Harnessing Macromolecular Crowding of Proteins for Engineering Sustainable Nanofiltration Membranes

Abstract

Membrane technology using advanced materials paves the way towards slashing industrial energy consumption. Protein-based separation membranes were engineered through macromolecular crowding of proteins induced by interactions with various green solvents to fabricate biodegradable, solvent-resistant nanofiltration membranes. Single-molecule tracking using quantum dots provided unprecedented insights into the real-time dynamics of membrane formation at the microscopic level. Molecular dynamics simulations corroborated these findings, highlighting the roles of macromolecular crowding and chain entanglement. The structural characteristics of the membranes by means of small- and wide-angle X-ray scattering spectroscopies were uncovered. The interpolymer chain distance, polymer conformation, and polymer chain arrangement were considerably influenced by interactions with different solvents. These structural modifications resulted in distinct membrane morphologies and mechanical properties. The biodegradable membranes exhibited excellent solvent resistance and molecular sieving performance in the nanofiltration range under high-pressure conditions. A case study demonstrated the successful removal of a carcinogenic impurity from a pharmaceutical. Given the abundance of proteins in biomass, our findings open the door for the development of sustainable and robust membranes through the manipulation of proteins.

Keywords

macromolecular crowding / membranes / nanofiltration / proteins / sustainability / zein

References

[1] G. Ignacz, A. K. Beke, V. Toth, and G. Szekely, “A Hybrid Modelling Approach to Compare Chemical Separation Technologies in Terms of Energy Consumption and Carbon Dioxide Emissions,” Nature Energy 10, no. 3 (2025): 308-317.

[2] G. Szekely, “The 12 Principles of Green Membrane Materials and Processes for Realizing the United Nations' Sustainable Development Goals,” RSC Sustain 2, no. 4 (2024): 871-880.

[3] A. K. Mohanty, F. Wu, R. Mincheva, et al., “Sustainable Polymers,” National Review Methods Primer 2, no. 1 (2022): 46.

[4] S. H. Park, C. Yang, N. Ayaril, and G. Szekely, “Solvent-Resistant Thin-Film Composite Membranes From Biomass-Derived Building Blocks: Chitosan and 2,5-Furandicarboxaldehyde,” ACS Sustainable Chemistry & Engineering 10, no. 2 (2022): 998-1007.

[5] X. Xu, Z. He, G. Van Eygen, et al., “Sealing Non-selective Subnanochannels of Chitosan Acetate Matrix of the Membrane to Valorise Glycerol From Biofuel Waste,” Journal of Membrane Science 713 (2025): 123294.

[6] V. Toth, R. Hardian, H. Vovusha, C. Yang, and G. Szekely, Interfacial Polymerization of Dopamine With Diamines for Ultrastable Janus Nanofiltration Membranes and Adhesives (Mater Today, 2025): 104-114.

[7] J. Cavalcante, D. G. Oldal, M. V. Peskov, et al., “Biobased Interpenetrating Polymer Network Membranes for Sustainable Molecular Sieving,” ACS Nano 18, no. 10 (2024): 7433-7443.

[8] A. Y. Gebreyohannes, S. L. Aristizábal, L. Silva, et al., “A Lignin-based Membrane Fabricated With a Deep Eutectic Solvent,” Green Chemistry 25, no. 12 (2023): 4769-4780.

[9] A. Alammar, R. Hardian, and G. Szekely, “Upcycling Agricultural Waste Into Membranes: From Date Seed Biomass to Oil and Solvent-resistant Nanofiltration,” Green Chemistry 24, no. 1 (2022): 365-374.

[10] M. H. Abdellah, C. Oviedo, and G. Szekely, “Controlling the Degree of Acetylation in Cellulose-based Nanofiltration Membranes for Enhanced Solvent Resistance,” Journal of Membrane Science 687 (2023): 122040.

[11] A. Torre-Celeizabal, F. Russo, F. Galiano, A. Figoli, C. Casado-Coterillo, and A. Garea, “Green Synthesis of Cellulose Acetate Mixed Matrix Membranes: Structure-Function Characterization,” ACS Sustainable Chemistry & Engineering 13, no. 3 (2025): 1253-1270.

