沙质微泡菌脯氨酰寡肽酶的异源表达及酶学性质

Heterologous expression and enzymatic characterization of a prolyl oligopeptidase from Microbulbifer arenaceous

摘要:

【背景】 脯氨酰寡肽酶是一类能在脯氨酸残基羧基侧水解多肽的丝氨酸蛋白酶，在食品工业领域有重要的应用价值，但现有酶种类少，产量水平低。【目的】 在大肠杆菌中异源表达沙质微泡菌(Microbulbifer arenaceous)肽酶S9家族脯氨酰寡肽酶基因(mapop)，研究其相关酶学性质。【方法】 MaPOP在大肠杆菌中异源表达，经亲和层析纯化后测定酶学性质，制备血管紧张素转换酶(angiotensin converting enzyme, ACE)抑制肽。【结果】 一步纯化获得了重组酶MaPOP。MaPOP最适反应pH和温度分别为7.5和37 ℃，在pH 3.5-11.0和低于40 ℃时保持稳定；在1.5 mol/L NaCl浓度时具有最高的酶活力，是无NaCl时的2倍；丝氨酸肽酶抑制剂苯甲基磺酰氟(phenylmethanesulfonyl fluoride, PMSF)和Co²⁺、Hg²⁺、Zn²⁺对MaPOP具有抑制作用。MaPOP特异性识别寡肽内部的Pro，切割L-Pro羧基侧的肽键，而不能切割D-Pro羧基侧和Pro-Pro之间的肽键。MaPOP分别与风味蛋白酶、中性蛋白酶、AopepA、AoproS8复合水解罗非鱼鱼鳞，ACE抑制率为55.85%-87.71%。【结论】 MaPOP的异源表达和酶学性质为MaPOP在食品工业中的应用提供了理论支持。

Abstract:

[Background] Prolyl oligopeptidases are serine proteases that cleave peptide bonds at the carboxyl side of proline. They have important applications in the food industry. However, the available prolyl oligopeptidases are limited, and their production levels are low. [Objective] To investigate the enzymatic properties of the peptidase family S9 prolyl oligopeptidase (MaPOP) from Microbulbifer arenaceous. [Methods] MaPOP was heterologously expressed in Escherichia coli. After purification by affinity chromatography, MaPOP was characterized and used for the preparation of ACE inhibitory peptides. [Results] MaPOP demonstrated the highest activity at pH 7.5 and 37 ℃. It was stable in the range of pH 3.5-11.0 and at temperatures up to 40 ℃. In the presence of 1.5 mol/L NaCl, MaPOP showed the highest enzyme activity, which was twice that in the absence of NaCl. The serine protease inhibitor PMSF and metal ions Co²⁺, Hg²⁺, and Zn²⁺ showed inhibitory effects on MaPOP. MaPOP specifically recognized proline residues in oligopeptides and cleaved the peptide bonds at the carboxyl side of internal L-prolines rather than those at the carboxyl side of D-prolines or between two proline residues. MaPOP was used in combination with flavor protease, neutral protease, AopepA, and AoproS8 to hydrolyze tilapia scales, achieving ACE inhibition rates of 55.85%-87.71%. [Conclusion] The heterologous expression and enzymatic characterization of MaPOP provide theoretical support for its application in the food industry.