Prolyl aminopeptidases (PAPs) are exopeptidases that cleave proline residues at the N-terminus of proteins or peptides and have wide applications in food and biomedical fields. Here, PAP was expressed recombinantly using Bacillus subtilis, and the batch fermentation conditions, including the agitation speed, pH and temperature, were systematically optimized based on a kinetic analysis. Finally, the agitation speed was controlled at 200 rpm from 0-6 h, 400 rpm from 6-12 h, 500 rpm from 12-28 h, and 400 rpm after 28 h. The pH was not adjusted during the first 12 h and from 16-28 h, and then, the pH was fixed at 7.0 from 12-16 h and after 28 h. The temperature was controlled at 40 degrees C for the first 8 h, then at 35 degrees C from 8-12 h, and at 33 degrees C after 12 h. The yield of PAP reached 174.8 U/mL under the optimized conditions, which was 1.66 times higher than that of the original production. The structure and storage stability of the lyophilized enzyme was significantly increased when protectants were added, and PAP together with alkaline protease and leucine aminopeptidase was used to hydrolyze rice protein. The amount of hydrophobic amino acids was significantly increased, which contributed to a reduction in the bitterness.