Single‐Particle and Single‐Molecule Characterization of Immobilized Enzymes: A Multiscale Path toward Optimizing Heterogeneous Biocatalysts

Abstract

Heterogeneous biocatalysis is highly relevant in biotechnology as it offers several benefits and practical uses. To leverage the full potential of heterogeneous biocatalysts, the establishment of well‐crafted protocols, and a deeper comprehension of enzyme immobilization on solid substrates are essential. These endeavors seek to optimize immobilized biocatalysts, ensuring maximal enzyme performance within confined spaces. To this aim, multidimensional characterization of heterogeneous biocatalysts is demanded. In this context, spectroscopic and microscopic methodologies conducted at different space and temporal scales can inform about the intraparticle enzyme kinetics, the enzyme spatial distribution, and mass transport issues. In this minireview, we identify enzyme immobilization, enzyme activity, and enzyme inactivation as the three fundamental processes for which advanced characterization tools unveil fundamental information. Recent advances in operando characterization of immobilized enzymes at the single‐particle (SP) and single‐molecule (SM) levels reveal valuable information on their functional properties unlocking the full potential of heterogeneous biocatalysis toward biotechnological applications.