Abstract

Psychrophilic cellobiose 2-epimerase (CE) is a promising biocatalyst for cold-process dairy manufacturing, yet its industrial application is hampered by the inherent trade-off between low-temperature activity and substrate affinity. To overcome this limitation, a semi-rational design strategy was implemented on a psychrophilic CE (psyCE). A GFP-based fluorescence assay was developed to normalize mutant expression, enabling precise identification of activity improvements. Structure analysis pinpointed six key residues for targeted mutagenesis, and a saturation mutagenesis library was constructed accordingly. Screening of the library yielded a robust double mutant (FR) with superior kinetics. The FR variant exhibited a record-breaking catalytic turnover (245.7 s −1 vs. 219.9 s −1 ) and an improved substrate affinity (208.9 mM vs. 261.9 mM), resulting in a 40% increase in overall catalytic efficiency. In a practical biocatalysis trial at 8 °C, FR achieved a 21% epilactose yield within 3 h, outperforming the wild type (20% within 4 h). Molecular dynamics simulations revealed the structural basis of this enhancement by showing the FR mutant evolves an enlarged binding pocket, while simultaneously establishing a strengthened hydrogen-bond network. This study not only provides a highly efficient cold-active biocatalyst but also establishes a screening platform for engineering psychrophilic enzymes.