阳离子交换纳晶胶分离乳过氧化物酶的研究

doi:10.11949/0438-1157.20240031

摘要/Abstract

摘要：

乳过氧化物酶（Lactoperoxidase，LP）因具有重要的生物学功能及广谱的抑菌活性而在食品、医药和化工等领域受到广泛关注。针对目前LP分离纯化方法中普遍存在的选择性差、成本高以及分离材料易污染等问题，以甲基丙烯酸羟乙酯（HEMA）及聚甲基丙烯酸缩水甘油酯纳凝胶（PGN）为复合基材，利用低温冷冻聚合和结晶致孔原理制备了pHEMA/PGN纳晶胶基质，然后接枝功能单体2-丙烯酰氨基-2-甲基-1-丙磺酸（AMPSA）得到具有阳离子交换功能的pHEMA/PGN纳晶胶介质，以用于从乳清中层析分离LP。考察了纳晶胶介质的基础性能，并重点探究了缓冲液pH值对层析性能的影响。结果表明：制备所得纳晶胶介质具有分布均匀的超大孔结构、优良的渗透性能和传质性能，对模型蛋白溶菌酶的吸附容量为4.41 mg·ml^-1；在最优分离条件下所得目标蛋白LP纯度约为96.0%，比活力为27.03 U·mg^-1，酶活性回收率为86.5%，分离效果良好，表明该纳晶胶介质在层析分离方面具有较大的应用潜力。

关键词: 乳过氧化物酶, 晶胶介质, 分离, 离子交换

Abstract:

Lactoperoxidase is a natural oxidoreductase, which has unique biological functions and broad-spectrum antibacterial activity, and has received extensive attention in the fields of food, medicine and chemical industry. In view of the shortcomings of traditional LP separation and purification methods, such as poor selectivity, high cost and easy contamination of separated materials, 2-hydroxyethyl methacrylate and poly(glycidyl methacrylate) nanogels(PGN) were used as composite substrates to prepare poly(2-hydroxyethyl methacrylate) composite cryogel embedded with PGN nanogels(pHEMA/PGN-based cryogel) by using the principle of low-temperature freezing polymerization. And then the pHEMA/PGN-based cryogel was grafted with the functional monomer 2-acrylamino-2-methyl-1-propanesulfonic acid to obtain pHEMA/PGN cation exchange nano-cryogel(pHEMA/PGN nano-cryogel) for the chromatography separation of LP from whey. The basic properties of pHEMA/PGN nano-cryogel were investigated, and the effect of buffer pH on chromatography performance was emphatically explored. The results showed that the obtained pHEMA/PGN nano-cryogel had large pore structure with uniform distribution, high permeability, excellent mass transfer performance, and strong adsorption capacity for lysozyme reaching 4.41 mg·ml^-1. The pHEMA/PGN nano-cryogel was used to isolate LP in whey, the purity was about 96.0%, the specific enzyme activity was 27.03 U·mg^-1, and enzyme activity recovery was 86.5% under the optimal conditions. The excellent separation performance indicated that the pHEMA/PGN nano-cryogel has good potential in the field of lactoperoxidase separation from whey.

Key words: lactoperoxidase, cryogel, separation, ion exchange

中图分类号:

TQ 028.8

张颂红, 赵欣怡, 楼小玲, 沈绍传, 贠军贤. 阳离子交换纳晶胶分离乳过氧化物酶的研究[J]. 化工学报, DOI: 10.11949/0438-1157.20240031.

Songhong ZHANG, Xinyi ZHAO, Xiaoling LOU, Shaochuan SHEN, Junxian YUN. Separation of lactoperoxidase from bovine whey milk by cation exchange nano-cryogels[J]. CIESC Journal, DOI: 10.11949/0438-1157.20240031.

图/表 7

图1 pHEMA/PGN纳晶胶基质接枝AMPSA的反应机理示意图

Fig.1 The reaction scheme of grafting AMPSA onto pHEMA/PGN-based cryogels

图2 晶胶介质扫描电镜图

Fig.2 Scanning electron microscope photographs of cryogels

图3 纳晶胶介质在不同流速下的停留时间分布

Fig.3 Residence time distributions in nano-cryogels at various flow rates

图4 纳晶胶介质在不同流速下的HETP及Dax值

Fig.4 HETP and Dax values of nano-cryogels at various flow rates

图5 纳晶胶介质压降与流量的关系

Fig.5 Flow rate vs. pressure drop in nano-cryogels

图6 纳晶胶介质在不同缓冲液pH值下的乳清层析过程

Fig.6 Chromatographic profiles in the separation of LP from bovine milk whey at different buffer pH values by the nano-cryogels

图7 纳晶胶介质在不同缓冲液pH值下乳清层析收集液的SDS-PAGE电泳图

Fig.7 SDS-PAGE analysis of chromatographic isolation process of LP from bovine milk whey at different buffer pH values by the nano-cryogels

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