Humer Diana, Ebner Julian
TU Wien, Institute of Chemical, Environmental and Bioscience Engineering, Research Area Biochemical Engineering, Vienna, Austria.
Methods Mol Biol. 2023;2617:227-237. doi: 10.1007/978-1-0716-2930-7_16.
In the following chapter a purification process for recombinant Horseradish peroxidase (HRP) produced in Escherichia coli is described. This enzyme is a secretory plant oxidoreductase belonging to the large peroxidase family III within the peroxidase-catalase superfamily of enzymes. It has high biotechnological significance, however, the isolation of the enzyme from its natural source, the horseradish root, has several shortcomings, which makes the development of a recombinant production strategy interesting. The presented protocol covers all process steps from isolation to the final chromatography step; the enzyme is solubilized from insoluble inclusion bodies, refolded and concentrated to yield a high purity enzyme preparation which is comparable to the commercially available plant-derived HRP. Moreover, we believe that this procedure can also be used to process other peroxidases of family II and III of the plant peroxidase superfamily, as they all share the same relevant features like disulfide bonds and a heme group.
在接下来的章节中，将描述一种用于纯化在大肠杆菌中产生的重组辣根过氧化物酶（HRP）的方法。这种酶是一种分泌型植物氧化还原酶，属于过氧化物酶 - 过氧化氢酶超家族中的大型过氧化物酶家族III。它具有很高的生物技术意义，然而，从其天然来源辣根中分离这种酶存在几个缺点，这使得开发重组生产策略变得很有意义。所展示的方案涵盖了从分离到最终色谱步骤的所有工艺步骤；该酶从不溶性包涵体中溶解、复性并浓缩，以产生一种高纯度的酶制剂，其质量可与市售的植物源HRP相媲美。此外，我们认为该方法也可用于处理植物过氧化物酶超家族II和III家族的其他过氧化物酶，因为它们都具有相同的相关特征，如二硫键和血红素基团。
