MOs, defined as starch-derived oligosaccharides containing 3–10 glucose units connected by α-(1–4) glycosidic linkages (Yip et al., 2024) have emerged as multifunctional biomaterials with expanding applications in food technology and pharmaceutical formulations (Shinde and Vamkudoth, 2022, Jaafar et al., 2021). Their commercial significance stems from a unique combination of physicochemical properties including moderate sweetness (approximately 60% sucrose equivalence), thermal stability, low hygroscopicity, and enhanced solubility compared to mono- and disaccharides (Jaafar et al., 2021, Xie et al., 2020). Beyond their role as natural sweeteners; MOs demonstrate remarkable functional versatility: (i) as bio-preservatives in processed foods through moisture regulation (Shinde and Vamkudoth, 2022); (ii) as prebiotic agents modulating gut microbiota composition (Vaze et al., 2024, Hashim, 2020); and (iii) as molecular scaffolds in diagnostic applications (e.g., maltoheptaose (G7) in diagnostics for recognizing protein and cell markers) (Shinde and Vamkudoth, 2022). Recent advancements have further extended their utility to cosmetic formulations and specialty chemical production (Hashim et al., 2005, Rodríguez Gastón et al., 2012).

However, the production process of MOs is complex and can be economically challenging (Yip et al., 2024, Shinde and Vamkudoth, 2022). Since the beneficial activity of MOs is closely linked to their chemical structure and DP, it is crucial to obtain MOs from starch with specific and precise DP values to achieve the desired beneficial effects (Zhong et al., 2025). Current production paradigms employ two complementary enzymatic approaches: MOS-forming amylases and CD-hydrolyzing enzymes (Ji et al., 2024).

CDs, cyclic oligosaccharides comprising six (α-CD), seven (β-CD), and eight (γ-CD) glucose units linked via α-(1–4) glycosidic bonds, represent ideal precursors for MOs synthesis (Li et al., 2024). Produced through cyclodextrin glycosyltransferases (EC 2.4.1.19) starch cyclization (Li et al., 2024); CDs combine thermal stability with amphiphilic architecture—a hydrophilic exterior surface enclosing hydrophobic cavity (Rodríguez Gastón et al., 2012, Hamilton et al., 2000). These characteristics underpin their widespread use as molecular encapsulation agents in drug delivery systems and food additives (Rodríguez Gastón et al., 2012). Recent process innovations have significantly enhanced CD production efficiency (Biwer et al., 2002); positioning enzymatic CDs ring-opening as a sustainable route to high-value MOs (maltohexaose to maltooctaose (G8)) with precise chain lengths (Yang et al., 2006).

Because the glycosidic bond oxygens of CDs are buried within their structure, the number of enzymes exhibiting catalytic activity toward CDs is limited, significantly fewer than those that act on starch (Li et al., 2024). Currently, only a few enzymes demonstrate significant ability to hydrolyze CDs (Li et al., 2024), including certain α-amylases (EC 3.2.1.1) (Wang et al., 2020), neopullulanases (EC 3.2.1.135), amylopullulanases (EC 3.2.1.1/41) (Li et al., 2013, Li et al., 2018, Li and Li, 2015), maltogenic amylases (EC 3.2.1.133) (Rahmati et al., 2017, Li et al., 2018), and cyclodextrinase (EC 3.2.1.54) (Sun et al., 2015, Ji et al., 2020). The GH57 family amylopullulanases have been characterized for their activity in hydrolyzing CDs (Li et al., 2013, Li and Li, 2015, Peng et al., 2021); demonstrating significant potential for the preparation of MOs through the ring-opening reaction of CDs.

Understanding enzymes-substrates-products interactions represents a critical frontier in biocatalysis, with particular relevance to: (i) activity modulation, (ii) mechanistic elucidation, and (iii) rational enzyme engineering (Roda et al., 2020). AaApu is an amylopullulanase from the GH57 family (Peng et al., 2021). This study combines biochemical and structural approaches to decipher the molecular mechanism of interactions between CDs as inhibitor or substrate with AaApu. Through comparative analysis of wild-type and D352N mutant (acid/base catalyst variant) complexes with all three CD types, we reveal the molecular determinants governing substrate specificity and competitive inhibition in GH57 amylopullulanases.