The aim of this study was to investigate covalent immobilization of horseradish peroxidase (HRP) on magnetic nanoparticles (Mag) encapsulated in calcium alginate beads (MABs) for color degradation, combining easy and fast removal of biocatalyst from the reaction mixture due to its magnetic properties and strong binding due to surface alginate functional groups. MABs obtained by extrusion techniques were analyzed by optical microscopy, FEG-SEM and characterized regarding mechanical properties, magnetization and HRP binding. HRP with initial concentration of 10 mg/g_carrier was successfully covalently bonded on MABs (diameter ~1 mm, magnetite/alginate ratio 1:4), with protein loading of 8.9 mg/g_carrier, immobilization yield 96.9% and activity 32.8 U/g. Immobilized HRP on MABs (HRP-MABs) was then used to catalyze degradation of two anthraquinonic dyes, Acid Blue 225 (AB225) and Acid Violet 109 (AV109), as models for wastewater pollutants. HRP-MABs decolorized 77.3% and 76.1% of AV109 and AB225, respectively after 15 min under optimal conditions (0.097 mM H₂O₂, 200 mg of HRP-MABs (8.9 mg/g_carrier), 0.08 and 0.1 g/mg beads/dye ratio for AV109 and AB225, respectively). Biocatalyst was used for 7 repeated cycles retaining 75% and 51% of initial activity for AB225 and AV109, respectively, showing potential for use in large scale applications for colored wastewater treatment.

Enzymatic hydrolysis of food proteins is convenient method to improve their functional properties and physiological activity. Herein, the successful covalent attachment of alcalase on alginate micron and submicron beads using the carbodiimide based chemistry reaction and the subsequent application of the beads for egg white and soy proteins hydrolysis were studied. In addition to the electrostatic extrusion technique (EE) previously used by others, the potential utilization of a novel ultrasonic spray atomization technique without drying (UA) and with drying (UAD) for alginate submicron beads production has been attempted. The immobilization parameters were optimized on microbeads obtained by EE technique (803 ± 23 µm) with respect to enzyme loading and alcalase activity. UA and UAD techniques resulted in much smaller particles (607 ± 103 nm and 394 ± 51 nm in diameter, respectively), enabling even higher enzyme loading of 671.6 ± 4 mg g⁻¹ on the carrier and the highest immobilized alcalase activity of 2716.1 IU g⁻¹ in the standard reaction. The UAD biocatalyst exhibited also better performances in the real food system based on egg white or soy proteins. It has been shown that the immobilized alcalase can be reused in seven successive soy protein hydrolysis cycles with a little decrease in the activity.

Enzymes immobilization can efficiently solve limitations of their large-scale application, such as stability and reusability. In this study, Alcalase? 2.4L (protease from Bacillus licheniformis) was covalently immobilized onto chitosan beads obtained by inverse emulsion technique using 1.5% (m/v) of chitosan and 0.67% (v/v) or 1.0% (v/v) of glutaraldehyde (CTPP (1.5/0.67) and CTPP (1.5/1.0)). Afterward, the beads were additionally crosslinked by immersion into 10 % (m/v) tripolyphosphate solution. The parameters studied were enzyme loading, enzyme coupling yield, bead diameter, SEM, biocatalyst activity, and FTIR. The beads had adequate enzyme loading and enzyme coupling yield (Pgmax was 117.1 mg/g dry CTPP 1.5/0.67 and 90.1 mg/g dry CTPP 1.5/1.0, and ?max was 96.7% for both carriers). CTPP (1.5/1.00) beads were smaller (diameter 635.2 ?25.2 mm wet/ 230.4?12.5 mm dry beads) and showed a higher specific activity of 20.1 ? 0.23 IU/mgprotein. The immobilized Alcalase? 2.4L was tested for hydrolyzing egg white and soy proteins. Alcalase? 2.4L, covalently attached to CTTP (1.5/1.0) chitosan beads, is a promising choice for industrial processes involving egg white protein hydrolysis, as the enzyme achieved a notable hydrolysis rate of 26.34 ? 0.879% after 195 minutes. Additionally, it remained effective through five successive applications under practical conditions (50?C, pH 8).

The formation of interfacial charge transfer (ICT) complexes between phenolic ligands and metal oxide surfaces enables surface functionalization strategies with potential applications in catalysis and bioconjugation. In this study, magnetite (Fe₃O₄) nanoparticles were modified with two phenolic ligands, 5-aminosalicylic acid (5ASA) and caffeic acid (CA), to generate ICT complexes capable of covalent or non-covalent enzyme immobilization, respectively. The modified nanomaterials were structurally characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FTIR). Horseradish peroxidase (HRP) was immobilized on these functionalized supports using varying nanoparticle amounts (10–30 mg) and initial enzyme concentrations (25–250 µg mL⁻¹). Catalytic activity was evaluated using pyrogallol oxidation assays. The Fe₃O₄/5ASA–HRP system exhibited a maximum activity of 2.5 U per 20 mg of support (approximately 125 U g⁻¹), whereas Fe₃O₄/CA showed minimal activity under the same conditions. Enzyme loading studies confirmed that 5ASA-enabled covalent attachment resulted in significantly higher immobilization efficiency (up to 1068 mg g⁻¹) compared to the CA system. Reusability tests demonstrated that the Fe₃O₄/5ASA system retained high absolute catalytic activity during the initial reaction cycles and consistently outperformed the non-covalently immobilized Fe₃O₄/CA system upon repeated reuse. The magnetic properties of Fe₃O₄ allowed rapid recovery of the biocatalysts using an external magnetic field. These results highlight the effectiveness of ICT-based functionalization for enzyme immobilization, positioning Fe₃O₄/5ASA as a promising platform for robust and reusable biocatalysts in environmental and industrial applications.

The formation of interfacial charge transfer (ICT) complexes between phenolic ligands and metal oxide surfaces enables surface functionalization strategies with potential applications in catalysis and bioconjugation. In this study, magnetite (Fe₃O₄) nanoparticles were modified with two phenolic ligands, 5-aminosalicylic acid (5ASA) and caffeic acid (CA), to generate ICT complexes capable of covalent or non-covalent enzyme immobilization, respectively. The modified nanomaterials were structurally characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FTIR). Horseradish peroxidase (HRP) was immobilized on these functionalized supports. Catalytic activity was evaluated using pyrogallol oxidation assays, with systematic variations in nanoparticle mass and enzyme concentration. The Fe₃O₄/5ASA–HRP system exhibited a maximum activity of 2.5 U per 20 mg of support (approximately 125 U/g), whereas Fe₃O₄/CA showed minimal activity under the same conditions. Data from enzyme loading studies confirmed that 5ASA-enabled covalent attachment resulted in significantly higher immobilization efficiency (up to 1068 mg/g) compared to the CA system. The magnetic properties of Fe₃O₄ allowed for rapid recovery of the biocatalysts using an external magnetic field. These results highlight the effectiveness of ICT-based functionalization for enzyme immobilization, positioning Fe₃O₄/5ASA as a promising platform for robust and reusable biocatalysts in environmental and industrial applications.