One-Step Activation, Purification, and Immobilization of Bovine Chymosin via Adsorption on Magnetic Particles

Chymosin is an aspartyl protease widely used in the food industry for milk coagulation during cheesemaking. Although recombinant production has replaced natural extraction from rennet, current heterologous expression systems still face significant challenges, including low solubility, costly purification steps, and enzyme instability after activation. To address these limitations, we sought to develop a more efficient and economical production strategy for bovine chymosin by cloning its codon-optimized prochymosin A gene into Escherichia coli using the pBAD/His vector under the control of the L-arabinose-inducible PBAD promoter. Overexpression of the recombinant gene resulted in the formation of inclusion bodies, which were solubilized with NaOH and refolded by dilution and pH adjustment with glycine. The folded prochymosin was then activated by acidification. To simplify the downstream process and improve enzyme recovery, different immobilization strategies were explored to combine activation, purification, and immobilization in a single step. While polymeric agarose-based supports showed low immobilization efficiency (<20%) due to pore clogging, magnetic nanoparticles completely overcame these limitations, achieving nearly 100% immobilization yield and retaining about 85% of enzymatic activity. This integrated magnetic-based approach provides a cost-effective and scalable alternative for the production and stabilization of active chymosin.