Computational Analysis of Crosslinker Binding Affinity and Site Interactions with Fungal and Bacterial Laccases to Guide CLEA Development
DOI:
https://doi.org/10.11113/bioprocessing.v4n2.81Keywords:
Laccase, Molecular Docking, Enzyme ImmobilizationAbstract
Efficient cross-linked enzyme aggregate (CLEA) formation requires not only strong interactions between enzymes and crosslinkers but also proper binding orientation to prevent active site obstruction. In this study, molecular docking was employed to investigate the interactions between fungal (Trametes versicolor) and bacterial (Bacillus subtilis) laccases and various crosslinkers, including glutaraldehyde, ethylene glycol, dialdehyde starch, pectin, and chitosan. Docking simulations revealed that chitosan exhibited the strongest binding affinity with both enzymes (-7.1 and -7.0 kcal/mol), primarily due to extensive hydrogen bonding and hydrophobic interactions. Binding site analysis further demonstrated that chitosan binds at sites distant from the active sites of the enzymes, which are 32.0 Å from the catalytic center within domain 2 of the bacterial laccase and 34.6 Å from the catalytic center within domain 3 of the fungal laccase, minimizing the risk of active site obstruction and preserving catalytic accessibility. In contrast, glutaraldehyde and ethylene glycol were found near the fungal laccase active site, potentially influencing substrate access. This study suggests that molecular docking can serve as a valuable preliminary approach to explore crosslinker–enzyme interactions and support the rational design of CLEAs before experimental validation.
References
Ali, M., Husain, Q., Sultana, S., & Ahmad, M. (2018). Immobilization of peroxidase on polypyrrole-cellulose-graphene oxide nanocomposite via non-covalent interactions for the degradation of Reactive Blue 4 dye. Chemosphere, 202, 198–207.
Arregui, L., Ayala, M., Gómez-Gil, X., Gutiérrez-Soto, G., Hernández-Luna, C. E., Herrera De Los Santos, M., Levin, L., Rojo-Domínguez, A., Romero-Martínez, D., & Saparrat, M. C. N. (2019). Laccases: structure, function, and potential application in water bioremediation. Microbial Cell Factories, 18, 1–33.
Barati, F., Hosseini, F., Vafaee, R., Sabouri, Z., Ghadam, P., Arab, S. S., ... & Piroozmand, F. (2024). In silico approaches to investigate enzyme immobilization: a comprehensive systematic review. Physical Chemistry Chemical Physics, 26(7), 5744-5761.
Basso, A., & Serban, S. (2019). Industrial applications of immobilized enzymes—A review. Molecular Catalysis, 479, 110607.
Bié, J., Sepodes, B., Fernandes, P. C. B., & Ribeiro, M. H. L. (2022). Enzyme immobilization and co-immobilization: main framework, advances and some applications. Processes, 10(3), 494.
Chauhan, V., Kaushal, D., Dhiman, V. K., Kanwar, S. S., Singh, D., Dhiman, V. K., & Pandey, H. (2022). An insight in developing carrier-free immobilized enzymes. Frontiers in Bioengineering and Biotechnology, 10, 794411.
Davis, A. M., & Teague, S. J. (1999). Hydrogen bonding, hydrophobic interactions, and failure of the rigid receptor hypothesis. Angewandte Chemie International Edition, 38(6), 736–749.
Debnath, R., & Saha, T. (2020). An insight into the production strategies and applications of the ligninolytic enzyme laccase from bacteria and fungi. Biocatalysis and Agricultural Biotechnology, 26, 101645. https://doi.org/https://doi.org/10.1016/j.bcab.2020.101645
Eberhardt, J., Santos-Martins, D., Tillack, A. F., & Forli, S. (2021). AutoDock Vina 1.2. 0: New docking methods, expanded force field, and python bindings. Journal of Chemical Information and Modeling, 61(8), 3891–3898.
Fan, J., Fu, A., & Zhang, L. (2019). Progress in molecular docking. Quantitative Biology, 7(2), 83–89.
Hakulinen, N., & Rouvinen, J. (2015). Three-dimensional structures of laccases. Cellular and Molecular Life Sciences, 72(5), 857–868.
Hanwell, M. D., Curtis, D. E., Lonie, D. C., Vandermeersch, T., Zurek, E., & Hutchison, G. R. (2012). Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. Journal of Cheminformatics, 4, 1–17.
Jailani, N., Jaafar, N. R., Suhaimi, S., Mackeen, M. M., Bakar, F. D. A., & Illias, R. M. (2022). Cross-linked cyclodextrin glucanotransferase aggregates from Bacillus lehensis G1 for cyclodextrin production: Molecular modeling, developmental, physicochemical, kinetic and thermodynamic properties. International Journal of Biological Macromolecules, 213, 516–533.
