Separation of lactoperoxidase using cation exchange nano-cryogels

doi:10.11949/0438-1157.20240031

Abstract

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

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

CLC Number:

TQ 028.8

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.

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.

Figures/Tables 7

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

Fig.2 Scanning electron microscope images of cryogels

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

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

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

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

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

References 39

40	Guan J T, Guan Y X, Yun J X, et al. Chromatographic separation of phenyllactic acid from crude broth using cryogels with dual functional groups[J]. Journal of Chromatography A, 2018, 1554: 92-100.
41	Savina I N, Mattiasson B, Galaev I Y. Graft polymerization of vinyl monomers inside macroporous polyacrylamide gel, cryogel, in aqueous and aqueous-organic media initiated by diperiodatocuprate(Ⅲ) complexes[J]. Journal of Polymer Science Part A: Polymer Chemistry, 2006, 44(6): 1952-1963.
42	Dong S S, Chen L, Dai B, et al. Isolation of immunoglobulin G from bovine milk whey by poly(hydroxyethyl methacrylate)-based anion-exchange cryogel[J]. Journal of Separation Science, 2013, 36(15): 2387-2393.
43	周英爽, 樊凤娇, 刘猛, 等. 乳铁蛋白与牛乳中其他蛋白质相互作用机制研究进展[J]. 食品科学, 2015, 36(5): 244-249.
	Zhou Y S, Fan F J, Liu M, et al. Interaction mechanism between lactoferrin and other proteins in bovine milk[J]. Food Science, 2015, 36(5): 244-249.
1	Althaus R L, Molina M P, Rodríguez M, et al. Analysis time and lactation stage influence on lactoperoxidase system components in dairy ewe milk[J]. Journal of Dairy Science, 2001, 84(8): 1829-1835.
2	Boots J W, Floris R. Lactoperoxidase: from catalytic mechanism to practical applications[J]. International Dairy Journal, 2006, 16(11): 1272-1276.
3	Wit J N, Hooydonk A V. Structure, functions and applications of lactoperoxidase in natural antimicrobial systems[J]. Netherlands Milk and Dairy Journal, 1996, 50(2): 227-244.
4	Nandini K E, Rastogi N K. Single step purification of lactoperoxidase from whey involving reverse micelles-assisted extraction and its comparison with reverse micellar extraction[J]. Biotechnology Progress, 2010, 26(3): 763-771.
5	Gruden Š, Oberčkal J, Bogovič Matijašić B, et al. Insights into factors affecting lactoperoxidase conformation stability and enzymatic activity[J]. International Dairy Journal, 2023, 138: 105537.
6	Maciel K S, Mól P C G, Verissimo L A A, et al. Synthesis and characterization of supermacroporous cryogel with immobilized p-aminobenzenesulfonamide as affinity ligand for the purification of lactoperoxidase from whey[J]. Journal of Separation Science, 2023, 46(3): e2200639.
7	Köksal Z, Kalın R, Gülçin İ, et al. Impact of some avermectins on lactoperoxidase in bovine milk[J]. International Journal of Food Properties, 2016, 19(6): 1207-1216.
8	Barrett N E, Grandison A S, Lewis M J. Contribution of the lactoperoxidase system to the keeping quality of pasteurized milk[J]. Journal of Dairy Research, 1999, 66(1): 73-80.
9	Koksal Z, Alim Z, Beydemir S, et al. Potent inhibitory effects of some phenolic acids on lactoperoxidase[J]. Journal of Biochemical and Molecular Toxicology, 2016, 30(11): 533-538.
