This README_Supplementary_Data.txt file was generated on 2025-03-04 by Kayla Reata Dittmer (kayla.dittmer@tuhh) ------------------- GENERAL INFORMATION ------------------- Name: Kayla Reata Dittmer (ORCID: 0009-0005-4841-9438) Role/Function: Data collector (alternative contact person) Institution: Institute of Technical Biocatalysis, Hamburg University of Technology Address: Denickestraße 15, 21073 Hamburg, Germany Email: kayla.dittmer@tuhh.de Name: Kathrin Marina Eckert (ORCID: 0000-0002-8454-4886) Role/Function: Data collector Institution: Institute of Thermal Separation Processes, Hamburg University of Technology Address: Eißendorfer Straße 38, 21073 Hamburg, Germany Email: kathrin.eckert@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: Patrick André Kißling (ORCID: 0000-0001-5596-9976) Role/Function: Data collector Institution: Institute of Chemical Reaction Engineering, Hamburg University of Technology Address: Eißendorfer Straße 38, 21073 Hamburg, Germany Email: patrick.kissling@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: 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 Name: Andreas Liese (ORCID: 0000-0003-4503-4039) Role/Function: Principal Investigator (main contact person) Institution: Institute of Technical Biocatalysis, Hamburg University of Technology Address: Denickestraße 15, 21073 Hamburg, Germany Email: liese@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 A" DOI of data set: https://doi.org/10.15480/882.16752 This data set is part of a second data set: https://doi.org/10.15480/882.16797 Keywords: biocatalysis, stimuli-responsive, hydrogels, smart carriers, enzyme immobilization --------------------- DATA & FILE OVERVIEW --------------------- ### 1. Table_S1.csv (creation date: 27.02.2026, version: 01) - **Description:** Degree of swelling and elastic modulus of hydrogels equilibrated at 25 °C in distilled water. - **Columns:** - Hydrogel: Polymer formulation considered - Degree of Swelling (-): Ratio of equilibrated gel mass to dry polymer mass - Elastic Modulus (kPa): Elastic modulus determined by uniaxial compression ### 2. Table_S2.csv (creation date: 27.02.2026, version: 01) - **Description:** Relative swelling ratios of hydrogels under temperature- and pH-responsive conditions. - **Columns:** - Hydrogel: Polymer formulation considered - Stimulus: The stimulus applied during the analysis (temperature / pH) - Swelling Ratio (-): Relative mass between the two states considered ### 3. Table_S3.csv (creation date: 27.02.2026, version: 01) - **Description:** Immobilization yield of formate dehydrogenase (FDH) on hydrogel monoliths and particles. - **Columns:** - Hydrogel: Polymer formulation considered - Shape: Hydrogel geometry (monolith / particles) - Binding Mechanism: The enzyme immobilization method applied for this analysis (adsorption / covalent binding (3 h) / covalent binding (24 h)) - Immobilization Yield (%): Mass of immobilized enzyme on the distinct carrier matrices (hydrogels) relative to the bulk solution. ### 4. Table_S4.csv (creation date: 27.02.2026, version: 01) - **Description:** Enzyme loading of FDH immobilized on hydrogel monoliths and particles. - **Columns:** - Hydrogel: Polymer formulation considered - Shape: Hydrogel geometry (monolith / particles) - Binding Mechanism: The enzyme immobilization method applied for this analysis (adsorption / covalent binding (3 h) / covalent binding (24 h)) - Enzyme Loading (%): Mass of immobilized enzyme on the distinct carrier matrices (hydrogels) relative to the dry mass of the matrix. ### 5. Table_S5.csv (creation date: 27.02.2026, version: 01) - **Description:** Activity yield of FDH immobilized on hydrogel monoliths. - **Columns:** - Hydrogel: Polymer formulation considered - Shape: Hydrogel geometry (monolith) - Binding Mechanism: The enzyme immobilization method applied for this analysis (adsorption / covalent binding (3 h) / covalent binding (24 h)) - Activity Yield (%): Specific activity of immobilized enzyme relative to free enzyme ### 6. Table_S6.csv (creation date: 27.02.2026, version: 01) - **Description:** Relative enzyme leaching from hydrogel monoliths. - **Columns:** - Hydrogel: Polymer formulation considered - Shape: Hydrogel geometry (monolith) - Binding Mechanism: The enzyme immobilization method applied for this analysis (adsorption / covalent binding (3 h) / covalent binding (24 h)) - Relative Leaching (%): Percentage of enzyme released during activity assay relative to immobilized enzyme mass ### 7. Table_S7.csv (creation date: 27.02.2026, version: 01) - **Description:** Hydrogel composition for synthesis. - **Columns:** - Chemicals: Each component used in the synthesis. - p(HEMA-co-IA) [pH responsive]: The mass of each component within the synthesis of pH-responsive formulation (p(HEMA-IA)) - p(NIPAM-co-IA) [dual responsive]: The mass of each component within the synthesis of dual-responsive formulation (p(NIPAM-IA)) - pNIPAM [temperature responsive]: The mass of each component within the synthesis of temperature-responsive formulation (pNIPAM) --------------------------- METHODOLOGICAL INFORMATION --------------------------- 1. Gel Synthesis: The responsive hydrogels were synthesized by radical polymerization, according to the formulation in Table_S7.csv. In the copolymeric formulations, the amount of itaconic acid corresponded to 10 mol.