壳聚糖锌离子固定化亲和层析填料的制备与性质

中国生物工程杂志

2012, Vol. 32

Issue (05): 85-90

技术与方法

壳聚糖锌离子固定化亲和层析填料的制备与性质

包海生¹, 高秀峰¹, 郑雪妮¹, 李永生²

1. 四川大学华西基础医学与法医学院成都 610041;
2. 四川大学化学工程学院成都 610065

Preparation and Properties of Chitosan Immobilized Zinc Ion Affinity Chromatography Matrix

BAO Hai-sheng¹, GAO Xiu-feng¹, ZHENG Xue-ni¹, LI Yong-sheng²

1. West China School of Preclinical and Forensic Medicine,Sichuan University,Chengdu 610041, China;
2. School of Chemical Engineering,Sichuan University,Chengdu 610065, China

全文: PDF(537 KB) HTML

摘要：

目的:制备了壳聚糖Zn²⁺固定化亲和层析填料,并对其性能进行了研究。方法:采用反相悬浮法制备了交联壳聚糖;再以环氧氯丙烷为活化剂,乙二胺为螯合配基,制备了固定化亲和层析填料;表征了其有效粒径以及均匀系数、含水量、失重率、氨基含量、骨架密度、堆积密度以及孔度值。从时间、加入ZnCl₂的浓度、温度、pH方面对Zn²⁺固定化条件进行了优选,并确定了Zn²⁺的固定化量。含组氨酸标签的乙醛脱氢酶粗酶液,经硫酸铵盐析后,考察了壳聚糖Zn²⁺固定化亲和层析填料的亲和性能。结果:制备的填料有效粒径为105μm;均匀系数为1.46;含水量为58.03%;失重率为85.43%;氨基含量为9.20mmol/g;骨架密度为1.217 8g/ml;堆积密度为0.843 2g/ml;孔度值为36.40%。固定化Zn²⁺的最佳条件是:时间3 h、加入ZnCl₂溶液浓度0.1 mol/L、温度28℃、pH 5.5;且此条件下,亲和层析填料中Zn²⁺固定化量为3.35mmol/g。壳聚糖Zn²⁺固定化亲和层析填料对乙醛脱氢酶的亲和性能为4.14IU/g(干重)。结论:制备了壳聚糖Zn²⁺固定化亲和层析填料,可用于带有组氨酸标签重组蛋白的快速分离与纯化。

关键词： 壳聚糖; 固定化金属亲和层析; 制备; 固定化量; 乙醛脱氢酶

Abstract:

Purpose:Chitosan immobilized zinc ion affinity chromatography matrixes were prepared,and its properties were studied.Methods:Immobilized metal affinity chromatography (IMAC) matrixes were successfully prepared with cross-linked chitosan prepared by reverse phase suspension polymerization,and 3-Chloro-1,2-epoxypropane as activated agent, ethylenediamine as chelating ligand and Zn²⁺ as center ions.Its effective grain size and uniformity coefficient,moisture content,rate of lostmass, amino content,skeletaldensity,bulk density and porosity were characterized.Time,concentration of ZnCl₂solution,temperature,pH of Zn²⁺ immobilized conditions were optimized to determine the amount of Zn²⁺immobilized.After purifying with salting out,aldehyde dehydrogenase containing a histidine-tag was used to examine the affinity of chitosan immobilized Zn²⁺affinity chromatography matrix.Results:the effective grain size,uniformity coefficient,moisture content,rate of lostmass,amino content,skeletaldensity,bulk density and porosity of the prepared matrix is 105μm,1.46,58.03%,85.43%,9.20 mmol/g, 1.2178 g/ml, 0.8432 g/ml and 36.40% respectively.The optimum conditions of Zn²⁺immobilized were:time 3h,ZnCl₂concentration 0.1 mol/L,temperature 28℃,pH 5.5.And the amount of Zn²⁺immobilized is 3.35 mmol/g under these optimum conditions.The affinity of chitosan immobilized zinc ion affinity chromatography matrix for aldehyde dehydrogenase is 4.14 IU/g(dry weight).Conclusions: The high-efficiency chitosan immobilized zinc ion affinity chromatography matrixes were prepared,and it can be used for quickly separating and purifying the recombinant protein containing a His-tag.

Key words: Chitosan Immobilized metal ion affinity chromatography Preparation The amount of immobilized Aldehyde dehydrogenase

收稿日期: 2012-01-04 出版日期: 2012-05-25

ZTFLH:

Q819

通讯作者: 高秀峰, 李永生 E-mail: xiufengg@163.com; lysgxf2005@yanhoo.cn.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章

引用本文:

包海生, 高秀峰, 郑雪妮, 李永生. 壳聚糖锌离子固定化亲和层析填料的制备与性质[J]. 中国生物工程杂志, 2012, 32(05): 85-90.