10	Viswanathan V, Ahmad M I, Singh P K, et al. Structural evidence of the conversion of nitric oxide ( N O ) to nitrite ion ( N O 2 - ) by lactoperoxidase (LPO): structure of the complex of LPO with N O 2 - nitrite ion at 1.89 Å resolution[J]. Journal of Inorganic Biochemistry, 2023, 247: 112311.
11	Shariat S Z A S, Borzouee F, Mofid M R, et al. Immobilization of lactoperoxidase on graphene oxide nanosheets with improved activity and stability[J]. Biotechnology Letters, 2018, 40(9/10): 1343-1353.
12	Ng P K, Yoshitake T. Purification of lactoferrin using hydroxyapatite[J]. Journal of Chromatography B, 2010, 878(13/14): 976-980.
13	Atasever A, Ozdemir H, Gulcin I, et al. One-step purification of lactoperoxidase from bovine milk by affinity chromatography[J]. Food Chemistry, 2013, 136(2): 864-870.
14	Li T Q, Ma L, Sun D X, et al. Purification of lactoperoxidase from bovine milk by integrating the technique of salting-out extraction with cation exchange chromatographic separation[J]. Journal of Food Measurement and Characterization, 2019, 13(2): 1400-1410.
15	Nandini K E, Rastogi N K. Reverse micellar extraction for downstream processing of lipase: effect of various parameters on extraction[J]. Process Biochemistry, 2009, 44(10): 1172-1178.
16	刘丽丽, 程利明, 车红霞, 等. 双水相萃取技术提取牛乳过氧化物酶[J]. 中国食品学报, 2016, 16(10): 93-98.
	Liu L L, Cheng L M, Che H X, et al. Extraction of lactoperoxidase in bovine milk using coupling aqueous two-phase[J]. Journal of Chinese Institute of Food Science and Technology, 2016, 16(10): 93-98.
17	Barba D, Beolchini F, Veglió F. Minimizing water use in diafiltration of whey protein concentrates[J]. Separation Science and Technology, 2000, 35(7): 951-965.
18	Priyanka B S, Rastogi N K. Downstream processing of lactoperoxidase from milk whey by involving liquid emulsion membrane[J]. Preparative Biochemistry & Biotechnology, 2018, 48(3): 270-278.
19	Morrison M, Allen P Z. Lactoperoxidase: identification and isolation from Harderian and lacrimal glands[J]. Science, 1966, 152(3729): 1626-1628.
20	卢蓉蓉, 许时婴, 王璋. 乳过氧化物酶的分离纯化和酶学性质研究[J]. 食品科学, 2006, 27(2): 100-104.
	Lu R R, Xu S Y, Wang Z. Isolation and purification of lactoperoxidase and its enzymatic properties[J]. Food Science, 2006, 27(2): 100-104.
21	Pan M M, Shen S C, Chen L, et al. Separation of lactoperoxidase from bovine whey milk by cation exchange composite cryogel embedded macroporous cellulose beads[J]. Separation and Purification Technology, 2015, 147: 132-138.
22	曲兴, 楼小玲, 张颂红, 等. 半疏水基质阴离子交换晶胶对α-酮异己酸的层析吸附特性[J]. 高校化学工程学报, 2022, 36(1): 46-52.
	Qu X, Lou X L, Zhang S H, et al. Chromatographic and adsorption characteristics of α-ketoisocaproate in semi-hydrophobic anion exchange croygel[J]. Journal of Chemical Engineering of Chinese Universities, 2022, 36(1): 46-52.
23	刘月娜, 程秀红, 沈绍传, 等. 离子交换晶胶层析分离甘草多糖的研究[J]. 高校化学工程学报, 2015, 29(6): 1507-1512.
	Liu Y N, Cheng X H, Shen S C, et al. Chromatographic separation of glycyrrhiza polysaccharides using ion-exchange cryogels[J]. Journal of Chemical Engineering of Chinese Universities, 2015, 29(6): 1507-1512.
24	Plieva F M, Galaev I Y, Mattiasson B. Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate-containing fluids and cell culture applications[J]. Journal of Separation Science, 2007, 30(11): 1657-1671.
25	Lozinsky V I, Galaev I Y, Plieva F M, et al. Polymeric cryogels as promising materials of biotechnological interest[J]. Trends in Biotechnology, 2003, 21(10): 445-451.
