壳聚糖-硅基凝胶微球作为固定化金属螯合亲和色谱基质的研究  

Chitosan-silica Hybrid Matrix for Immobilized Metal Chelated Affinity Chromatography

作　　者：钟莺莺[1] 叶俊超[1] 邬建敏[1] 

机构地区：[1]浙江大学理学院化学系,杭州310027

出　　处：《分析化学》2007年第11期1581-1585,共5页Chinese Journal of Analytical Chemistry

摘　　要：采用溶胶凝胶及微乳液技术制备了以壳聚糖-硅基杂化材料为骨架并带有金属离子螯合官能团的球形基质(CSHB),并对该基质的制备条件及结构形貌进行了研究与表征。实验表明,当微乳液反应体系的组分为:100mL壳聚糖溶液(2%m/V)、100mL Span乳化剂、250mL环己烷、13.3mL四乙氧基硅烷(TEOS)、1.33mL 3-缩水甘油丙氧基三甲氧基硅烷(GPTMS)和亚氨基二乙酸(IDA)0.802g时,可获得粒径均匀,刚性较好的微球。红外光谱证明了该基质是一种多组分的杂合材料,差热分析数据表明该杂合材料的热稳定性随反应体系中GPTMS的含量增加而增大。CSHB通过动态吸附金属离子Cu2+与Ni2+后,可对金属螯合蛋白产生配位吸附作用。Cu2+-CSHB柱对牛血清蛋白(BSA)具良好的可逆吸附能力,蛋白能被咪唑等金属离子螯合剂洗脱,回收率达76.6%。BSA在CSHB柱上的吸附率只有4.7%,表明CSHB对蛋白的非特异吸附较低。Ni2+-CSHB柱对过氧化氢酶(CAT)也显示出初步的纯化效果,一步纯化倍数为2.43倍。该基质有望用于具有组氨酸纯化标签的基因工程表达蛋白的分离与纯化。Chitosan-silica hybrid beads （CSHB）bearing with metal ion chelating group was prepared by solgel method and micro-emulsion technique. The condition for the preparation of CSHB, as well as its structure and morphology were investigated. The experimental results show that rigid beads with narrow size distribution could be obtained, when the component in micro-emulsion system was controlled in the following conditions： 100 mL chitosan （2% , m/V） , 100 mL span emulsifier, 250 mL cyclohexane, 13.3 mL Tetraethoxysilane （TEOS）, 1.33 mL 3-Glycidoxypropyhrimrthoxysilane （GVTMS）and 0. 802 g Iminodiacetic acid （IDA）. FTIR verified that the prepared beads were multi-component hybrid materials. The data of DSC-TG show that phase transition peak of the beads shift to higher temperature with the increasing of GVITMS content, which indicate that GVTMS plays an important role in enhancing the interaction of different component. The CSHB could adsorb metal chelating protein through coordinating force after the beads was loaded with heavy metal ions such as Cu^2＋ and Ni^2＋. Bovine serum albumin （BSA） could be reversibly adsorbed on Cu^2＋-CSHB column and eluted by imidazole with recovery rate of 76.6% ; meanwhile only 4.7% BSA was adsorbed on CSHB column, indicating the low nonspecific adsorption of BSA on the column. Ni^2＋ -CSHB column also show some purification effect for the catalase （CAT）. The enzyme purification fold could reach 2.43 after one step of process. This novel hybrid beads could be potentially use in the purification of his-tagged protein in bioengineering process.

关 键 词：溶胶凝胶 壳聚糖-硅基杂化凝胶 金属螯合亲和层析 蛋白纯化 

分 类 号：O657.7[理学—分析化学]