Interactions of Polydopamine\nNanoparticles with Serine\nand Cysteine Proteases: Implications for Enhancing Protein Stabilization\nand Enzyme Activity

The interactions between polydopamine nanoparticles (PDA\nNPs) with\nserine protease α-chymotrypsin (α-CT) and cysteine protease\nstem bromelain (SB) have prompted significant emphasis due to their\npotential implications in biocatalysis and therapeutic applications.\nThis study addresses the structural integrity, binding kinetics, and\ncatalytic potential of α-CT and SB in the presence of PDA NPs\nby employing a combination of spectroscopic techniques and thermodynamic\nand kinetic analyses. The spectroscopic results reveal that PDA NPs\nbind to both enzymes (α-CT and SB) to create a stable complex\nwithout disrupting their active sites. Furthermore, fluorescence,\ncircular dichroism (CD), and dynamic light scattering (DLS) analysis\nclearly demonstrate that the binding between α-CT and PDA NPs\nis strong compared to SB + PDA NPs. The thermodynamic data implies\nthat PDA NPs significantly enhance the thermal stability of both α-CT\nand SB, as evidenced by the increased thermal transition temperature\n(<i>T</i><sub>m</sub>) by 2.68 ± 0.50 °C in α-CT\nand 3.35 ± 0.50 °C in SB. Interestingly, ζ-potential\nand microscopic results such as TEM and FESEM pointed toward the adsorption\nof both proteases over the surface of PDA NPs. Additionally, spectrophotometric\nassay and kinetic studies confirm the retention of enzyme activity\nof both proteases upon conjugation with PDA NPs. Further, kinetic\n(<i>V</i><sub>max</sub>, <i>K</i><sub>m</sub>)\nand thermodynamic (Δ<i>G</i><sub>u</sub>, Δ<i>H</i><sub>m</sub>, Δ<i>S</i><sub>m</sub>, ΔC<sub>p</sub>) parameters also concluded the formation of a stable enzyme–NP\ncomplex. This study conveys the potential of PDA NPs in developing\nadvanced enzyme–NPs integrated for enhancing protein stability\nand activity in diverse biomedical and industrial applications.