Enhancing the Operational Stability of Recombinant Phenylalanine Ammonia-Lyase Immobilized on Magnetic Nanoparticles by Post-Entrapment

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.