were analyzed: pH-responsive poly(HEMA-co-IA), temperature-responsive pNIPAM, and dual-responsive poly(NIPAM-co-IA). The materials are applied in a PFR under constant and fluctuating temperature and pH conditions to investigate the impact of responsivity of the carrier matrix on the conversion in biocatalytic processes. ---------------------------------- EXPLANATION OF MEASURED VARIABLES ---------------------------------- Yield (-): Ratio of product concentration (c_NADH) to initial substrate concentration (c_NAD⁺). Specific Yield y(t): Defined as follows y_spec(t) = c_NADH(t) / (c_NAD⁺ · m_imm.Enzyme). y(t) normalizes the product formation to the mass of immobilized enzyme. Relative Yield Y(t) (-): Defined as follows: Y(t) = y_spec(t) / y_spec(t = 1 h). The relative yield is normalized by the yield measured after 1 h. Residual Yield (-): Relative yield measured after 6 h of reactor operation (Y(6 h)). Flow Rate (mL/min): Volumetric feed rate of substrate solution through the PFR. Time (h): Continuous reactor operation time during PFR experiments.