The purpose of this study was to investigate the effect of pretreatments of heat (HP), ultrasound (UP), and combinations of heat/ultrasound (HPUP) and ultrasound/heat (UPHP) on protamex-catalyzed enzymolysis of corn gluten meal (CGM). Enzymolysis kinetics, molecular conformation and microstructure of CGM protein were monitored. Enzymolysis reaction rate increased with increasing substrate concentration and 80 g/L resulted in the highest hydrolysis reaction rate. The best improvement in CGM enzymolysis was obtained by HPUP (KM value decreased by 11.56%) and then followed by UPHP (KM value decreased by 9.97%). Fluorescence spectra and fourier transform infrared (FTIR) spectra indicated that the CGM pretreatments induced molecular unfolding, exposing the hydrophobic groups. HPUP and UPHP pretreatments both decreased the a-helix content by 12.2% and increased the random coil content by 2.23 and 2.60%, respectively. Scanning electron microscopy and atomic force microscopy showed that HPUP and UPHP changed surface topography and distribution of protein particles, enhancing the rate of enzymolysis. In conclusion, combined ultrasound and heat pretreatment and controlled enzymolysis could be an effective method for the functionality modification of globular proteins. (C) 2016 Elsevier Ltd. All rights reserved.