Poly(styrene- alt -maleic acid)-assisted Membrane Solubilization for Improved Immobilization and Catalytic Performance of Soybean Lipolytic Enzymes in Electrospun Poly(vinyl alcohol) Fibers

Efficient extraction and stabilization of plant-derived enzymes remain challenging due to their susceptibility to denaturation during processing. Soybean lipases, while exhibiting intrinsically high activity, lose functionality rapidly in the presence of salts, organic solvents, or elevated temperatures, thereby limiting their direct industrial use. To address these challenges, we developed a poly(styrene-alt-maleic acid) (PSMA)-assisted extraction and immobilization platform that simultaneously disrupts membranes and forms stable catalytic nanoparticles suitable for nanofiber fabrication. When applied to Glycine max (soybean) extracts, the PSMA-assisted process yielded the highest specific lipase activity of 16 mU/mg under optimized conditions (pH 7.5; mass-to-buffer volume ratio 1:25). Proteomic profiling identified 16 proteins showing significant abundance differences between conventional MOPS-buffered and PSMA/MOPS-assisted extractions, confirming selective stabilization of lipolytic enzymes. Morphological characterization revealed that the immobilized enzymes self-assembled into spherical, homogeneous nanoparticles with an average diameter of 227 nm. Incorporating 1% (w/v) of these nanoparticles into electrospun poly(vinyl alcohol) (PVA) fibers enhanced the enzyme activity by nearly 3-fold relative to the prespun solution, while maintaining comparable fiber size to the unloaded membranes (174 ± 65 nm vs 138 ± 31 nm, p > 0.05). By integrating the self-assembly behavior of PSMA with electrospun PVA nanofibers, this work demonstrates a scalable and effective route for preserving enzymatic function and fabricating ultrafine catalytic membranes for industrial biocatalysis.