Heterogeneous biocatalysis by magnetic nanoparticle immobilized biomass-degrading enzymes derived from microbial cultures

Abstract

Recombinant enzymes have become increasingly popular and are frequently used as environmentally safe biocatalysts due to their wide range of applications and high specificity. Purifying these enzymes from the host cells, media, and other contaminants is essential for their characterization and applications. The widely utilized method for protein purification by nickel–nitrilotriacetic acid (Ni–NTA) resin-based affinity chromatography is a time-consuming, labor-intensive, and resource-demanding technique. In this study, we synthesized NTA–Ni@Fe₃O₄ nanoparticles (NPs) to capture enzymes from cell lysates and microbial culture media and developed a model system to show the efficacy of immobilizing and recycling biomass-degrading enzymes known as cellulases. Cellulases, which play an important role in biomass degradation and biofuel production, were baited with NTA–Ni@Fe₃O₄ NPs and purified in a single step. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicates efficient purification of the immobilized enzymes on the NPs from the cell lysate and extracellular media. Additionally, we successfully showed that cellulase-immobilized NTA–Ni@Fe₃O₄NPs can serve as a heterogeneous catalyst for the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (pNPGlc) and carboxymethylcellulose (CMC). The NTA–Ni@Fe₃O₄ NPs immobilized with enzymes showed recyclability for up to five cycles. The applications of this methodology may be extended to various industries requiring efficient enzyme purification and recycling for promising advancements in biotechnology and sustainable biomanufacturing processes.