[12] C. Alfano, Y. Fichou, K. Huber, et al., “Molecular Crowding: The History and Development of a Scientific Paradigm,” Chemical Review 124, no. 6 (2024): 3186-3219.

[13] D. de Sancho, A. Sirur, and R. B. Best, “Molecular Origins of Internal Friction Effects on Protein-folding Rates,” Nature Communications 5, no. 1 (2014): 4307.

[14] A. K. Hołda and I. F. J. Vankelecom, “Understanding and Guiding the Phase Inversion Process for Synthesis of Solvent Resistant Nanofiltration Membranes,” Journal of Applied Polymer Science 132, no. 27 (2015): 42130.

[15] J. A. Doolan, L. S. Alesbrook, K. Baker, et al., “Next-generation Protein-based Materials Capture and Preserve Projectiles From Supersonic Impacts,” Nature Nanotechnology 18, no. 9 (2023): 1060-1066.

[16] A. Miserez and J. Yu, and P. Mohammadi, “Protein-Based Biological Materials: Molecular Design and Artificial Production,” Chemical Review 123, no. 5 (2023): 2049-2111.

[17] L. Koziej, F. Fatehi, M. Aleksejczuk, et al., “Dynamic Assembly of Pentamer-Based Protein Nanotubes,” ACS Nano 19, no. 9 (2025): 8786-8798.

[18] M. Rütten, L. Lang, H. Wagler, et al., “Assembly of Differently Sized Supercharged Protein Nanocages Into Superlattices for Construction of Binary Nanoparticle-Protein Materials,” ACS Nano 18, no. 36 (2024): 25325-25336.

[19] H. Qi, Y. Teng, S. Chen, et al., “Major Structural Protein in Locust Mandible Capable of Forming Extraordinarily Stiff Materials via Hierarchical Self-assembly,” Matter 7, no. 3 (2024): 1314-1329.

[20] M. Peydayesh, M. Bagnani, W. L. Soon, and R. Mezzenga, “Turning Food Protein Waste Into Sustainable Technologies,” Chemical Reviews 123, no. 5 (2023): 2112-2154.

[21] R. Shukla and M. Cheryan, “Zein: The Industrial Protein From Corn,” Industrial Crops and Products 13, no. 3 (2001): 171-192.

[22] X. Zhang, C. Dong, Y. Hu, M. Gao, and G. Luan, “Zein as a Structural Protein in Gluten-free Systems: An Overview,” Food Science and Human Wellness 10, no. 3 (2021): 270-277.

[23] X. Yan, M. Li, X. Xu, X. Liu, and F. Liu, “Zein-based Nano-delivery Systems for Encapsulation and Protection of Hydrophobic Bioactives: A Review,” Frontiers in Nutrition 9 (2022): 999373.

[24] L. Vogt, L. Liverani, J. A. Roether, and A. R. Boccaccini, “Electrospun Zein Fibers Incorporating Poly(Glycerol Sebacate) for Soft Tissue Engineering,” Nanomaterials 8, no. 3 (2018): 150.

[25] M. Demir, L. Ramos-Rivera, R. Silva, S. N. Nazhat, and A. R. Boccaccini, “Zein-based Composites in Biomedical Applications,” Journal of Biomedical Materials Research: Part A 105, no. 6 (2017): 1656-1665.

[26] J. L. de Oliveria, E. V. R. Campos, A. E. S. Pereira, et al., “Zein Nanoparticles as Eco-Friendly Carrier Systems for Botanical Repellents Aiming Sustainable Agriculture,” Journal of Agricultural and Food Chemistry 66, no. 6 (2018): 1330-1340.

[27] A. Altan, Z. Aytac, and T. Uyar, “Carvacrol Loaded Electrospun Fibrous Films From Zein and Poly(Lactic Acid) for Active Food Packaging,” Food Hydrocolloids 81 (2018): 48-59.

[28] B. Cao, Q. Qu, B. Jiang, et al., “Zein Improved GF Separator for Dendrite-Free Aqueous Zinc-Ion Batteries,” Journal of Materials Chemistry A 12, no. 45 (2024): 31185-31194.

