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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 enzymatic combined with D500 macroporous resin on the separation and purification of crude heparin to replace the traditional oxidation method. Methods Crude heparin was used as raw material, and the protein impurities in raw materials were removed by trypsin hydrolysis. The crude heparin was purified by adsorption and exchange with macroporous resin. The titer of fine heparin was determined by the chromogenic substrate method and the titer recovery was calculated. Results The optimum conditions for the separation and purification of the hydrolyzate with D500 macroporous resin were as follows: the adsorption temperature was 55 ℃; the salt concentration in heparin solution was 2.8 ° Bé; the heparin solution was pH 8.5; the injection concentration was 2.5 mg / ml; the flow rate was 1.5 ml / min. The optimal desorption conditions were as follows: desorption temperature 50 ℃, elution speed 1.0 ml / min, eluent concentration 19 ° Bé, p H 8.5. The best heparin titer was 189 IU / mg, which was 2.42 times higher than that of the raw material (78 IU / mg), and the titer of recovery was 97.2%. Conclusion Purification of crude heparin by the method of protease combined with macroporous resin adsorption exchange can reduce the damage of heparin biological potency during the purification process and improve the yield of fine heparin. This method can replace the traditional oxidation method to purify heparin. Purification of crude heparin provides a new method.