基于foldon介导的寡聚化以提高阿魏酸酯酶催化效率

日期：2021-11-29

信息摘要：

将foldon结构域与阿魏酸酯酶C-末端进行融合表达并用组氨酸标签对融合蛋白进行纯化。实现基于foldon的寡聚化阿魏酸酯酶及单体阿魏酸酯酶在毕赤酵母GS115中表达，并应用目标蛋白与foldon结构域融合可自发形成三聚体结构的特性对阿魏酸酯酶进行改造，以提高阿魏酸酯酶的催化性能。经纯化获得寡聚化及单体阿魏酸酯酶，寡聚化阿魏酸酯酶表观分子量约为110 kDa，单体阿魏酸酯酶表观分子量为40 kDa；寡聚化阿魏酸酯酶的适反应温度和pH分别为50 ℃和5.0，而单体阿魏酸酯酶则分别为50 ℃和6.0。

Abstract: A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 ℃, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency (k_cat/K_m) of the oligomerized FAE increased 7.57-folds. The catalytic constant (k_cat) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.

Keywords: feruloyl esterase oligomerization foldon catalytic efficiency enzyme engineering

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基于foldon介导的寡聚化以提高阿魏酸酯酶催化效率

日期：2021-11-29