[29] R. Qu, B. Xia, M. Hou, et al., “Preparation of High Antibacterial, Easy Coating, and Easy Cleaning Pea Protein Isolate/Zein-Carboxymethyl Cellulose Composite Coating Material and Its Preservation Application,” ACS Sustainable Chemistry & Engineering 13, no. 9 (2025): 3728-3740.

[30] X. Fan, J. Zhao, H. Pu, et al., “Self-Modification of Dynamic Network Valence Bonds in ZeinNFs via Post-Heat Treatment: Preparation of an Efficient and Environmentally Friendly Ethylene Adsorbent,” ACS Sustainable Chemistry & Engineering 12, no. 42 (2024): 15643-15650.

[31] P. Vandezande, L. E. Gevers, and I. F. J. Vankelecom, “Solvent Resistant Nanofiltration: Separating on a Molecular Level,” Chemical Society Reviews 37, no. 2 (2008): 365-405.

[32] R. Kecili, J. Billing, M. Leeman, et al., “Selective Scavenging of the Genotoxic Impurity Methyl p-Toluenesulfonate From Pharmaceutical Formulations,” Separation and Purification Technology 103 (2013): 173-179.

[33] R. Kecili, J. Billing, D. Nivhede, B. Sellergren, A. Rees, and E. Yilmaz, “Fast Identification of Selective Resins for Removal of Genotoxic Aminopyridine Impurities via Screening of Molecularly Imprinted Polymer Libraries,” Journal of Chromatography A 1339 (2014): 65-72.

[34] H. Xiao, Y. Feng, W. R. F. Goundry, and S. Karlsson, “Organic Solvent Nanofiltration in Pharmaceutical Applications,” Organic Process Research & Development 28, no. 4 (2024): 891-923.

[35] Z. Jiang, R. Dong, A. M. Evans, et al., “Aligned Macrocycle Pores in Ultrathin Films for Accurate Molecular Sieving,” Nature 609, no. 7925 (2022): 58-64.

[36] B. Alhazmi, G. Ignacz, M. Di Vincenzo, M. N. Hedhili, G. Szekely, and S. P. Nunes, “Ultraselective Macrocycle Membranes for Pharmaceutical Ingredients Separation in Organic Solvents,” Nature Communications 15, no. 1 (2024): 7151.

[37] S. Morita, F. Tsunomori, and H. Ushiki, “Polymer Chain Conformation in the Phase Separation Process of a Binary Liquid Mixture,” European Polymer Journal 38, no. 9 (2002): 1863-1870.

[38] J. U. Garcia, T. Iwama, E. Y. Chan, D. R. Tree, K. T. Delaney, and G. H. Fredrickson, “Mechanisms of Asymmetric Membrane Formation in Nonsolvent-Induced Phase Separation,” ACS Macro Lett 9, no. 11 (2020): 1617-1624.

[39] J. Yamamoto, A. Matsui, F. Gan, et al., “Quantitative Evaluation of Macromolecular Crowding Environment Based on Translational and Rotational Diffusion Using Polarization Dependent Fluorescence Correlation Spectroscopy,” Scientific Reports 11, no. 1 (2021): 10594.

[40] L. Heo, Y. Sugita, and M. Feig, “Protein Assembly and Crowding Simulations,” Current Opinion in Structural Biology 73 (2022): 102340.

[41] C. Pelosi, M. Tiné, and F. Wurm, “Main-Chain Water-Soluble Polyphosphoesters: Multi-Functional Polymers as Degradable PEG-alternatives for Biomedical Applications,” European Polymer Journal 141 (2020): 110079.

[42] I. W. Hamley and V. Castelletto, “Small-Angle Scattering of Block Copolymers: In the Melt, Solution and Crystal States,” Progress in Polymer Science 29, no. 9 (2004): 909-948.

[43] B. Sutisna, G. Polymeropoulos, V. Musteata, et al., “Functionalized Nanochannels From Self-Assembled and Photomodified Poly(Styrene-b-Butadiene-b-Styrene),” Small 14, no. 18 (2017): e1701885.

