Shinomiya Kazufusa, Kobayashi Hiroko, Inokuchi Norio, Nakagomi Kazuya, Ito Yoichiro
School of Pharmacy, Nihon University, Chiba, Japan.
J Liq Chromatogr Relat Technol. 2011 Jan;34(3):182-194. doi: 10.1080/10826076.2011.546151.
Partition efficiency of the high-pitch locular multilayer coil was evaluated in countercurrent chromatographic (CCC) separation of proteins with an aqueous-aqueous polymer phase system using the small-scale cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory. The separation column was specially made by high-pitch (ca 5 cm) winding of 1.0 mm I.D., 2.0 mm O.D. locular tubing compressed at 2 cm intervals with a total capacity of 29.5 mL. The protein separation was performed using a set of stable proteins including cytochrome C, myoglobin, and lysozyme with the 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate system (pH 9.2) under 1000 rpm of column revolution. This high-pitch locular tubing yielded substantially increased stationary phase retention than the normal locular tubing for both lower and upper mobile phases. In order to demonstrate the capability of the high-pitch locular tubing, the purification of collagenase from the crude commercial sample was carried out using an aqueous-aqueous polymer phase system. Using the 16.0% (w/w) PEG 1000 - 6.3% (w/w) dibasic potassium phosphate - 6.3% (w/w) monobasic potassium phosphate system (pH 6.6), collagenase I, II, V and X derived from Clostridium hystolyticum were separated from other proteins and colored small molecular weight compounds present in the crude commercial sample, while collagenase N-2 and S-1 from Streptomyces parvulus subsp. citrinus were eluted with impurities at the solvent front with the upper phase. The collagenase from C. hystolyticum retained its enzymatic activity in the purified fractions. The overall results demonstrated that the high-pitch locular multilayer coil is effectively used for the CCC purification of bioactive compounds without loss of their enzymatic activities.
使用我们实验室制造的小型交叉轴盘管行星离心机（X轴CPC），在蛋白质的逆流色谱（CCC）分离中，采用水-水聚合物相系统，评估了高螺距分室多层盘管的分配效率。分离柱是通过对内径1.0 mm、外径2.0 mm的分室管进行高螺距（约5 cm）缠绕制成的，每隔2 cm压缩一次，总容量为29.5 mL。使用一组稳定的蛋白质，包括细胞色素C、肌红蛋白和溶菌酶，在12.5%（w/w）聚乙二醇（PEG）1000和12.5%（w/w）磷酸氢二钾系统（pH 9.2）中，以1000 rpm的柱转速进行蛋白质分离。对于上下流动相，这种高螺距分室管产生的固定相保留量比普通分室管显著增加。为了证明高螺距分室管的性能，使用水-水聚合物相系统对粗制商业样品中的胶原酶进行了纯化。使用16.0%（w/w）PEG 1000 - 6.3%（w/w）磷酸氢二钾 - 6.3%（w/w）磷酸二氢钾系统（pH 6.6），将溶组织梭菌来源的胶原酶I、II、V和X与粗制商业样品中存在的其他蛋白质和有色小分子化合物分离，而短小链霉菌亚种柠檬黄变种的胶原酶N - 2和S - 1则与杂质一起在上相的溶剂前沿被洗脱。溶组织梭菌的胶原酶在纯化组分中保留了其酶活性。总体结果表明，高螺距分室多层盘管可有效地用于生物活性化合物的CCC纯化，而不会损失其酶活性。
