Polyethylenimine-Assisted Interfacial Modulation Based on Electrostatic Balancing and Hierarchical Channel to the Cytidine 5'-Monophosphate Conversion Performance of Uridine-Cytosine Kinase.

Interfacial modulation of protein microenvironments plays a pivotal role in enhancing enzyme immobilization and catalysis. In this study, we proposed a polyethylenimine (PEI)-assisted strategy that combines electrostatic and affinity interactions to improve the performance of Cytidine 5'-Monophosphate (CMP) conversion by uridine-cytidine kinase (UCK). The PEI-modified interface creates an optimal local microenvironment that maintains a balanced charge distribution, stabilizes UCK's conformation, and prevents denaturation. Electrostatic interactions promote product adsorption, enhance diffusion, and reduce substrate accumulation, boosting reaction efficiency. The kinetic assays revealed an increase in the maximum reaction rate from 16.8 to 113.2 μM·min with a remarkable increase in substrate affinity and enzyme activity. The relative enzyme activity at the optimal substrate concentration increased from 70.4 to 106.9%, and by 113.3% under conditions of substrate inhibition. This study provides theoretical and technical support for the efficient production of CMP with promising applications in food, feed, and medical fields.