Hierarchical biocatalytic membranes embedded with trypsin–inorganic hybrid nanoflowers for effective β-lactoglobulin hydrolysis

Abstract

β-lactoglobulin (β-LG) is a significant allergen in milk. Enzymatic hydrolysis of β-LG produces peptides with low allergenicity and improved functionality. The biocatalytic membranes combine enzyme function and membrane separation in one step, but is still challenging for proteolysis. Herein, the biocatalytic membranes embedded with trypsin-inorganic hybrid nanoflowers are developed by a facile and green method for β-LG hydrolysis. Ethylene vinyl alcohol copolymer (EVAL) nanofibers are fabricated as 3D scaffolding on the membrane surface and trypsin-copper phosphate nanoflowers are in-situ grown in the porous structure of membranes. The nanoflowers are uniformly entrapped in the EVAL nanofiber layer and preferentially grow in the EVAL nanofibers layer, leading to the hierarchical structure of membranes. The trypsin loading of the nanoflowers membrane is approximate to 282.1 µg/cm2. Lower Michaelis-Menten constants Km prove the better affinity of Cu3(PO4)2-TR nanoflower membrane with substrates during filtration than that in static mode, even than free trypsin. The hierarchical structure of the membranes ensures both the enhanced trypsin-substrate contacts and low mass transfer resistance during the β-LG filtration. This work not only presents a novel concept of enzyme immobilized biocatalytic membranes, but also offers a sustainable solution for high-performance proteolysis.