牛胰蛋白酶制备工艺的改进及其对基因工程人胰岛素前体的酶切作用

Innovation of the preparative process of bovine trypsin and its enzymatic degradation effect on the precursor of genetic engineering human insulin

目的对牛胰蛋白酶制备工艺进行改进,制备高纯度牛胰蛋白酶,并对其酶切基因工程人胰岛素前体的作用进行考察。方法采用粗制后先色谱分离再活化的方法对传统工艺进行改进,对传统工艺制备样品和改进工艺制备样品中的胰蛋白酶和胰凝乳蛋白酶的比活力进行测定和比较;进一步采用这2种样品对基因工程人胰岛素前体进行酶切,对酶切作用进行比较。结果传统工艺制备样品中胰蛋白酶比活力为212.5 U·mg-1,胰凝乳蛋白酶比活力为20.4 U·mg-1;而改进工艺样品中胰蛋白酶比活力为240.4 U·mg-1,胰凝乳蛋白酶比活力为0.14 U·mg-1。传统工艺制备样品酶切基因工程人胰岛素前体目标产物纯度质量分数为16.3%,改进工艺制备样品酶切基因工程人胰岛素前体目标产物纯度质量分数为32.2%。结论粗制后先进行SP色谱可实现胰蛋白酶原和胰凝乳蛋白酶原较好的分离,该方法适合于高纯度胰蛋白酶的制备,制备的胰蛋白酶适合作为工具酶进行基因工程人胰岛素前体的酶切。

Objective To isolate and purify bovine trypsin with high purity and determine the activation of the genetic engineering human insulin prosoma with it. Methods The traditional preparative process of bo-vine trypsin was improved. The crude product was firstly separated by column chromatography and then was activated. The activities of trypsin and chymotrypsin of the samples, from the traditional method and the im-proved way, were assayed and compared. Then the genetic engineering human insulin precursor was enzy-matically degradated by these 2 samples and the target product was detected by HPLC. The effects of these two samples were compared. Results The specific activity was 212.5 and 20. 4 U·mg^-1 for trypsin and chy- motrypsin in traditionally prepared sample, while that was 240. 4 and 0. 14 U·mg^-1 in the improved method. Conclusions Trypsinogen and chymotrypsinogen can be better separated by SP column chromatography. This method is suitable for the preparation of trypsin with higher purity, and the product is better for the degrada-tion of the precursor of genetic engineering human insulin.