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 (LP) has received widespread attention in the fields of food, medicine and chemical industry because of its important biological functions and broad-spectrum antibacterial activity. 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. 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, 2024, 75(7): 2574-2582.

Songhong ZHANG, Xinyi ZHAO, Xiaoling LOU, Shaochuan SHEN, Junxian YUN. Separation of lactoperoxidase using cation exchange nano-cryogels[J]. CIESC Journal, 2024, 75(7): 2574-2582.

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 images 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

Fig.4 HETP and Dax 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 by the nano-cryogels

图7 纳晶胶介质在不同缓冲液pH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 by the nano-cryogels

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