Mechanistic insights into substrate-targeting modification effects on interfacial kinetics of raw starch-degrading amylases

Current research on interfacial enzyme catalysis of starch granules largely overlooks interactions with molecules of specific spatial conformations. Here, we demonstrated that the CBM20-truncated mutant AmyM-TR2 exhibited over 30-fold greater hydrolysis efficiency toward starch granules compared to wild-type AmyM, with a shifted preference toward amylopectin. Molecular docking revealed that the exposed domain C in AmyM-TR2 enables binding to double-helical α-glucan molecules by three key residues (R351, W374, and W392). Interfacial catalytic analysis revealed that the discrepancy of AmyM and AmyM-TR2 in hydrolysis efficiency toward starch granules was related to substrate-targeting specificity and corresponding targeted productive binding sites (PBSs). AmyM-TR2 demonstrated a superior sustained catalytic capacity toward its targeted PBSs within multi-component starch. The processive catalytic capacity of AmyM-TR2 toward amylopectin double helices in starch granules is the fundamental determinant of its highly efficient degradation, providing new insights for interfacial catalytic mechanisms and modification of interface enzymes.IMPORTANCEInterfacial enzyme reactions are ubiquitous in both natural processes and industrial applications. Understanding the mechanistic aspects of enzymatic action on insoluble substrates is crucial for advancing sustainable biomass conversion and waste treatment technologies. Starch granule degradation mediated by raw starch-digesting enzymes (RSDEs) is a classical interfacial enzymatic reaction. The mechanism of RSDEs on starch granules is attractive, as it effectively circumvents the high energy consumption and viscosity challenges associated with conventional high-concentration starch hydrolysis processes. In this study, we demonstrate interfacial catalysis in starch granule depolymerization by substrate-targeting modification in raw starch-degrading amylase AmyM by exposing novel starch-binding modules, thus establishing a critical link between catalytic characteristics of amylase and starch substrate properties, which provides new insight for the contributions to insoluble substrate degradation within heterogeneous systems.