The preparation of antioxidant starch with the activity of glutathione peroxidase (GPx) for scavenging free radicals can not only enrich the types of modified starch but also alternate native GPx to overcome its drawbacks. In this work, an antioxidant cassava starch (AO-ca-starch) was prepared by the sequential esterification and selenylation of cassava starch. The process was optimized based on the selenium content. Various characterizations for the AO-ca-starch indicated that the catalytic center of GPx, the selenium, was anchored on the starch. The catalytic activity of AO-ca-starch, a starch-based biomimetic GPx, was about 4.95 × 105 times higher than that of the typical artificial selenoenzyme (diphenyl diselenide, PhSeSePh), and it exhibited a typical saturation kinetic catalytic behavior. The surface changes of the starch during the modification were conducive to the formation of hydrophobic microenvironments, which played an important role in the catalytic reaction of biomimetic GPx due to the binding of the hydrophobic substrates. The match of the catalytic center and the hydrophobic microenvironments was the key factor for maintaining the high catalytic activity of AO-ca-starch. Without cytotoxicity, AO-ca-starch exemplified a new and promising modified starch as a selenium-enriched functional food and antioxidant drug.
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