氨肽酶YwaD的异源表达及其在玉米肽脱苦中的应用

Heterologous Expression of Aminopeptidase YwaD and Its Application in Debittering Zein Peptides

摘要:

目的 制备一种性能优良的新型氨肽酶，并初步评价其对玉米肽的脱苦能力。方法 选取地衣芽孢杆菌（Bacillus licheniformis，B. licheniformis）SQ1来源的氨肽酶YwaD在枯草芽孢杆菌（Bacillus subtilis，B. subtilis）WB600中进行表达，纯化后探究其酶学性质及其对玉米肽的脱苦效果。结果 氨肽酶YwaD均在B. subtilis WB600中分泌表达，其相对分子质量约为49 000。氨肽酶YwaD最适反应温度为80 ℃，半衰期为24 h，且在4.0 mol/L的NaCl条件下相对酶活力高于90%，表明该酶具有较强的耐热和耐盐性能；氨肽酶YwaD具有底物特异性，以Leu-pNA为底物时，K_m为0.093 mol/L，V_max为18.975 μg/（L·min）。与碱性蛋白酶单酶酶解法制备的玉米肽（ZA）相比，氨肽酶YwaD联合碱性蛋白酶分步双酶酶解法制备的玉米肽（ZAAP）的水解度增加了12.14%；苦味氨基酸质量浓度提高了4.85倍，鲜味氨基酸质量浓度提高了4.74倍；相对分子质量小于500的肽段占比增加了21.14%；DPPH自由基清除率增加了34.53%，羟基自由基清除率增加了16.59%；苦味值降低了约80%，鲜味值提高了约27%。结论 氨肽酶YwaD可将苦味肽N端疏水性氨基酸（多为苦味氨基酸）释放出来，显著去除玉米肽的苦味并增强其抗氧化性，该酶在功能性植物肽制备中有良好的应用前景。

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 K_m of 0.093 mol/L and the V_max 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.