酶法结合D500大孔树脂分离纯化粗品肝素

Enzymatic-D500 macroporous resin synergistic separation and purification of crude heparin

目的探讨酶法结合D500大孔树脂吸附交换分离纯化粗品肝素的效果,以替代传统的氧化法。方法以肝素粗品为原料,采用胰蛋白酶水解法去除原料中的蛋白杂质,结合大孔树脂吸附交换精制粗品肝素。通过生色底物法测定精品肝素的效价,并计算效价回收率。结果酶解液用D500大孔树脂分离纯化的最佳动态吸附条件为:吸附温度55℃,肝素溶液的盐浓度2.8°Bé,肝素溶液p H 8.5,进样浓度2.5 mg/ml,流速1.5 ml/min;最佳解吸条件为:解吸温度50℃,洗脱速度1.0 ml/min,洗脱剂浓度19°Bé,p H 8.5。在此条件下制得的精品肝素效价最高达189 IU/mg,比原料(78 IU/mg)提高2.42倍,且效价回收率为97.2%。结论利用蛋白酶结合大孔树脂吸附交换的方法对粗品肝素进行精制,可减少对精制过程中肝素生物效价的破坏,且提高了精品肝素的得率,该方法可替代传统氧化法精制肝素,为粗品肝素的分离纯化提供了新的方法。

Objective To investigate the effect of separation and purification of crude heparin by enzymatic hydrolysis combined with D500 macroporous resin synergistic absorption and exchange methods instead of the traditional oxidation process. Methods Crude heparin as a raw material was purified by hydrolysis with trypsin then by macroporous resin synergistic absorption and exchange. The potency of purified heparin was determined by chromogenic substrate method,and the recovery rate of potency was calculated. Results The optimal temperature, salt concentration, p H value, sample concentration for loading and flow rate for separation and purification of hydrolyzate by D500 macroporous resin were2. 8° Bé, 8. 5, 2. 5 mg / ml and 1. 5 ml / min respectively. However, the optimal condition for desorption was as follows：desorption temperature 50 ℃, elution rate 1. 0 ml / min, eluent concentration 19° Bé, p H 8. 5. Under the optimized condition, the potency of purified heparin reached 189 IU / mg, which was 2. 42 times higher than that of raw material（78 IU / mg）, and the recovery rate was 97. 2%. Conclusion The application of enzymatic hydrolysis and macroporous resin methods for purification of heparin decreased the loss of bioactive potency of heparin, and increased the yield of purified heparin. This research provided a new approach for separation and purification of crude heparin.