Understanding the\nEffect of Ionic Liquid–Mediated\nSolvent Engineering on the Kinetics and Thermodynamic Stability of\nPhenylalanine Ammonia-Lyase

Phenylalanine ammonia-lyase (PAL) plays a central role\nin the phenylpropanoid\npathway and in the treatment of phenylketonuria. However, the integration\nof PAL into sustainable industrial biocatalysis is hampered by its\ninstability under harsh conditions. This study demonstrates that ionic\nliquid (IL)–assisted solvent (Tris-HCl buffer) engineering\nenables improvement of the reaction kinetics and thermodynamic stability\nof Rhodotorula glutinisPAL (<i>Rg</i>PAL) under various stresses. Under optimized conditions,\na 66.2% higher K<sub>cat</sub> value, >60% remaining activity after\n5 weeks of storage at room temperature, and >80% activity of <i>Rg</i>PAL after incubation at 60 °C for 1 h were obtained\nin the [Ch][Ac]-blended Tris-HCl solvent compared to pristine Tris-HCl.\nThe spectroscopic and molecular docking results suggest that the higher\nextent of hydration and the soft interactions complemented by the\nILs with the D-chain residues of <i>Rg</i>PAL jointly contributed\nto achieving more stable and active conformations of <i>Rg</i>PAL. The enzyme showed a higher melting temperature (<i>T</i><sub>m</sub>) in ILs+Tris-HCl compared to that in pristine Tris-HCl,\nwith less change in enthalpy (Δ<i>H</i><sub>fu</sub>) and entropy (Δ<i>S</i><sub>fu</sub>) of unfolding.\nOverall, IL-mediated solvent engineering alters the microenvironment\nof <i>Rg</i>PAL and allows the development of a robust PAL-based\nbiocatalytic system.