26	Yun J X, Dafoe J T, Peterson E, et al. Rapid freezing cryo-polymerization and microchannel liquid-flow focusing for cryogel beads: adsorbent preparation and characterization of supermacroporous bead-packed bed[J]. Journal of Chromatography A, 2013, 1284: 148-154.
27	Yao K J, Yun J X, Shen S C, et al. In-situ graft-polymerization preparation of cation-exchange supermacroporous cryogel with sulfo groups in glass columns[J]. Journal of Chromatography A, 2007, 1157(1/2): 246-251.
28	Yan L D, Shen S C, Yun J X, et al. Isolation of lysozyme from chicken egg white using polyacrylamide-based cation-exchange cryogel[J]. Chinese Journal of Chemical Engineering, 2011, 19(5): 876-880.
29	Yun J X, Cheng X H, Ye J L, et al. Chromatographic adsorption of serum albumin and antibody proteins in cryogels with benzyl-quaternary amine ligands[J]. Journal of Chromatography A, 2015, 1381: 173-183.
30	沈哲明, 沈绍传, 贠军贤, 等. 大尺寸弱阴离子交换晶胶层析一步法分离ATP的实验研究[J]. 高校化学工程学报, 2009, 23(5): 768-773.
	Shen Z M, Shen S C, Yun J X, et al. Purification of adenosine triphosphate from crude fermentation broth by one-step isolation using a large-size weak anion-exchange supermacroporous cryogel[J]. Journal of Chemical Engineering of Chinese Universities, 2009, 23(5): 768-773.
31	刘杰, 沈绍传, 陈平, 等. 内嵌纳米粒阴离子交换聚甲基丙烯酸羟乙酯复合晶胶分离三磷酸胞苷[J]. 化工学报, 2014, 65(10): 3938-3945.
	Liu J, Shen S C, Chen P, et al. Separation of cytidine triphosphate from Saccharomyces cerevisiae broth by anion exchange poly(2-hydroxyethyl methacrylate) composite cryogel embedded with SiO₂ nanoparticles[J]. CIESC Journal, 2014, 65(10): 3938-3945.
32	贺亚维, 张颂红, 黄杰, 等. 内嵌纳凝胶阴离子交换聚甲基丙烯酸羟乙酯复合晶胶分离苯乳酸研究[J]. 化工学报, 2020, 71(12): 5636-5643.
	He Y W, Zhang S H, Huang J, et al. Separation of phenyllactic acid from transformation broth by anion exchange poly(2-hydroxyethyl methacrylate) composite cryogel embedded with nanogels[J]. CIESC Journal, 2020, 71(12): 5636-5643.
33	黄杰. 半疏水纳晶胶的制备及性能研究[D]. 杭州: 浙江工业大学, 2019.
	Huang J. Preparation and properties of semi-hydrophobic nano-cryogels[D]. Hanzhou: Zhejiang University of Technology, 2019.
34	刘流, 张颂红, 贠军贤, 等. 纳凝胶的制备、性能及应用进展[J]. 化工进展, 2018, 37(12): 4726-4734.
	Liu L, Zhang S H, Yun J X, et al. Recent research progress on preparation methods, properties and applications of nanogels[J]. Chemical Industry and Engineering Progress, 2018, 37(12): 4726-4734.
35	Neamtu I, Rusu A G, Diaconu A, et al. Basic concepts and recent advances in nanogels as carriers for medical applications[J]. Drug Delivery, 2017, 24(1): 539-557.
36	Sasaki Y, Akiyoshi K. Nanogel engineering for new nanobiomaterials: from chaperoning engineering to biomedical applications[J]. Chemical Record, 2010, 10(6): 366-376.
37	Mishra N, Wani T U, Rashid M, et al. Targeting aspects of nanogels: an overview[J]. International Journal of Pharmaceutical Sciences and Nanotechnology, 2014, 7(4): 2612-2630.
38	黄杰, 张颂红, 贠军贤, 等. 聚甲基丙烯酸缩水甘油酯疏水纳凝胶的制备与表征[J]. 化工进展, 2019, 38(12): 5435-5441.
	Huang J, Zhang S H, Yun J X, et al. Preparation and characterization of poly(glycidyl methacrylate) hydrophobic nanogels[J]. Chemical Industry and Engineering Progress, 2019, 38(12): 5435-5441.
39	王良华, 陈芳, 贠军贤, 等. 阳离子交换型连续床用超大孔晶胶的制备[J]. 高校化学工程学报, 2009, 23(3): 480-485.
	Wang L H, Chen F, Yun J X, et al. Preparation of supermacroporous cryogel used in cation-exchange monolith[J]. Journal of Chemical Engineering of Chinese Universities, 2009, 23(3): 480-485.