BAO Hai-sheng, GAO Xiu-feng, ZHENG Xue-ni, LI Yong-sheng. Preparation and Properties of Chitosan Immobilized Zinc Ion Affinity Chromatography Matrix. China Biotechnology, 2012, 32(05): 85-90.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I05/85

[1] Porath J,Carlsson J,Olsson I,et al.Metal chelate affinity chromatography:a new approach to protein fractionation.Nature, 1975, 258(5536): 598-599.

[2] 宋超,吉爱国,梁浩.金属螯合亲和层析的应用研究进展.现代生物医学进展, 2008, 8(6): 1178-1180. Song C,Ji A G,Liang H.Research progress of metal-chelate affinity chromatography.Progress in Modern Biomedicine,2008,8(6): 1178-1180.

[3] Hutchinson M H, Chase H A. Adsorptive refolding of histidine-tagged glutathione S-transferase using metal affinity chromatography. Journal of Chromatography A, 2006, 1128(1-2): 125-132.

[4] 张怀,袁其朋,朱亚平,等. 重组Hepcidin融合蛋白的金属螯合亲和层析纯化.北京化工大学学报(自然科学版), 2005, 32(6): 15-19. Zhang H,Yuan Q P,Zhu Y P,et al.Purification of recombinant hepcidin fusion protein by immobilized metal ion affinity chromatography.Journal of Beijing University of Chemical Technology(Natural Science),2005,32(6):15-19.

[5] 李艳利,史永昶,姜涌明,等. 壳聚糖多孔珠的制备及其作为亲和吸附剂载体的研究. 药物生物技术, 1998, 5(4): 241 - 244. Li Y L,Shi Y C,Jiang Y M,et al.Preparation of chitosan porous beads(CPB) and studies on CPB as affinity adsorbents support.Pharmaceutical Biotechnology,1998,5(4):241-244.

[6] 丁明,施建军,皇甫立霞,等.壳聚糖微球的制备研究.化学世界, 1998,(12): 636-640. Ding M, Shi J J, Huang F L X, et al. Preparation of chitosan microspheres. Chemistry World, 1998, (12): 636-640.

[7] 易喻,崔国艳,王鸿,等.一种新型壳聚糖分离介质的制备.化工进展,2011, 30(9): 2049-2954. Yi Y, Cui G Y, Wang H, et al. Study the preparation of a novel crossl-linked chitosan support. Chemical Industry and Engineering Progress, 2011, 30(9): 2049-2054.

[8] Porath J,Olin B.Immobilized metal ion affinity adsorption and affinity chramatography of biomaterials.Serum protein affinity for gel-immobilized metal ion and nickel ions. Biochemistry, 1983, (22): 1621-1630.

[9] 刘颖,嘎日迪.李向清,等.1-(2-吡啶偶氮)-2-萘酚水相分光光度法测定水中微量锌.内蒙古石油化工, 1996, 22(4): 7-9. Liu Y,Ga R D,Li X Q,et al.1-(2-pyridylazo)-2-naphthol aqueous phase spectrophotometric determination of trace zinc of water. Inner Mongulia Petrochemical Industry,1996,22(4):7-9.

[10] 陈国珍,黄贤智,刘文远,等. 紫外-可见光分光光度法(下册).北京: 原子能出版社,1987.530-531. Chen G Z, Huang X Z, Liu W Y, et al. UV-visible Spectrophotometry. Beijing: Atomic Energy Press, 1987.530-531.

[11] 姜志新,谌竟清,宋正孝.离子交换分离工程.天津: 天津大学出版社, 1992. 344-346. Jiang Z X, Zhan J Q, Song Z X. Ion Exchange Separation Engineering.Tianjin: Tianjin University Press,1992. 344-346.

[12] 中国电力企业联合会标准部.电厂化学(火电卷第六分册). 北京:中国电力出版社,477-478. China Electricity Council Standards. Plant Chemistry(Thermal power volume sixth volumes).China Electric Power Press, 477-478.

[13] 袁彦超,陈炳稔,王瑞香.甲醛、环氧氯丙烷交联壳聚糖树脂的制备及性能.高分子材料科学与工程, 2004, 20(1): 53-57. Yuan Y C,Chen B R,Wang R X.Studies of properties and preparation of chitosan resin crosslinked by formaldehyde and epichlorohydrin.Polymeric Materials Science & Engineering,2004,20(1):53-57.

