ENHANCING THE OPERATIONAL STABILITY OF RECOMBINANT PHENYLALANINE AMMONIA-LYASE IMMOBILIZED ON MAGNETIC NANOPARTICLES BY POST-ENTRAPMENT

Bálint ALÁCS; Anna ZRINYI; Evelin BELL

doi:10.24193/subbchem.2026.1.04

Authors

Bálint ALÁCS Department of Organic Chemistry and Technology, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Műegyetem rkp. 3., H-1111 Budapest, Hungary
Anna ZRINYI Department of Organic Chemistry and Technology, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Műegyetem rkp. 3., H-1111 Budapest, Hungary
Evelin BELL Department of Organic Chemistry and Technology, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Műegyetem rkp. 3., H-1111 Budapest, Hungary. *Corresponding author: bell.evelin@vbk.bme.hu https://orcid.org/0000-0002-9083-7757

DOI:

https://doi.org/10.24193/subbchem.2026.1.04

Keywords:

immobilized metal ion affinity chromatography, enzyme immobilization, sol-gel, alginate

Abstract

Recombinant Petroselinum crispum phenylalanine ammonia-lyase (PcPAL) was selectively immobilized on magnetic nanoparticles by metal affinity binding (IMAC) to create a well applicable biocatalyst. To overcome the stability limitations of coordination bond, two post-immobilization entrapment strategies were investigated: macroscopic entrapment in calcium-alginate hydrogel beads and also in sol–gel matrix. The catalytic efficiency and operational stability of the composite biocatalysts were evaluated in the ammonia elimination reaction of l-phenylalanine. The concentration of immobilized biocatalyst was optimized in the calcium-alginate stabilization. In the sol–gel shell formation the amount of tetraethyl ortosilicate (TEOS) and the combination with a less crosslinking capability dimethyldiethoxysilane (DMDEOS) was investigated. In the latter case the TEOS was used in 4 different ratios in the silane precursor mixture. While the best calcium-alginate beads (5 m/m% loading) provided a biocompatible environment, they suffered from mechanical instability and physical disintegration occur after four reaction cycles. In contrast, the optimized silica-coated nanobiocatalyst exhibited superior mechanical and chemical stability, preventing enzyme leaching and retaining over 80% of its initial activity after seven consecutive reaction cycles. These results demonstrate that individual particle encapsulation via a silica shell offers a more robust solution for the design of reusable magnetic biocatalysts than macroscopic hydrogel entrapment.

References

1. J. M. Guisan; F. López-Gallego; L. Betancor; C. Mateo; V. Grazu; G. Fernandez-Lorente; J. Rocha-Martin; J. M. Bolivar; K. Ovsejevi; C. Manta; et al. Immobilization of Enzymes and Cells; Guisan, J.M., Ed.; Methods in Molecular Biology; Humana Press: Totowa, NJ, 2013; Vol. 1051; ISBN 978-1-62703-549-1.

2. Sheldon, R.A. Fundamentals of Green Chemistry: Efficiency in Reaction Design. Chem. Soc. Rev. 2012, 41, 1437–1451, doi:10.1039/C1CS15219J.

3. Bornscheuer, U.T.; Huisman, G.W.; Kazlauskas, R.J.; Lutz, S.; Moore, J.C.; Robins, K. Engineering the Third Wave of Biocatalysis. Nature, 2012, 485, 185–194, doi:10.1038/nature11117.

4. Cui, J.D.; Qiu, J.Q.; Fan, X.W.; Jia, S.R.; Tan, Z.L. Biotechnological Production and Applications of Microbial Phenylalanine Ammonia Lyase: A Recent Review. Crit. Rev. Biotechnol. 2014, 34, 258–268, doi:10.3109/07388551.2013.791660.

5. Datta, S.; Christena, L.R.; Rajaram, Y.R.S. Enzyme Immobilization: An Overview on Techniques and Support Materials. 3 Biotech, 2013, 3, 1–9, doi:10.1007/s13205-012-0071-7.

6. Mateo, C.; Palomo, J.M.; Fernandez-Lorente, G.; Guisan, J.M.; Fernandez-Lafuente, R. Improvement of Enzyme Activity, Stability and Selectivity via Immobilization Techniques. Enzyme Microb. Technol., 2007, 40, 1451–1463, doi:10.1016/j.enzmictec.2007.01.018.

7. Gupta, A.K.; Gupta, M. Synthesis and Surface Engineering of Iron Oxide Nanoparticles for Biomedical Applications. Biomaterials, 2005, 26, 3995–4021, doi:10.1016/j.biomaterials.2004.10.012.

8. Lu, A.-H.; Salabas, E.L.; Schüth, F. Magnetic Nanoparticles: Synthesis, Protection, Functionalization, and Application. Angewandte Chemie International Edition, 2007, 46, 1222–1244, doi:10.1002/anie.200602866.

9. Lucena, G.N.; Santos, C.C. dos; Pinto, G.C.; Piazza, R.D.; Guedes, W.N.; Jafelicci Júnior, M.; de Paula, A. V.; Marques, R.F.C. Synthesis and Characterization of Magnetic Cross-Linked Enzyme Aggregate and Its Evaluation of the Alternating Magnetic Field (AMF) Effects in the Catalytic Activity. J. Magn. Magn. Mater., 2020, 516, 167326, doi:10.1016/j.jmmm.2020.167326.

10. Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Metal Chelate Affinity Chromatography, a New Approach to Protein Fractionation. Nature, 1975, 258, 598–599, doi:10.1038/258598a0.

11. Sánta-Bell; Molnár; Varga; Nagy; Hornyánszky; Paizs; Balogh-Weiser; Poppe “Fishing and Hunting”—Selective Immobilization of a Recombinant Phenylalanine Ammonia-Lyase from Fermentation Media. Molecules, 2019, 24, 4146, doi:10.3390/molecules24224146.

12. Cassimjee, K.E.; Kourist, R.; Lindberg, D.; Wittrup Larsen, M.; Thanh, N.H.; Widersten, M.; Bornscheuer, U.T.; Berglund, P. One‐step Enzyme Extraction and Immobilization for Biocatalysis Applications. Biotechnol. J., 2011, 6, 463–469, doi:10.1002/biot.201000357.

13. Weiser, D.; Nagy, F.; Bánóczi, G.; Oláh, M.; Farkas, A.; Szilágyi, A.; László, K.; Gellért, Á.; Marosi, G.; Kemény, S.; et al. Immobilization Engineering – How to Design Advanced Sol–Gel Systems for Biocatalysis? Green Chemistry, 2017, 19, 3927–3937, doi:10.1039/C7GC00896A.

14. Appert, C.; Logemann, E.; Hahlbrock, K.; Schmid, J.; Amrhein, N. Structural and Catalytic Properties of the Four Phenylalanine Ammonia-Lyase Isoenzymes from Parsley (Petroselinum Crispum Nym.). Eur. J. Biochem., 1994, 225, 491–499, doi:10.1111/j.1432-1033.1994.00491.x.

15. Poppe, L.; Rétey, J. Friedel–Crafts‐Type Mechanism for the Enzymatic Elimination of Ammonia from Histidine and Phenylalanine. Angewandte Chemie International Edition, 2005, 44, 3668–3688, doi:10.1002/anie.200461377.