[1]	Xiaoping LUO, Yuntian HOU, Yijie FAN. Flow boiling heat transfer and temperature uniformity in micro-channel with countercurrent phase separation structure [J]. CIESC Journal, 2024, 75(7): 2474-2485.
[2]	Xiaoqiao QIN, Hongbo TAN, Na WEN. Thermodynamic and economic analysis of air separation unit with energy storage and generation [J]. CIESC Journal, 2024, 75(7): 2409-2421.
[3]	Wenxuan ZHOU, Zhen LIU, Fujian ZHANG, Zhongqiang ZHANG. Mechanism of water treatment by high permeability-selectivity time dimension membrane method [J]. CIESC Journal, 2024, 75(7): 2583-2593.
[4]	Junyong HU, Yali HU, Xueyi TAN, Jiaxin HUANG, Lewei ZHANG, Junli ZENG, Xiaoyi LIU, Yuan TAO. Experimental study on the performance of multi-stage reverse electrodialysis based on LiCl-NH₄Cl aqueous solution [J]. CIESC Journal, 2024, 75(7): 2670-2679.
[5]	Zongwei HUO, Yabin NIU, Yanqiu PAN. Behavior of high viscosity oil droplets in oil-water membrane separation and its influencing factors [J]. CIESC Journal, 2024, 75(6): 2262-2273.
[6]	Yiqi ZHANG, Xuesong TAN, Wuhuan LI, Quan ZHANG, Changlin MIAO, Xinshu ZHUANG. Efficient fractionation of sugarcane bagasse with phenoxyethanol under mild condition [J]. CIESC Journal, 2024, 75(6): 2274-2282.
[7]	Wei WANG, Xu BAI, Xiang ZHAO, Xueliang MA, Wei LIN, Jiuyang YU. Optimization of air flotation cyclone separation conditions based on response surface methodology [J]. CIESC Journal, 2024, 75(5): 1929-1938.
[8]	Rufeng XU, Yucheng CHEN, Dan GAO, Jingyu JIAO, Dong GAO, Haibin WANG, Shanjing YAO, Dongqiang LIN. Model-assisted process optimization of ion-exchange chromatography for monoclonal antibody charge variant separation [J]. CIESC Journal, 2024, 75(5): 1903-1911.
[9]	Zijia ZHANG, Xinyue QIU, Xiang SUN, Zhibin LUO, Haizhong LUO, Gaohong HE, Xuehua RUAN. Progress in molecular structure design for polyimide membrane materials to enhance CO₂ permeation ability [J]. CIESC Journal, 2024, 75(4): 1137-1152.
[10]	Binyu MO, Yaxin ZHANG, Guozhen LIU, Gongping LIU, Wanqin JIN. Recent progress of metal-organic framework membranes for mono/divalent ions separation [J]. CIESC Journal, 2024, 75(4): 1183-1197.
[11]	Tianyi LI, Yutai WU, Yongsheng WANG, Jiarui GU, Yiheng SONG, Fengcheng YANG, Guangping HAO. Advances in light isotopes separation and catalytic labeling [J]. CIESC Journal, 2024, 75(4): 1284-1301.
[12]	Jun LI, Liang ZHAO, Jinsen GAO, Chunming XU. Research progress of extraction technology in processing different distillate by grade and composition [J]. CIESC Journal, 2024, 75(4): 1065-1080.
[13]	Tiantian LYU, Min YUAN, Jiang WANG, Meizhen GAO, Jiahui YANG, Hong XU, Jinxiang DONG, Qi SHI. Preparation of ZTIF based hydrophobic micro-mesoporous carbon and their adsorption and separation performance of 5-hydroxymethylfurfural [J]. CIESC Journal, 2024, 75(4): 1642-1654.
[14]	Yiru WEN, Jia FU, Dahuan LIU. Advances in machine learning-based materials research for MOFs: energy gas adsorption separation [J]. CIESC Journal, 2024, 75(4): 1370-1381.
[15]	Xiao DONG, Zhishan BAI, Xiaoyong YANG, Wei YIN, Ningpu LIU, Qifan YU. Research and industrial application of coupled impurity removal technology in CHPPO process oxidation liquids [J]. CIESC Journal, 2024, 75(4): 1630-1641.