Preparation of gelatin-based cationic exchange resin microspheres and separation performance for lactoferrin

doi:10.16085/j.issn.1000-6613.2024-1871

Abstract

Abstract:

Ion exchange chromatography is one of the common methods for large-scale purification of proteins. In this study, a large-sized agarose gel bead (BB) (100—300μm) was used as the matrix to prepare cation exchange chromatography media by chemical cross-linking and ligand coupling. The separation and purification effect of lactoferrin (LF) in bovine colostrum was investigated. By optimizing the type and amount of emulsifier, transparent and spherical agarose microbeads were obtained. Subsequently, the matrix microbeads were chemically cross-linked and sulfonic acid-based ligand-coupled to prepare a high-rigidity cation exchange chromatography medium (SP-BB) with a pressure resistance of up to 0.45MPa. The results of scanning electron microscopy (SEM) and nitrogen adsorption-desorption tests showed that SP-BB microbeads contained rich pore structures, which were beneficial to the adsorption of protein molecules. Meanwhile, the adsorption kinetics, dynamics, and dynamic loading capacity (DBC) of SP-BB were tested using lysozyme (LYS) and bovine serum albumin (BSA) as model proteins, and the results showed that the performance of the self-prepared medium was better than that of domestic products and comparable to that of imported products. Finally, the exploration of the separation and purification of lactoferrin (LF) in bovine colostrum using SP-BB was conducted, and the results showed that SP-BB successfully captured LF from the mixture, and the purity of the LF in the first purification eluate was 88.8%, with a recovery rate of 71.2%. This work provides a new idea for the preparation of large-sized cation exchange media and the separation and purification of lactoferrin in bovine colostrum.

Key words: agarose microbeads, SP cation exchange medium, chromatography resin, protein adsorption, lactoferrin purification

摘要：

离子交换色谱是蛋白质大规模纯化的常见方法之一。本文以大粒径琼脂糖凝胶微球（BB）（100~300μm）为基质，进行化学交联与配体偶联制备阳离子交换色谱介质，并考察对牛初乳中乳铁蛋白（LF）的分离纯化效果。通过对乳化剂种类及用量探究，得到透明圆整且均一的大粒径琼脂糖微球。随后，对基质微球进行化学交联与磺酸基配体偶联，制备耐压达0.45MPa的高刚性磺酸基阳离子交换介质（SP-BB）。通过扫描电镜（SEM）和氮气吸脱附测试结果表明SP-BB微球中含有丰富的孔道结构，有利于蛋白分子的吸附。同时，以溶菌酶（LYS）和牛血清白蛋白（BSA）为模型蛋白对SP-BB开展吸附静力学、动力学以及动态载量（DBC）测试，结果表明自制SP-BB性能优于国产介质，略低于进口介质。最后应用自制SP-BB对牛初乳中乳铁蛋白（LF）分离纯化，结果表明SP-BB成功从牛初乳中捕获LF，液相色谱结果表明经一次纯化分离出的乳铁蛋白纯度高达88.8%，回收率为71.2%。本工作为一种大粒径阳离子交换介质的制备及其在牛初乳中乳铁蛋白的分离纯化提供了新思路。

关键词: 琼脂糖微球, SP阳离子交换介质, 色谱填料, 蛋白吸附, 乳铁蛋白纯化

CLC Number:

TQ033

WANG Wenjin, WANG Yixuan, ZENG Zeyang, YANG Jianhai, XU Cai, GUO Jia, DAI Liyan. Preparation of gelatin-based cationic exchange resin microspheres and separation performance for lactoferrin[J]. Chemical Industry and Engineering Progress, 2025, 44(12): 7141-7151.

王文瑾, 王意萱, 曾泽阳, 杨建海, 徐偲, 郭佳, 戴立言. 琼脂糖阳离子交换介质微球的制备及对乳铁蛋白的分离性能[J]. 化工进展, 2025, 44(12): 7141-7151.

Figures/Tables 19

References 9

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模型	参数	BSA			LYS
模型	参数	自制SP	国产SP	进口SP	自制SP	国产SP	进口SP
Langmuir	Q_m1/mg·g^-1	221.7	245.7	322.7	177.5	175.6	199.7
	K_d/mg·g^-1	0.027	0.029	0.173	0.0057	0.0056	0.005
	R²	0.921	0.987	0.974	0.94	0.937	0.985
Freundlich	K_f/mg^1-^1/n·L ^1/n ·g^-1	225.6	243.2	274.5	193.9	192.7	222.7
	n	5.15	5.22	3.19	6.76	6.73	7.96
	R²	0.887	0.9	0.97	0.876	0.885	0.939

模型	参数	BSA			LYS
模型	参数	自制SP	国产SP	进口SP	自制SP	国产SP	进口SP
Langmuir	Q_m1/mg·g^-1	221.7	245.7	322.7	177.5	175.6	199.7
	K_d/mg·g^-1	0.027	0.029	0.173	0.0057	0.0056	0.005
	R²	0.921	0.987	0.974	0.94	0.937	0.985
Freundlich	K_f/mg^1-^1/n·L ^1/n ·g^-1	225.6	243.2	274.5	193.9	192.7	222.7
	n	5.15	5.22	3.19	6.76	6.73	7.96
	R²	0.887	0.9	0.97	0.876	0.885	0.939

模型	参数	BSA			LYS
模型	参数	自制SP	国产SP	进口SP	自制SP	国产SP	进口SP
伪一阶方程	Q_e1/mg·g^-1	89.8	81.4	63.6	120.2	124.4	140.1
	k₁/min^-1	0.0287	0.0275	0.0266	0.0468	0.0457	0.0419
	R²	0.926	0.964	0.97	0.958	0.972	0.952
伪二阶方程	Q_e2/mg·g^-1	112.9	105	82.2	140.4	146.6	166.5
	k₂/min^-1	2.65	2.54	3.13	0.0004	0.0003	0.0003
	R²	0.948	0.975	0.982	0.984	0.995	0.979

模型	参数	BSA			LYS
模型	参数	自制SP	国产SP	进口SP	自制SP	国产SP	进口SP
伪一阶方程	Q_e1/mg·g^-1	89.8	81.4	63.6	120.2	124.4	140.1
	k₁/min^-1	0.0287	0.0275	0.0266	0.0468	0.0457	0.0419
	R²	0.926	0.964	0.97	0.958	0.972	0.952
伪二阶方程	Q_e2/mg·g^-1	112.9	105	82.2	140.4	146.6	166.5
	k₂/min^-1	2.65	2.54	3.13	0.0004	0.0003	0.0003
	R²	0.948	0.975	0.982	0.984	0.995	0.979