Abstract:Objective The aim of this study was to prepare a novel aminopeptidase with excellent properties and evaluate its performance in debittering zein peptides.Method Aminopeptidase aminopeptidase YwaD derived from Bacillus licheniformis (B. licheniformis) SQ1 was expressed in Bacillus subtilis (B. subtilis) WB600, and the enzymatic properties and debittering effect on zein peptides were investigated after purification.Result Aminopeptidase YwaD was expressed in a secretory form in B. subtilis WB600, with the relative molecular mass of about 49 000. Aminopeptidase YwaD showed the optimum reaction temperature of 80 ℃, the half-life of 24 h, and the relative activity higher than 90% in the presence of 4.0 mol/L NaCl, which indicated that aminopeptidase YwaD had strong tolerance to high temperatures and salt. Aminopeptidase YwaD had substrate specificity, with the Km of 0.093 mol/L and the Vmax of 18.975 μg/(L·min) in the case of Leu-pNA as the substrate. Compared with zein peptides produced by single-enzyme hydrolysis using alkaline protease alone (ZA), the hydrolysis degree of zein peptides produced by aminopeptidase YwaD combined with alkaline protease in two steps (ZAAP) increased by 12.14%, the mass concentration of bitter amino acids by 4.85 times, the mass concentration of umami amino acids by 4.74 times, the proportion of peptides with molecular mass less than 500 by 21.14%, the DPPH free radical scavenging rate by 34.53%, and the hydroxyl radical scavenging rate increased by 16.59%, while decreasing the bitter value by approximately 80% and increasing the umami value by 27%.Conclusion Aminopeptidase YwaD could release hydrophobic amino acids (mostly bitter amino acids) at the N-terminus of bitter peptides to significantly remove the bitterness and enhance the antioxidant activity of zein peptides, demonstrating a good application prospect in the preparation of functional plant peptides.
