双水相萃取结合硼酸亲和吸附分离纯化辣根过氧化物酶

doi:10.7506/spkx1002-6630-20201209-105

食品科学 ›› 2022, Vol. 43 ›› Issue (2): 55-61.doi: 10.7506/spkx1002-6630-20201209-105

双水相萃取结合硼酸亲和吸附分离纯化辣根过氧化物酶

吴雅娇，邢又元，韩娟，吴嘉聪，李媛媛，王赟

（1.江苏大学化学化工学院，江苏镇江 212003；2.江苏大学食品与生物工程学院，江苏镇江 212003；3.江苏大学京江学院，江苏镇江 212003）

出版日期:2022-01-25 发布日期:2022-01-29
基金资助:
国家自然科学基金面上项目（21878131；22078133；21676124）

An Integrated Method Combining Aqueous Two-phase Extraction and Boric Acid Affinity Adsorption for Separation and Purification of Horseradish Peroxidase from Horseradish Roots

WU Yajiao, XING Youyuan, HAN Juan, WU Jiacong, LI Yuanyuan, WANG Yun

(1. School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212003, China; 2. School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212003, China; 3. Jingjiang College, Jiangsu University, Zhenjiang 212003, China)

Online:2022-01-25 Published:2022-01-29

摘要/Abstract

摘要： 开发一种结合两步双水相萃取和亲和吸附分离的新型集成化方法，用于从辣根中分离和纯化辣根过氧化物酶（horseradish peroxidase，HRP）。首先，将两步双水相萃取工艺用于糖和色素的分离，然后将“多价”苯硼酸亲和磁性石墨烯复合材料（d-PBA-GO@Fe3O4@PEI）用作进一步吸附分离，实现从杂质蛋白中选择性分离HRP。通过研究温敏聚合物17R4-盐双水相体系的液液相平衡行为，以及9 种模型化合物在双水相体系中的分配行为，最终确定8% 17R4-13.2% (NH4)2SO4双水相体系作为初步纯化的工艺体系。通过构建的集成化方法，成功地从辣根中分离出HRP，而对HRP的结构没有影响，HRP的比活力和纯化因子分别达到28.93 U/mg和1.92。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。

关键词: 辣根过氧化物酶；纯化；双水相体系；亲和吸附；苯硼酸

Abstract: The aim of the present work was to develop a novel method integrating two-step aqueous two-phase extraction and affinity adsorption for the separation and purification of horseradish peroxidase (HRP) from horseradish roots. Two-step aqueous two-phase extraction was used to remove sugars and pigments, and then “multivalent” phenylboronic acid affinity magnetic graphene composite (d-PBA-GO@Fe3O4@PEI) was to selectively isolate HRP from impurity proteins. The liquid-liquid equilibrium behaviors of 17R4-(NH4)2SO4, K3C6H5O7 or K2HPO4 aqueous two-phase systems (ATPSs) and the partition behaviors of nine model compounds in these R4-salt ATPSs were investigated, indicating that the optimum ATPS was 8% 17R4-13.2% (NH4)2SO4. The successful isolation of HRP from horseradish roots was achieved by the integrated method without negative effect on the structure of HRP, and the specific activity and purification factor of HRP were 28.93 U/mg and 1.92, respectively. The method has excellent separation and purification performance and thus holds great promise for application in the industrial production of plant glycoproteins.

Key words: horseradish peroxidase; purification; aqueous two-phase system; affinity adsorption; phenylboronic acid

中图分类号:

TS201.2

吴雅娇，邢又元，韩娟，吴嘉聪，李媛媛，王赟. 双水相萃取结合硼酸亲和吸附分离纯化辣根过氧化物酶[J]. 食品科学, 2022, 43(2): 55-61.

WU Yajiao, XING Youyuan, HAN Juan, WU Jiacong, LI Yuanyuan, WANG Yun. An Integrated Method Combining Aqueous Two-phase Extraction and Boric Acid Affinity Adsorption for Separation and Purification of Horseradish Peroxidase from Horseradish Roots[J]. FOOD SCIENCE, 2022, 43(2): 55-61.

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双水相萃取结合硼酸亲和吸附分离纯化辣根过氧化物酶

An Integrated Method Combining Aqueous Two-phase Extraction and Boric Acid Affinity Adsorption for Separation and Purification of Horseradish Peroxidase from Horseradish Roots

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