Efficient biomanufacturing of vitamin E succinate in non-aqueous media via synergistic computational evolution and immobilization of lipase.

Biocatalysts exhibit broad application prospects in industrial production. However, the inherent instability of natural enzymes in non-aqueous media severely limits their practical utilization. Using Candida rugosa lipase as a model, a synergistic evolution strategy integrating ancestral sequence reconstruction, free energy optimization and surface hydration shell modulation was developed in this study. The optimal mutant CM5 exhibited simultaneously enhanced stability and activity, showing a 4.78-fold increase in tolerance to 65% (v/v) dimethyl sulfoxide, a 1.67-fold extension in half-life at 50 ℃ and a 2.30-fold increase in activity compared to the wild type. Molecular dynamics simulations indicated that the enhanced stability of CM5 originates from improved structural rigidity and the stabilized hydration shell. Meanwhile, its increased activity is attributed to a reduced energy barrier for the lid conformational transition and an optimal enzyme substrate affinity that significantly boosts substrate turnover efficiency. Moreover, a volumetric activity of 142.71 U/mL in a 5 L bioreactor was obtained via optimization of the chassis cells, and the resulting culture was then used for the immobilization. The lipase CM5 immobilized on amino octadecyl bifunctional macroporous resin (CM5@AOMR) achieved a vitamin E succinate yield of > 99.9% and maintained an outstanding yield of 98.2% even after ten consecutive batch reactions. Overall, this study developed a comprehensive workflow from computational design and efficient fermentation to immobilization and practical applications of lipase, providing a promising new approach for developing next generation biocatalysts suited for extreme industrial environments.