Tong X D, Xue B, Sun Y
Department of Biochemical Engineering, Tianjin University, Tianjin 300072, People's Republic of China.
Biotechnol Prog. 2001 Jan-Feb;17(1):134-9. doi: 10.1021/bp000134g.
A novel magnetic support was prepared by an oxidization-precipitation method with poly(vinyl alcohol) (PVA) as the entrapment material. Transmission electron microscopy indicated that the magnetic particles had a core-shell structure, containing many nanometer-sized magnetic cores stabilized by the cross-linked PVA. The particles showed a high magnetic responsiveness in magnetic field, and no aggregation of the particles was observed after the particles had been treated in the magnetic field. These facts indicated that the particles were superparamagnetic. Cibacron blue 3GA (CB) was coupled to the particles to prepare a magnetic affinity support (MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption properties of the MAS. The adsorption equilibrium of lysozyme to the MAS was described by the Langmuir-type isotherm. The capacity for lysozyme adsorption was more than 70 mg/g MAS (wet weight) at a relatively low CB coupling density (3-5 micromol/g). In addition, 1.0 M NaCl solution could be used to dissociate the adsorbed lysozyme. Finally, the MAS was recycled for the purification of alcohol dehydrogenase (ADH) from clarified yeast homogenates. Under proper conditions, the magnetic separation yielded over 5-fold purification of the enzyme with 60% recovery of the enzyme activity.
采用氧化沉淀法，以聚乙烯醇（PVA）作为包埋材料制备了一种新型磁性载体。透射电子显微镜表明，磁性颗粒具有核壳结构，包含许多由交联PVA稳定的纳米级磁核。这些颗粒在磁场中表现出高磁响应性，并且在磁场中处理后未观察到颗粒聚集。这些事实表明这些颗粒是超顺磁性的。将汽巴克隆蓝3GA（CB）偶联到颗粒上，制备用于蛋白质吸附的磁性亲和载体（MAS）。以溶菌酶作为模型蛋白来测试MAS的吸附特性。溶菌酶对MAS的吸附平衡用朗缪尔型等温线描述。在相对较低的CB偶联密度（3 - 5 μmol/g）下，溶菌酶的吸附容量超过70 mg/g MAS（湿重）。此外，1.0 M NaCl溶液可用于解离吸附的溶菌酶。最后，将MAS循环用于从澄清的酵母匀浆中纯化乙醇脱氢酶（ADH）。在适当条件下，磁性分离使该酶的纯化倍数超过5倍，酶活性回收率为60%。