[44] P. Argos, K. Pedersen, M. D. Marks, and B. A. Larkins, “A Structural Model for Maize Zein Proteins,” Journal of Biological Chemistry 257, no. 17 (1982): 9984-9990.

[45] N. Matsushima, G. Danno, H. Takezawa, and Y. Izumi, “Three-Dimensional Structure of Maize α-Zein Proteins Studied by Small-Angle X-Ray Scattering,” Biochimica Et Biophysica Acta 1339, no. 1 (1997): 14-22.

[46] L. A. Forato, A. C. Doriguetto, H. Fischer, Y. P. Mascarenhas, A. F. Craievich, and L. A. Colnago, “Conformation of the Z19 Prolamin by FTIR, NMR, and SAXS,” Journal of Agricultural and Food Chemistry 52, no. 8 (2004): 2382-2385.

[47] A. G. Kikhney and D. I. Svergun, “A Practical Guide to Small Angle X-Ray Scattering (SAXS) of Flexible and Intrinsically Disordered Proteins,” FEBS Letters 589, no. 19PartA (2015): 2570-2577.

[48] F. S. Tekin and P. Z. Çulfaz-Emecen, “Controlling Cellulose Membrane Performance via Solvent Choice During Precursor Membrane Formation,” ACS Applied Polymer Materials 5, no. 3 (2023): 2185-2194.

[49] K. Shi, J. L. Kokini, and Q. Huang, “Engineering Zein Films With Controlled Surface Morphology and Hydrophilicity,” Journal of Agricultural and Food Chemistry 57, no. 6 (2009): 2186-2192.

[50] A. Christiansen, Q. Wang, M. S. Cheung, and P. Wittung-Stafshede, “Effects of Macromolecular Crowding Agents on Protein Folding in Vitro and in Silico,” Biophysical Reviews 5, no. 2 (2013): 137-145.

[51] S. Mukherjee, M. M. Waegele, P. Chowdhury, L. Guo, and F. Gai, “Effect of Macromolecular Crowding on Protein Folding Dynamics at the Secondary Structure Level,” Journal of Molecular Biology 393, no. 1 (2009): 227-236.

[52] A. Sadat, M. G. Corradini, and I. J. Joye, “Vibrational and Fluorescence Spectroscopy to Study Gluten and Zein Interactions in Complex Dough Systems,” Current Research Food Science 5 (2022): 479-490.

[53] S. Babitha and P. S. Korrapati, “Biodegradable Zein-Polydopamine Polymeric Scaffold Impregnated With TiO₂ Nanoparticles for Skin Tissue Engineering,” Biomedical Materials 12, no. 5 (2017): 055008.

[54] K. M. Martinez, R. Hardian, G. Ignacz, and G. Szekely, “Automating the Determination of Pore Size Distribution in Liquid Separation Membranes via Solute Retention Experiments,” Journal of Membrane Science 726 (2025): 124015.

[55] C. Guindani, G. Candiotto, P. H. H. Araújo, et al., “Controlling the Biodegradation Rates of Poly(Globalide-co-ε-Caprolactone) Copolymers by Post Polymerization Modification,” Polymer Degradation and Stability 179 (2020): 109287.

[56] G. Székely, J. Bandarra, W. Heggie, B. Sellergren, and F. C. Ferreira, “Organic Solvent Nanofiltration: A Platform for Removal of Genotoxins From Active Pharmaceutical Ingredients,” Journal of Membrane Science 381, no. 1 (2011): 21-33.

[57] M. F. Serag and S. Habuchi, “Conserved Linear Dynamics of Single-Molecule Brownian Motion,” Nature Communications 8, no. 1 (2017): 15675.

[58] M. F. Serag, M. Abadi, and S. Habuchi, “Single-Molecule Diffusion and Conformational Dynamics by Spatial Integration of Temporal Fluctuations,” Nature Communications 5, no. 1 (2014): 5123.

[59] K. Jaqaman, D. Loerke, M. Mettlen, et al., “Robust Single-Particle Tracking in Live-Cell Time-Lapse Sequences,” Nature Methods 5, no. 8 (2008): 695-702.

RIGHTS & PERMISSIONS

2026 The Author(s). SusMat published by Sichuan University and John Wiley & Sons Australia, Ltd.