Khan, M., Husain, Q., & Ahmad, N. (2019). Elucidating the binding efficacy of β‐galactosidase on polyaniline–chitosan nanocomposite and polyaniline–chitosan–silver nanocomposite: activity and molecular docking insights. Journal of Chemical Technology & Biotechnology, 94(3), 837–849.
Khongkomolsakul, W., Yang, E., Dadmohammadi, Y., Dong, H., Lin, T., Huang, Y., & Abbaspourrad, A. (2025). Enzyme immobilization with plant-based polysaccharides through complex coacervation. LWT, 219, 117537.
Laskowski, R. A., & Swindells, M. B. (2011). LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. ACS Publications, 2788-2786.
Loi, M., Glazunova, O., Fedorova, T., Logrieco, A. F., & Mulè, G. (2021). Fungal laccases: The forefront of enzymes for sustainability. Journal of Fungi, 7(12), 1048.
Maier, J. A., Martinez, C., Kasavajhala, K., Wickstrom, L., Hauser, K. E., & Simmerling, C. (2015). ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. Journal of Chemical Theory and Computation, 11(8), 3696–3713.
Masjoudi, M., Golgoli, M., Nejad, Z. G., Sadeghzadeh, S., & Borghei, S. M. (2021). Pharmaceuticals removal by immobilized laccase on polyvinylidene fluoride nanocomposite with multi-walled carbon nanotubes. Chemosphere, 263, 128043.
Meng, X.-Y., Zhang, H.-X., Mezei, M., & Cui, M. (2011). Molecular docking: a powerful approach for structure-based drug discovery. Current Computer-Aided Drug Design, 7(2), 146–157.
Moreno, A. D., Ibarra, D., Eugenio, M. E., & Tomás‐Pejó, E. (2020). Laccases as versatile enzymes: from industrial uses to novel applications. Journal of Chemical Technology & Biotechnology, 95(3), 481–494.
Panwar, V., Lzaod, S., & Dutta, T. (2023). Thermostable bacterial laccase: catalytic properties and its application in biotransformation of emerging pollutants. ACS Omega, 8(38), 34710–34719.
Robescu, M. S., & Bavaro, T. (2025). A comprehensive guide to enzyme immobilization: All you need to know. Molecules, 30(4), 939.
Rodrigues, R. C., Barbosa, O., Ortiz, C., Berenguer-Murcia, Á., Torres, R., & Fernandez-Lafuente, R. (2014). Amination of enzymes to improve biocatalyst performance: Coupling genetic modification and physicochemical tools. RSC Advances, 4(72), 38350–38374.
Roy, K., Kar, S., & Das, R. N. (2015). Chapter 5 - Computational Chemistry. In K. Roy, S. Kar, & R. N. Das (Eds.), Understanding the Basics of QSAR for Applications in Pharmaceutical Sciences and Risk Assessment (pp. 151–189). Academic Press. https://doi.org/https://doi.org/10.1016/B978-0-12-801505-6.00005-3
Sahoo, R. N., Pattanaik, S., Pattnaik, G., Mallick, S., & Mohapatra, R. (2022). Review on the use of Molecular Docking as the First Line Tool in Drug Discovery and Development. Indian Journal of Pharmaceutical Sciences, 84(5).
Sampaio, C. S., Angelotti, J. A. F., Fernandez-Lafuente, R., & Hirata, D. B. (2022). Lipase immobilization via cross-linked enzyme aggregates: Problems and prospects - A review. International Journal of Biological Macromolecules, 215, 434–449. https://doi.org/https://doi.org/10.1016/j.ijbiomac.2022.06.139
Shapovalov, M. V, & Dunbrack, R. L. (2011). A smoothed backbone-dependent rotamer library for proteins derived from adaptive kernel density estimates and regressions. Structure, 19(6), 844–858.
Wen, X., Zeng, Z., Du, C., Huang, D., Zeng, G., Xiao, R., Lai, C., Xu, P., Zhang, C., & Wan, J. (2019). Immobilized laccase on bentonite-derived mesoporous materials for removal of tetracycline. Chemosphere, 222, 865–871.
Yip, Y. S., Manas, N. H. A., Jaafar, N. R., Rahman, R. A., Puspaningsih, N. N. T., & Illias, R. M. (2023). Combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase and maltogenic amylase from Bacillus lehensis G1 for maltooligosaccharides synthesis. International Journal of Biological Macromolecules, 242, 124675.
Yu, D., Wang, N., Gong, Y., Wu, Z., Wang, W., Wang, L., Wu, F., & Jiang, L. (2022). Screening of active sites and study on immobilization of Bacillus cereus phospholipase C. LWT, 159, 113245. https://doi.org/https://doi.org/10.1016/j.lwt.2022.113245