% of the total monomer content. 2. Swelling Equilibria: After synthesis, the hydrogels were characterized with respect to swelling behavior and mechanical properties. The degree of swelling (DoS) was determined as the ratio of the mass of the equilibrated gel to the dry polymer mass. For this purpose, hydrogels were equilibrated in deionized water for 24 h at 25 °C. The dry mass was obtained after vacuum drying at 40 °C for 48 h. Stimuli-responsive swelling was evaluated under pH- and temperature-varying conditions. For both analyses, monoliths were pre-equilibrated in deionized water (24 h, 25 °C). For pH-responsiveness, samples were equilibrated in 50 mM potassium phosphate buffer at pH 8 (state I), followed by stepwise transfer to buffers of pH 7, 6, and 4 (state II), with equilibration and gravimetric determination at each step. The pH 4 buffer was adjusted using 85 wt.% ortho-phosphoric acid. Temperature-responsiveness was assessed by comparing swelling at 25 °C (state I) and 40 °C (state II) in deionized water. The relative swelling S was calculated as the mass ratio between state I and state II. 3. Mechanical Analysis: The elastic modulus was determined by uniaxial compression under ambient conditions using a universal testing machine (nominal force: 2.5 kN). A flat cylindrical stamp (40 mm diameter) was used to ensure full surface contact with the hydrogel samples. The measurement started at a contact force of 0.02 N and compression was applied at a constant rate of 50 mm*min⁻¹. The test was terminated at 75% strain or upon reaching the load cell limit (50 N). Force and displacement were continuously recorded. The elastic modulus E_C was calculated from the linear elastic region of the force–deformation curve using the slope of elastic deformation and the sample cross-sectional area. 4. Enzyme Immobilization: Adsorptive immobilization was performed by incubating hydrogels in an FDH solution (0.5 U·mL⁻¹ final concentration) at 4 °C for 24 h under gentle shaking. Samples were withdrawn over time to monitor immobilization. Protein concentration in the supernatant was determined using the Bradford assay (595/450 nm) with bovine serum albumin as calibration standard (0–200 µg·mL⁻¹). For covalent immobilization, an EDC/NHS coupling protocol was applied. Hydrogels were pre-equilibrated in water (24 h), activated with EDC (52 mM) and NHS (86.8 mM) in 100 mM MES buffer (pH 5) for 3 h at 25 °C, rinsed, and subsequently incubated in enzyme solution as described for adsorption. Silica beads were used as non-responsive reference material and functionalized by adsorption only. To prevent fracture due to capillary forces, beads were pre-wetted via water vapor exposure at 40 °C before immersion in enzyme solution. 5. FDH Activity Assay: Enzyme activity was determined by monitoring NADH formation at 340 nm. Immobilizates were added to a reaction solution containing 200 mM sodium formate and 1 mM NAD⁺ in 50 mM potassium phosphate buffer (pH 8). Samples were taken over 5 h and analyzed spectrophotometrically. NADH concentration was calculated using the Lambert–Beer law. The effective microplate pathlength (100 µL volume) was determined by comparison with a 1 cm cuvette using absorbance differences at 975 and 900 nm. 6. Immobilization Characterization: Immobilization performance was evaluated using four parameters: Immobilization yield (Y_immo): Percentage of enzyme mass bound relative to the initially added enzyme mass, determined via mass balance of the supernatant. Enzyme loading (E): Mass of immobilized enzyme per dry carrier mass. Activity yield (Y_EA): Specific activity of immobilized enzyme relative to free enzyme under identical conditions. Enzyme leaching: Percentage of enzyme mass released during reaction relative to the initially immobilized enzyme mass. ----------------- 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 were characterized in terms of swelling behavior, mechanical properties, and morphology to confirm their stimulus-dependent responses. Formate dehydrogenase (FDH) from Candida boidinii was immobilized using adsorption and EDC/NHS-mediated covalent binding on two carrier geometries (monoliths and particles). Immobilization yield, enzyme loading, activity yield, and enzyme leaching were systematically evaluated to compare the influence of polymer chemistry, carrier size, and immobilization strategy. ---------------------------------- EXPLANATION OF MEASURED VARIABLES ---------------------------------- Degree of Swelling (Dos) (m_gel / m_dry_polymer) : Ratio of equilibrated gel mass to dry polymer mass. Swelling Ratio (S) (-): Relative mass ratio between two stimulus states (temperature or pH), comparing 40 °C / 25 °C and pH4 / pH8. Immobilization Yield (Y_immo) (%): Percentage of enzyme bound relative to initial enzyme mass. Enzyme Loading (E) (mg_enzyme/g_polymer): Mass of immobilized enzyme per dry mass of polymer carrier. Activity Yield (Y_EA) (%): Specific activity of immobilized enzyme relative to free enzyme. Relative Leaching (%): Percentage of enzyme released during activity assay relative to immobilized enzyme mass.