Herewith we report the first attempt towards non-covalent immobilization of Trametes versicolor laccase on neat and magnetically responsive highly porous polyamide 6 (PA6) microparticles and their application for catechol oxidation. Four polyamide supports, namely neat PA6 and such carrying Fe, phosphate-coated Fe and Fe₃O₄ cores were synthesized in suspension by activated anionic ring-opening polymerization (AAROP) of ε-caprolactam (ECL). Enzyme adsorption efficiency up to 92% was achieved in the immobilization process. All empty supports and PA6 laccase complexes were characterized by spectral and synchrotron WAXS/SAXS analyses. The activity of the immobilized laccase was evaluated using 2,2’-Azino-bis-(3- ethylbenzothiazoline-6-sulfonic acid (ABTS) and compared to the native enzyme. The PA6 laccase conjugates displayed up to 105% relative activity at room temperature, pH 4, 40 °C and 20 mM ionic strength (citrate buffer). The kinetic parameters of the ABTS oxidation were also determined. The reusability of the immobilized laccase-conjugates was proven for five consecutive oxidation cycles of catechol.

The use of free pectinases as clarification biocatalysts constitutes a well-established practice in the large-scale production of various types of wines. However, when in the form of free enzymes, the recovery and reusability of pectinases is difficult if not impossible. To address these limitations, the present study focuses on the noncovalent adsorption immobilization of a commercial pectinolytic preparation onto highly porous polyamide 6 (PA6) microparticles, both with and without magnetic properties, prepared via activated anionic polymerization. The two pectinase complexes resulting after immobilization underwent comparative activity and kinetic studies, contrasting them with the free enzyme preparation. In comparison with the free enzyme, the PA6-immobilized pectinase complexes exhibited more than double the specific activity toward the pectin substrate. They displayed a slightly higher affinity to the substrate while acting as faster catalysts that were more resistant to inhibition. Furthermore, the immobilized complexes were applied in the clarification process of industrial rosé must, whereby they demonstrated accelerated performance as compared with the free enzyme. Moreover, the PA6-immobilized pectinase biocatalysts offered the potential for three consecutive cycles of reuse, achieving complete rosé must clarification within relevant timeframes in the range of 3–36 h. All these results suggest the potential industrial application of the pectinases noncovalently immobilized upon PA6 microparticles.

Free pectinase is commonly employed as a biocatalyst in wine clarification; however, its removal, recovery, and reuse are not feasible. To address these limitations, this study focuses on the immobilization of a commercial pectinolytic preparation (Pec) onto highly porous polymer microparticles (MPs). Seven microparticulate polyamide (PA) supports, namely PA4, PA6, PA12 (with and without magnetic properties), and the copolymeric PA612 MP, were synthesized through activated anionic ring-opening polymerization of various lactams. Pectinase was non-covalently immobilized on these supports by adsorption, forming Pec@PA conjugates. Comparative activity and kinetic studies revealed that the Pec@PA12 conjugate exhibited more than twice the catalytic efficiency of the free enzyme, followed by Pec@PA6-Fe and Pec@PA4-Fe. All Pec@PA complexes were tested in the clarification of industrial rosé must, demonstrating similar or better performance compared to the free enzyme. Some immobilized biocatalysts supported up to seven consecutive reuse cycles, maintaining up to 50% of their initial activity and achieving complete clarification within 3–30 h across three consecutive cycles of application. These findings highlight the potential for industrial applications of noncovalently immobilized pectinase on various polyamide microparticles, with possibilities for customization of the conjugates’ properties.

Free pectinase is commonly employed as a biocatalyst in wine clarification; however, its removal, recovery, and reuse are not feasible. To address these limitations, this study focuses on the immobilization of a commercial pectinolytic preparation (Pec) onto highly porous polymer microparticles (MP). Seven microparticulate polyamide (PA) supports, namely PA4, PA6, PA12 (with and without magnetic properties), and the copolymeric PA612 MP, were synthesized through activated anionic ring-opening polymerization of various lactams. Pectinase was noncovalently immobilized on these supports by adsorption, forming Pec@PA conjugates. Comparative activity and kinetic studies revealed that the Pec@PA12 conjugate exhibited more than twice the catalytic efficiency of the free enzyme, followed by Pec@PA6-Fe and Pec@PA4-Fe. All Pec@PA complexes were tested in the clarification of industrial rosé must, demonstrating similar or better performance compared to the free enzyme. Some immobilized biocatalysts supported up to seven consecutive reuse cycles, maintaining up to 50% of their initial activity and achieving complete clarification within 3-30 hours across three consecutive cycles of application. These findings highlight the potential for industrial application of noncovalently immobilized pectinase on various polyamide microparticles, with possibilities for customization of the conjugates' properties.

Lyophilized pectinase from Aspergillus niger (PeL) was immobilized onto polyamide 6 (PA6) microparticles (MPs) through an adsorption-based procedure within the pH range of 5–8, yielding four PeL@PA6 complexes. In contrast to commercial enological preparations that are complex enzymatic cocktails with unspecified exact compositions, the use of PeL with known specific activity enabled a more reliable evaluation and improvement of the immobilization process and of the structure–activity relationships of the resulting biocatalysts. Thermogravimetric analysis demonstrated better thermal stability of the PeL@PA6 complexes compared to neat PA6 MPs. UV-CD studies revealed that the secondary structure and conformational stability of PeL before and after immobilization were strongly pH-dependent, with maximum stability observed at pH 6–7. All four PeL@PA6 complexes retained significant catalytic activity and showed good tolerance to ethanol-rich media relevant to enological applications. Kinetic analysis indicated increased apparent K_m values after immobilization, suggesting diffusional limitations associated with the porous PA6 support. Clarification experiments of industrial white and rosé wine musts confirmed the practical applicability of the immobilized system. All PeL@PA6 complexes preserved the color and phenolic integrity of the musts, displayed good operational stability during reuse, and exhibited higher long-term storage stability than the free enzyme. These results demonstrate that PA6 MPs are promising supports for pectinase immobilization in wine clarification and related biotechnological applications.