表面接枝法制备大孔强阴离子交换层析介质  

Preparation of Macroporous Strong Anion Exchange Chromatography Media by Surface Grafting

作　　者：李子炀 侯恒扬 柴振博 张恺 乔娟 马磊 靳海波[1] 张荣月[1] LI Ziyang;HOU Hengyang;CHAI Zhenbo;ZHANG Kai;QIAO Juan;MA Lei;JIN Haibo;ZHANG Rongyue(Beijing Key Laboratory of Clean Fuel and Efficient Catalytic Emission Reduction Technology,College of New Materials and Chemical Engineering,Beijing University of Petrochemical Technology,Beijing 102617,China)

机构地区：[1]北京石油化工学院新材料与化工学院燃料清洁化及高效催化减排技术北京市重点实验室,北京102617

出　　处：《中国生物工程杂志》2024年第10期64-74,共11页China Biotechnology

基　　金：国家自然科学基金(22074148);国家重点研发计划(2021YFC2103401)资助项目。

摘　　要：目的:开发一种表面接枝聚合制备大孔阴离子交换层析介质的方法,并对该类介质的色谱性能进行评价研究。方法:以聚丙烯酸酯类大孔微球为基质,通过氧化还原引发N,N,N-三甲基-3-(2-甲基烯丙酰氨基)-1-氯化丙铵接枝聚合的方法制备了一种大孔阴离子层析介质;通过单因素实验获得最佳制备工艺条件,考察各反应因素对该介质色谱性能的影响;对蛋白在吸附前后的酯酶样活性进行测定,判断介质与蛋白分子相互作用后活性变化;在不同流速条件下测试介质的耐压性能;使用1 mol/L NaOH溶液测试介质的稳定性能;将介质置于非吸附条件下对蛋白进行吸附,测试其非特异性吸附效果;通过计算不同流速下介质填充柱的塔板高度来研究其柱效,并使用阴离子交换层析法从鸡蛋清内提取高纯度蛋白。结果:在最佳制备条件下,介质蛋白静态结合容量最高可达138.94 mg/mL,介质的蛋白活性回收率可达98%,143~574 cm/h线速度范围内介质的蛋白动态结合容量从99.234 mg/mL减少至93.981 mg/mL;在143~3871 cm/h线速度范围内,柱压线性增加;通过扫描电子显微镜观察介质的表面形貌,发现接枝对介质的贯通孔径无影响;当洗脱液流速为535 cm/h时,其最小塔板高度为0.0599 mm;该阴离子交换介质在非吸附条件下,对牛血清白蛋白的吸附量为0.136 mg/mL,经过1 mol/L NaOH溶液浸泡96 h后,其蛋白静态结合容量仅下降2.4%;经该介质一步纯化鸡蛋清蛋白,可获得较高纯度的溶菌酶、卵白蛋白和卵铁传递蛋白。结论:介质具有较好的传质性能、较高的蛋白活性回收率和较低的非特异性吸附值,并且具有良好的稳定性和刚性。相较于传统介质,该类介质在蛋白高通量分离中展现了较大潜力。Objective:A surface grafting method was explored for the preparation of macroporous anion-exchange chromatography media.The chromatographic performance of the medium obtained was evaluated in the present study.Methods:A macroporous anionic chromatography medium was prepared by redox-initiated graft polymerization of 3-(methacryloylamino)propyl trimethylammonium chloride using polyacrylate-based macroporous microspheres as the substrate.The optimal preparation process was determined by single-factor experiments,and the effects of the reaction factors on the chromatographic performance of the medium were investigated in detail.The esterase-like activity of proteins was measured before and after adsorption to determine the change in activity due to the interaction of the medium with the protein molecule.The pressure resistance of the medium was tested under different flow conditions.The stability properties of the medium were tested using 1 mol/L NaOH solution.Th non-specific adsorption of proteins on the medium was tested under non-adsorptive conditions.Plate heights for media packed columns were determined at different flow rates to evaluate column efficiency.High-purity protein was purified from egg white using anion-exchange chromatography.Results:The static binding capacity of the protein on the prepared medium could reach up to 138.94 mg/mL under the optimal preparation conditions.Recovery of protein activity from the medium was up to 98%.The dynamic binding capacity of the medium decreased from 99.234 mg/mL to 93.981 mg/mL in the linear velocity range of 143 to 574 cm/h.The column pressure increased linearly in the range of 143 to 3871 cm/h linear velocity.The surface and internal morphology of the medium was examined using a scanning electron microscope and it was found that grafting polymers had no effect on the pore diameter of the medium.The minimum plate height was 0.0599 mm at an eluent flow rate of 535 cm/h.The anion-exchange chromatography medium adsorbed 0.136 mg/mL of bovine serum albumin under non-

关 键 词：大孔聚合物微球 阴离子交换层析介质 接枝法 蛋白结合容量 

分 类 号：Q816[生物学—生物工程]