Separation and purification of horseradish peroxidase from horseradish roots using a novel integrated method,New Journal of Chemistry

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Separation and purification of horseradish peroxidase from horseradish roots using a novel integrated method
New Journal of Chemistry ( IF 2.7 ) Pub Date : 2020-12-17 , DOI: 10.1039/d0nj04614k
Wei Ji _{1,

2,

3,

4} , Wenmei Ao _{2,

3,

4,

5} , Mengqiu Sun _{2,

3,

4,

6} , Chunlai Feng _{2,

3,

4,

6} , Yun Wang _{2,

3,

4,

5}

Affiliation

The aim of the present work was to develop a novel method integrating two-step aqueous two-phase extraction and temperature-controlled affinity precipitation for the separation and purification of horseradish peroxidase (HRP) from horseradish roots. First, a two-step aqueous two-phase extraction (ATPE) process was used as a primary separation process to remove sugars and pigments, and then a temperature-controlled affinity precipitation process based on a thermosensitive triblock copolymer (PEG₁₁₃-b-PVBA₄₉-b-PNIPAM₁₀₅) was exploited as a further purification process to selectively isolate HRP from other impurity proteins. The optimum PEG4000 (0.20, w/w) − (NH₄)₂SO₄ (0.18, w/w) aqueous two-phase system (ATPS) was obtained by studying the liquid–liquid equilibrium of PEG (400/1000/2000/4000) + (NH₄)₂SO₄/K₃C₆H₅O₇/K₂HPO₄ ATPSs and the distribution behaviors of 9 kinds of model compounds in these PEG-salt ATPSs. The successful isolation of HRP from horseradish roots was achieved by the integrated method without having a negative effect on the structure of HRP, and the extraction efficiency and purification factor of HRP reached 84.26 ± 0.15% and 9.15 ± 0.10, respectively. The integrated method shows outstanding separation and purification performances and has good prospects for the industrial production of plant glycoprotein.

中文翻译：

新型集成方法从辣根中分离纯化辣根过氧化物酶

本工作的目的是开发一种结合两步水两相萃取和温控亲和沉淀的新型方法，用于从辣根中分离和纯化辣根过氧化物酶（HRP）。首先，将两步水两相萃取（ATPE）工艺用作去除糖和色素的主要分离工艺，然后基于热敏三嵌段共聚物（PEG ₁₁₃ - b -PVBA）进行温度控制的亲和沉淀工艺₄₉ - b- PNIPAM ₁₀₅）被用作进一步纯化方法以从其他杂质蛋白中选择性分离HRP。最佳PEG4000（0.20，w / w）-（NH ₄）₂通过研究PEG（400/1000/2000/4000）+（NH ₄）₂ SO ₄ / K ₃ C ₆的液-液平衡，获得了SO ₄（0.18，w / w）水两相系统（ATPS）。H ₅ O ₇ / K ₂ HPO ₄这些PEG-盐ATPS中的ATPS和9种模型化合物的分布行为。通过一体化方法，成功地从辣根中分离出HRP，而对HRP的结构没有负面影响，HRP的提取效率和纯化因子分别达到84.26±0.15％和9.15±0.10。该方法具有优异的分离纯化性能，对植物糖蛋白的工业化生产具有良好的前景。

更新日期：2021-01-13

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