From gene to enzyme: Microbial engineering of bovine chymosin for efficient cheese coagulation.

Chymosin, an aspartic protease, specifically cleaves the Phe105-Met106 bond in κ-casein to initiate milk coagulation, making it indispensable for cheese production. Owing to ethical, economic, and supply limitations associated with calf-derived rennet, recombinant production in microbial hosts has become a preferred alternative. This study integrated molecular docking and molecular dynamics (MD) simulations with experimental heterologous expression to enhance structural understanding and recombinant production efficiency. MD simulation (20 ns) supported structural stability (RMSD ~ 0.4-0.5 nm; Rg 2.4-2.5 nm), while docking with κ-casein yielded a stable complex (lowest binding energy - 943.9 kcal/mol), consistent with catalytic functionality. A codon-optimized prochymosin gene (PylB promoter, YwbN signal peptide) was expressed in Escherichia coli BL21-CodonPlus (pET28a), Bacillus subtilis KO7 (pHFS-chy/PylB), and Bacillus thuringiensis 4Q7 (pPFS-chy/cyt). E. coli achieved high-level expression (40.61 kDa; optimized at 1.0 mM IPTG, 37 °C, 12 h), producing 800 OD₂₈₀ units of inclusion bodies from 5 L culture. In contrast, B. subtilis exhibited delayed and weak intracellular expression (~ 40.6 kDa at 120 h), and B. thuringiensis showed transient expression at 24 h. Inclusion bodies were solubilized with 95.25% recovery, refolded, purified via anion-exchange chromatography (~ 14.8% recovery), and activated by acid treatment (pH 2 → pH 6). The activated enzyme demonstrated milk-clotting activity comparable to the native counterpart (66.6 MCU/mL; 6 min clotting time). Collectively, these findings establish E. coli as an efficient platform for recombinant chymosin production, while indicating that Bacillus systems require further optimization for enhanced secretion and yield.