One-point covalent immobilization of a xylanase on elastomer: effects of surface stretching on enzymatic activity

• A two-step, highly selective protein immobilization method based on the Jo-In biomolecular welding system. In step 1, we take advantage of the mild conditions and selectivity of thiol-maleimide reaction to immobilize the Jo protein. In step 2, we bind the enzyme to the surface by using In-xylanase fusion protein. Importantly, we demonstrate that enzymatic activity is preserved comparable to free enzyme solutions. • Single molecule atomic force microscopy is used to evaluate the mode of binding of proteins to the surface. • A custom device for stretching surfaces to modulate enzyme spatial relationships. Protein immobilization on solid surfaces is a prevalent strategy in biotechnology and industry, offering advantages in terms of stability and reusability. Various methods, including covalent attachment, adsorption, covalent cross-linking, and entrapment, have been developed, with challenges in adapting these techniques to enzymes due to their catalytic pockets. In a previous study, we introduced a method for grafting the glycoside hydrolase Neocallimastix patriciarum endo -β-1,4-xylanase ( Np Xyn11A) onto paramagnetic beads, preserving its biological activity. Here, we extend this approach to polymeric surfaces, specifically polyisoprene (PI) and polydimethylsiloxane (PDMS), known for their elasticity. The surfaces are plasma-activated and functionalized with maleimide functions to immobilize the engineered protein Jo through a one-point thiol-maleimide click reaction. In a second step, the xylanase fused to the protein In is immobilized on the membrane by a spontaneous and specific covalent bond between Jo and In. A custom device is employed to stretch the elastomer surfaces, altering the distance between immobilized enzymes. The specific activity of the immobilized xylanase, evaluated with a chromogenic substrate, mirrors that of the free enzyme in solution. However, the use of natural polysaccharides reveals activity variations based on the extent of surface stretching, providing insights into the impact of spatial proximity on enzyme dynamics. Our study contributes to overcoming limitations in traditional enzyme immobilization, offering valuable insights into the dynamics of multi-enzymatic complexes.