Abstract: The combination of flocculation, ultrafiltration, and freeze drying method to extract the recombinant bacillus subtilis leucine aminopeptidase, with aminopeptidase removal terminal hydrophobic amino acids for debittering effect index, debittering effect effects of leucine aminopeptidase and proline aminopeptidase hydrolysis of rice peptide was investigated.The preparation conditions of the enzyme were as follows: adding 0.15% ± 0.008% flocculant, 2.5% ± 0.006% diatomite, adjusting p H8.0 for filtration, using 30 k Da PES roll membrane, ultrafiltration concentration 7 times, under this condition: the total recovery of aminopeptidase rate was 64.69% ± 1.29%. In the optimized enzymatic hydrolysis conditions, the total amount of leucine, arginine and free hydrophobic amino acids in the hydrolyzate were 3.39 ± 0.10, 16.48 ± 0.49 and 4.39 ± 0.13 times higher than that of the hydrolyzate.After the enzymolysis, the molecular weight was 500¹000 Da was reduced by 30.63% ± 0.61%, and the content of proline was 1.78 ± 0.07 times higher than that of single enzyme hydrolysis after double enzyme synergistic hydrolysis.The optimized process is simple and the enzyme has good application in the peeling of rice peptides.