Rational Engineering of Cellobiose 2-Epimerase Through Flexible Loop Modulation and Structure-Guided Sequence Alignment for Enhanced Lactulose Synthesis

Lactulose, a valuable functional disaccharide with pharmaceutical and food applications, is efficiently synthesized via enzymatic isomerization of lactose. This study developed an integrated strategy combining protein engineering of cellobiose 2-epimerase (CsCE) from Caldicellulosiruptor saccharolyticus and process optimization to enhance lactulose production. A dual-track engineering approach—incorporating flexible loop modulation (residues 161–193) and structure-guided sequence alignment with N-acetyl-D-glucosamine-2-epimerase—enabled the creation of two superior mutants, R17Q/L184S and R17Q/S142T. The R17Q/L184S variant exhibited a 37% increase in crude enzyme activity, improved thermostability (half-life of 200 min at 80 °C), and enhanced substrate affinity (Km reduced by 23.2%). R17Q/S142T achieved a 21% higher specific activity (24.08 U/mg), the highest among all variants. Structural and molecular dynamics analyses revealed that L184S enriched hydrogen bonding and hydrophobic interactions, improving structural rigidity, while S142T introduced allosteric regulation that facilitated catalytic efficiency. Under optimized conditions (70 °C, pH 7.5, 40% lactose, 20 U/mL enzyme, 3 h), lactulose yield reached 75.6% with >95% purity. This work demonstrates the successful application of synergistic enzyme engineering and process intensification for high-efficiency lactulose biosynthesis, providing viable candidates and system solutions for industrial-scale production.