化工进展 ›› 2022, Vol. 41 ›› Issue (11): 6038-6044.DOI: 10.16085/j.issn.1000-6613.2022-0096

• 生物与医药化工 • 上一篇    下一篇

减电荷聚甲基丙烯酸钠接枝介质的制备及蛋白质吸附性能

李宪秀1(), 何涛1, 毛建卫1,2, 沙如意1()   

  1. 1.浙江科技学院生物与化学工程学院,浙江省农产品化学与生物加工技术重点实验室,浙江省农业生物资源 生化制造协同创新中心,浙江 杭州 310023
    2.浙江工业职业技术学院,浙江 绍兴 312000
  • 收稿日期:2022-01-13 修回日期:2022-02-08 出版日期:2022-11-25 发布日期:2022-11-28
  • 通讯作者: 沙如意
  • 作者简介:李宪秀(1992—),女,博士,讲师,研究方向为生物分离。E-mail:lixianxiu@zust.edu.cn
  • 基金资助:
    浙江省重点研发计划(2017C02009);浙江科技学院科研启动基金(F701103K12)

Preparation and protein adsorption performance of charge-reduced poly(methacrylate)-grafted sepharose FF

LI Xianxiu1(), HE Tao1, MAO Jianwei1,2, SHA Ruyi1()   

  1. 1.School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Zhejiang Provincial Key Laboratory for Chemical & Biological Processing Technology of Farm Product, Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical Manufacturing, Hangzhou 310023, Zhejiang, China
    2.Zhejiang Industry Polytechnic College, Shaoxing 312000, Zhejiang, China
  • Received:2022-01-13 Revised:2022-02-08 Online:2022-11-25 Published:2022-11-28
  • Contact: SHA Ruyi

摘要:

离子交换容量(IC)为320mmol/L的聚甲基丙烯酸钠(pMA)接枝型介质(FF-pMA-320)对溶菌酶和γ-球蛋白具有较高的吸附容量,但其传质速率较低。在保持聚合物链长度的前提下,通过乙醇胺与接枝链上的羧基进行电荷中和反应,降低pMA接枝型介质的电荷密度,提升介质的蛋白质传质速率。将FF-pMA-320进行部分电荷中和修饰,制备得到离子交换容量分别为230mmol/L和170mmol/L的减电荷阳离子交换介质,分别命名为pMA-320-R230和pMA-320-R170。采用吸附平衡、吸附动力学和柱穿透实验,研究了溶菌酶和γ-球蛋白在这两种新型介质上的吸附行为,并与初始介质FF-pMA-320进行了比较。结果表明:随着介质的IC值(电荷密度)从320mmol/L降低到170mmol/L,介质对两种蛋白质吸附容量随之减少,这与电荷中和修饰降低蛋白质吸附位点有关。随着接枝聚合物电荷密度的降低,相邻聚合物链之间的静电排斥作用减弱,蛋白质吸附容量降低,造成接枝链的灵活性增加以及蛋白质排阻效应减弱。因此,溶菌酶和γ-球蛋白在pMA-320-R170上的传质速率分别是FF-pMA-320的1.6倍和5.5倍。柱穿透实验结果表明,由于pMA-320-R170对γ-球蛋白传质速率较高,使得介质对蛋白质的动态结合容量(DBC)高于其他两种介质,在流速为150~750cm/h时可保持在10mg/mL以上。研究结果对设计和开发高性能蛋白质色谱介质提供借鉴和指导。

关键词: 色谱, 蛋白质, 吸附, 动力学, 动态结合容量

Abstract:

Poly(methacrylate) (pMA)-grafted Sepharose FF with ionic capacity (IC) of 320mmol/L (FF-pMA-320) showed the high lysozyme and γ-globulin adsorption capacities, but its uptake rates for the two proteins were low. On the premise of maintaining polymer chain length, the charge density on pMA-grafted Sepharose FF was reduced by neutralization of the carboxyl groups on pMA chains with ethanolamine to improve protein uptake rate. FF-pMA-320 was partly neutralized to prepare the two charge-reduced cation exchangers with IC values (in mmol/L) of 230 and 170, which were respectively named as pMA-320-R230 and pMA-320-R170. Adsorption equilibrium, uptake kinetics and column breakthrough experiments were performed to study lysozyme and γ-globulin adsorption on to the two new resins, which were compared with the starting resin (FF-pMA-320). It was found that the adsorption capacities for the two proteins decreased with IC (charge density) decreasing from 320mmol/L to 170mmol/L, which was related to the decrease of adsorption site caused by the partial neutralization. With decreasing charge density of grafted polymer, there were weaker electrostatic repulsion between neighbouring polymer chains and lower protein adsorption capacity, thus increasing chain flexibility and reducing protein exclusion effect. Therefore, the uptake rates of lysozyme and γ-globulin on pMA-320-R170 were about 1.6 times and 5.5 times higher than those on FF-pMA-320, respectively. Column breakthrough results demonstrated that the dynamic binding capacity (DBC) of γ-globulin on pMA-320-R170 was higher than other two resins due to its high uptake rate, and kept over 10mg/mL in the flow rate range of 150—750cm/h. The findings in this work provided reference and direction to design and develop high-performance protein chromatography.

Key words: chromatography, protein, adsorption, kinetics, dynamic binding capacity

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