The enhancement mechanisms of chondroitin sulfate on α-amylase activity: Exploring the interaction using in vitro and in silico studies

Abstract

Glycosaminoglycans (GAG) are bioactive polysaccharide rich in -SO3- and -COO- groups, also known as acidic mucopolysaccharides. In this study, the feasibility of three structurally distinct forms of chondroitin sulfate (CS-A, CS-C, and CS-D) from the GAG family was explored as a potential strategy to enhance industrial α-amylase activity. All three CSs were found to increase α-amylase activity to varying degrees, with CS-D showing the most significant increase, exceeding 78 %. Furthermore, fluorescence quenching experiments indicated that the interaction between CS and α-amylase is primarily driven by hydrophobic interactions. In silico, molecular docking revealed that the sulfate groups of all three CSs form hydrogen bonds with α-amylase, with CS-D exhibiting the lowest binding energy due to its two sulfate groups. Kinetic simulations further suggested that binding to CS increases the flexibility of key active site residues (Asp197, Glu233, and Asp300), modifies the secondary structure, and enlarges the substrate-binding pocket, thereby promoting α-amylase's hydrolytic activity. Thus, this work revealed CS as an α-amylase activator and further elucidated its interaction mechanism using in vitro and in silico studies, which may be beneficial to apply CS in pharmaceutical or food industry.