[14] 黄文强,何炳林.离子交换与吸附树脂.上海: 科技教育出版社,1995. 96-100. Huang W Q,He B L. Ion Exchange and Adsorption Resins.Shanghai: Science and Technology Education Press, 1995. 96-100 .

[15] 余艺华,孙彦,何炳林.交联壳聚糖树脂的制备工艺及性能表征.天津大学学报,2000, 33(1): 113-117. Yu Y H, Sun Y, He B L. Preparation and characterization of crosslinked chitosan resin.Journal of Tianjin University, 2000, 33(1): 113-117.

[16] 赵绪光,高秀峰,郑雪妮,等.重组嗜碱耐盐芽孢杆菌(XJU-1)乙醛脱氢酶aldA基因在工程菌BL21(DE3)中的表达和酶学性质研究. 酿酒科技, 2010, 193(7): 21-23. Zhao X G, Gao X F, Zheng X N, et al.Expression and enzymological characters of aldehyde dehydrogenase gene (aldA) of Bacillus halodurans XJU-1 in E.coli BL21(DE3). Liquor-Making Science & Technology, 2010,193(7): 21-23.

[1]	肖云喜,张俊河,杨雯雯,程洪伟. 用于疫苗生产的人二倍体细胞研究进展^*[J]. 中国生物工程杂志, 2021, 41(11): 74-81.
[2]	杨威,宋方祥,王帅,张黎,王红霞,李焱. 药物输送系统中Janus纳米粒子的制备及应用 ^*[J]. 中国生物工程杂志, 2020, 40(7): 70-81.
[3]	陈春琳,秦松,宋宛霖,刘志丹,刘正一. 褐藻寡糖生物法制备研究进展 ^*[J]. 中国生物工程杂志, 2020, 40(10): 85-95.
[4]	武慧蓉,温朝辉. 壳聚糖在神经组织工程中的应用 ^*[J]. 中国生物工程杂志, 2019, 39(6): 73-77.
[5]	许嘉越,李紫倩,张革. 登革病毒3'UTRΔ30系列疫苗的研究进展[J]. 中国生物工程杂志, 2019, 39(3): 97-104.
[6]	程功,焦思明,任立世,冯翠,杜昱光. 枯草芽孢杆菌壳聚糖酶水解制备低脱乙酰度壳寡糖及其组分分析 ^*[J]. 中国生物工程杂志, 2018, 38(9): 19-26.
[7]	康肸,邓爱鹏,杨树林. 壳聚糖基温敏水凝胶的研究进展[J]. 中国生物工程杂志, 2018, 38(5): 79-84.
[8]	段思腾,李光然,马义勇,邱裕佳,李宇,王伟. 负载NGF的可注射壳聚糖透明质酸水凝胶材料理化性能及生物相容性研究[J]. 中国生物工程杂志, 2018, 38(4): 70-77.
[9]	张莉,丁涓,郝宇晨,叶城,蒲洋. 一株海洋微藻的鉴定及其原生质体制备条件优化 ^*[J]. 中国生物工程杂志, 2018, 38(11): 42-50.
[10]	张杨玲,汪园,张革. 埃博拉病毒疫苗rVSV-ZEBOV的研究进展[J]. 中国生物工程杂志, 2018, 38(1): 51-56.
[11]	李大为, 何进, 何凤利, 刘雅丽, 邓旭东, 叶雅静, 尹大川. 丝素蛋白/壳聚糖复合材料在组织工程中应用的研究进展[J]. 中国生物工程杂志, 2017, 37(10): 111-117.
[12]	王佃亮. 讲座细胞药物的制备工艺——细胞药物连载之二[J]. 中国生物工程杂志, 2016, 36(7): 127-133.
[13]	钱建瑛, 许正宏, 窦文芳. 融合蛋白GGH粉针剂制备工艺研究[J]. 中国生物工程杂志, 2016, 36(11): 48-53.
[14]	佘栋宇, 黄嘉慧, 刘东晨, 张途, 谢秋玲. Her2抗体与MMAE偶联物的制备及生物活性研究[J]. 中国生物工程杂志, 2015, 35(2): 66-71.
[15]	李婷, 孙静, 赵相哲, 连立强, 谢富强. 骨形成蛋白2/珍珠层粉/壳聚糖支架制备及生物性能研究[J]. 中国生物工程杂志, 2015, 35(11): 1-6.

Viewed

Full text

Abstract

Cited

Shared

Discussed