Enzymatic hydrolysis of starch granules forms the fundamental basis of how nature degrades starch in plant cells, how starch is utilized as an energy resource in foods, and develops efficient, low-cost saccharification of starch, such as bioethanol and sweeteners. However, most investigations on starch hydrolysis have focused on its rates of degradation, either in its gelatinized or soluble state. These systems are inherently more well-defined, and kinetic parameters can be readily derived for different hydrolytic enzymes and starch molecular structures. Conversely, hydrolysis is notably slower for solid substrates, such as starch granules, and the kinetics are more complex. The main problems include that the surface of the substrate is multifaceted, its chemical and physical properties are ill-defined, and it also continuously changes as the hydrolysis proceeds. Hence, methods need to be developed for analyzing such heterogeneous catalytic systems. Most data on starch granule degradation are obtained on a long-term enzyme-action basis from which initial rates cannot be derived. In this review, we discuss these various aspects and future possibilities for developing experimental procedures to describe and understand interfacial enzyme hydrolysis of native starch granules more accurately.

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBD_St1 and SBD_St2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBD_GA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBD_GA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2–2.4-fold on amylose and decreased 3–9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3–1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBD_GA-TuαGT and-SBD_St2-TuαGT showed K_d of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBD_St1-TuαGT had 3–5-fold lower affinity. SBD_St2 contributed more than SBD_St1 to activity, substrate binding, and the stability of TuαGT fusions.

Interfacial enzyme catalysis is widespread in both nature and industry. Granular starch is a sustainable and abundant raw material for which a rigorous correlation of the surface structure with enzymatic degradation is lacking. Here pullulanase-catalyzed debranching of 12 granular starches varying in amylopectin contents and branch chain contents and lengths is shown to present a biphasic relationship characteristic of the Sabatier principle. Introducing normalization of the specific rate (v ₀/E ₀) by a substrate-dependent constant C, related to the Arrhenius prefactor of k _cat, reveals that optimal activity according to the Sabatier principle occurs at moderate substrate binding strength. The density of pullulanase attack sites (^kinΓ_max), determined using combined conventional and inverse Michaelis-Menten kinetics, was increased by branching enzyme treatment. Medium ^kinΓ_max and branch chain length conferred the highest activity depending on substrate load. Correlation analysis demonstrated that starch granular crystallinity, surface order, and average branch chain length influence the enzymatic degradation by affecting the C constant. Therefore, C should be considered together with the enzyme binding strength to understand the degradation of starch granules. The Sabatier principle could serve as a diagnostic tool to characterize enzyme performance on substrates having different surface structures and guide rational modification of granular starches for specific purposes.