黑芝麻多酚氧化酶的重组表达及其酶学特性

Recombinant expression of black sesame polyphenol oxidase and its enzymatic properties

摘要:

为明确黑芝麻多酚氧化酶的酶学性质，利用大肠杆菌Escherichia coli原核表达了黑芝麻多酚氧化酶 (Black sesame polyphenol oxidase，BsPPO)。将合成的基因构建至pMAL-c5x载体，并在大肠杆菌中进行表达，对重组蛋白进行分离纯化及融合标签切除，获得的BsPPO蛋白用于酶学性质探究。结果表明，合成的Bsppo基因1 752 bp，编码585个氨基酸，理论蛋白分子量为65.3 kDa；构建的pMAL-c5x-Bsppo重组质粒在大肠杆菌Escherichia coli BL21(DE3) 中可溶表达了MBP-BsPPO蛋白；酶切去除MBP融合标签后对BsPPO进行了酶学性质研究，结果表明BsPPO的最适温度和pH分别为25 ℃和4.0，在低温和弱酸性环境中有较好的稳定性。短时间低强度的光照和Cu2+可激活BsPPO的活性，Zn2+和Ca2+能抑制其活性。BsPPO可催化单酚、二酚以及三酚类化合物，对l-酪氨酸以及香草酸表现出较高的催化活性，此外BsPPO还对黑芝麻中含有的2-甲氧基肉桂酸、吲哚3-羧酸和根皮素表现出良好的催化活性。研究结果为黑芝麻多酚氧化酶酶学特性的明确奠定了理论基础。

关键词:黑芝麻多酚氧化酶，大肠杆菌，原核表达，亲和纯化，酶学性质

Abstract:

To investigate the enzyme properties of the black sesame polyphenol oxidase (BsPPO), a synthesized Bsppo gene was cloned into the vector pMAL-c5x and expressed in E. coli. Subsequently, the MBP fusion label in the recombinant protein was removed by protease digestion after affinity purification. The synthesized Bsppo gene contained 1 752 bp which encodes 585 amino acids with a deduced molecular weight of 65.3 kDa. Transformation of the recombinant vector into E. coli BL21(DE3) resulted in soluble expression of the fusion protein MBP-BsPPO. The enzymatic properties of the recombinant BsPPO was investigated after MBP fusion tag excision followed by affinity purification. The results demonstrated that the optimal temperature and pH for BsPPO was 25°C and 4.0, respectively. BsPPO exhibited a good stability under low temperature and acidic environment. Low-intensity short-term light exposure increased the activity of BsPPO. Cu2+ could improve the activity of BsPPO while Zn2+ and Ca2+ showed the opposite effect. BsPPO could catalyze the oxidation of monophenols, diphenols, and triphenols, and exhibited good catalytic activity on l-tyrosine and vanillic acid. Moreover, BsPPO exhibited high catalytic activity on black sesame metabolites, including 2-methoxy cinnamic acid, indole-3-carboxylic acid and phloretin. These results may serve as a basis for further characterization of BsPPO.

Key words:black sesame polyphenol oxidase, Escherichia coli, prokaryotic expression, affinity purification, enzymatic properties