Protease prehydrolysis followed by subcritical water (SW) treatment was carried out to extract protein from heat-denatured soy meal. The composition, physicochemical, interfacial and emulsifying properties of soy protein isolate (SPI) were evaluated. A significant increase in extraction yield was observed although protein purity was decreased with prolonged hydrolysis. Due to contaminated beta-glucosidase in protease from Aspergillus oryzae, Maillard reaction and the conversion of isoflavone glycosides into aglycones occurred, leading to the enrichment of aglycones in SPI. Compared to native SPI, enzyme-assisted SW-prepared SPI exhibited higher hydrophobic amino acids, surface hydrophobicity, and interfacial adsorption due to protein unfolding, accompanied by the formation of small soluble aggregates. Additionly, remarkable improvements of emulsifying ability and physical stability of emulsion were probably associated with higher surface protein loads. These results could develop a feasible protocol for producing nutrient-enhanced soy proteins with excellent emulsifying properties as novel functional ingredients applied in food industry. (C) 2015 Elsevier Ltd. All rights reserved.