Enzymatic preparation and functional development of agaro-oligosaccharides: Advances in structure-function relationships and application challenges.

Agaro-oligosaccharides (AGOS), derived from agar depolymerization, are marine-derived functional carbohydrates with diverse structures and promising bioactivities, including antioxidant, anti-inflammatory, and metabolic regulatory effects. Enzymatic degradation is an attractive strategy for AGOS production due to its mild conditions, high selectivity, and ability to preserve functional groups. However, the rational preparation of structurally defined AGOS remains challenging because product profiles are determined by complex interactions among agarase catalytic architecture, substrate recognition, substrate properties, and reaction conditions. This review summarizes the catalytic characteristics of agarases and their roles in determining AGOS structural diversity. We then discuss the challenges in establishing reliable structure-function relationships, as many reported bioactivities remain based on phenotypic observations and lack direct mechanistic validation. Finally, we highlight key barriers limiting AGOS applications, including scalable production, quality control, gastrointestinal fate, and human validation. An enzyme-structure-function framework is proposed to guide the rational production and application of structurally defined AGOS.