Performance of an aqueous two-phase-based countercurrent chromatographic system for horseradish peroxidase purification
Performance of an aqueous two-phase-based countercurrent chromatographic system for horseradish peroxidase purification
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DOI：
10.1002/jssc.200301532
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发表时间：
2003-12-01
影响因子：
3.1
通讯作者：
Cascone, O
中科院分区：
文献类型：
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作者：
Magri, ML;Cabrera, RB;Cascone, O
采用聚合物-磷酸盐双水相系统（ATPS）对山毛榉根提取物中的辣根过氧化物酶（HRP）进行了逆流色谱（CCC）纯化。通过在1000 rpm下使用制备柱，获得了由10%w/w PEG 1540和14.8%w/w磷酸盐组成的ATPS的顶相的25-30%保留率，其中加入了2 mol/kg氯化钠。在标准分离运行时间（4 h）内，保留水平稳定。将辣根根提取物样品注入系统中（10-25 mL; 200-250 U/mL过氧化物酶; 2.0-4.0 mg/mL总蛋白）。在所选的ATPS中，在色谱运行过程中，HRP在CCC“柱”中的保留率达到，其中酶的分配系数K大于或等于8。用新鲜的但不加盐的流动相替换移动的相，得到产物洗脱。该级分中HRP的回收率大于或等于负载的总活性的45%，纯化因子为6。酶活性也被发现在通过馏分和在剩余的液体（固定）相，一个事实，应归因于多种过氧化物酶异构体的存在。活性级分的SDS-PAGE显示在44 kDa处的蛋白质条带，与HRP的存在相容。因此，优化的CCC系统允许直接从复杂的生物材料中分离HRP。这些结果开辟了实现蛋白质分离与CCC/ ATPS和放大工业分离器的过程的可能性。
Countercurrent chromatography (CCC) purification of horseradish peroxidase (HRP) from Armoracia rusticana root extracts was achieved by employing polymer-phosphate aqueous two-phase systems (ATPS). By using preparative columns at 1000 rpm, a 25-30% retention of the top phase of an ATPS composed of 10% w/w PEG 1540 and 14.8% w/w phosphate - with added 2 mol/kg sodium chloride - was obtained. The retention level was stable during the standard separation running time (4 h). Horseradish root extract samples were injected into the system (10-25 mL; 200-250 U/mL peroxidase; 2.0-4.0 mg/mL total protein). Retention of HRP in the CCC "column" during the chromatographic run was attained in the selected ATPS, where the partition coefficient K for the enzyme was greater than or equal to 8. Replacement of the mobile phase with a fresh one but in the absence of added salt brought about product elution. Recovery of HRP in this fraction accounts for greater than or equal to 45% of the total activity loaded, with a purification factor of 6. Enzyme activity was also found in the pass-through fraction and in the remaining liquid (stationary) phase, a fact that should be ascribed to the existence of multiple peroxidase isoforms. SDS-PAGE of the active fraction showed a protein band at 44 kDa, compatible with the presence of HRP. Thus, the optimised CCC system allowed the separation of HRP directly from a complex biological material. These results open up the possibility of achieving protein separation with CCC/ ATPS and of scaling-up processes in industrial separators.